Glycoproteins

GLYCOPROTEINS

.

By ROBERT G SPlRO Departments of Biological Chemistry and Medicine. Harvard Medical School. and the Elliott P Joslin Research Loboratory. Boston. Massachusetts

.

I. Introduction . . . . . . . . . . . . . I1. Definition . . . . . . . . . . . . . . I11. Distribution and Biological Properties . . . . . . . IV . Isolation . . . . . . . . . . . . . . V. Composition and Methods of Analysis . . . . . . . VI. Glycopeptides . . . . . . . . . . . . . VII . Carbohydrate-Peptide Linkages . . . . . . . . . A . Glycosylamine Bond Involving Asparagine . . . . . B. 0-Glycosidic Bond to Serine or Threonine . . . . . . C. 0-Glycosidic Bond to Hydroxylysine . . . . . . . D . Other Glycopeptide Bonds . . . . . . . . . VIII . Sequence of Amino Acids around Glycopeptide Bonds . . . A . Bond Involving Asparagine . . . . . . . . . B. Bond Involving Serine or Threonine . . . . . . . C . Bond Involving Hydroxylysine . . . . . . . . I X . Nature of the Carbohydrate Units . . . . . . . . A. Asparagine-Linked Units . . . . . . . . . . B. Serine(Thre0nine)-Linked Units . . . . . . . . C . Hydroxylysine-Linked Units . . . . . . . . . X . Proteins with More Than One Type of Carbohydrate Unit . . X I . Methods for Structural Analysis of Carbohydrate Units . . . XI1. Heterogeneity of Carbohydrate Units . . . . . . . XI11. Structure of Carbohydrate Units of Specific Glycoproteins . . . A . Plasma Glycoproteins . . . . . . . . . . B . Immunoglobulins . . . . . . . . . . . C. Hormones . . . . . . . . . . . . . D . Enzymes . . . . . . . . . . . . . E. Hen Egg Glycoproteins . . . . . . . . . . F. Glycoproteins of Mucous Secretions . . . . . . . G . Collagens and Basement Membranes . . . . . . . H. Proteoglycans . . . . . . . . . . . . I. Glycoproteins of Plasma Membranes . . . . . . . XIV . Physical Properties . . . . . . . . . . . . XV . Concepts of Glycoprotein Biosynthesis . . . . . . . XVI . Concepts of Glycoprotein Catabolism . . . . . . . XVII . Biological Role of the Carbohydrate Portion of Glycoproteins . References . . . . . . . . . . . . . .

349

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350 350 350 354 355 358 360 361 363 365 366 367 367 372 374 375 375 378 382 382 385 393 398 398 403 407 411 416 419 424 430 434 440 445 447 449 455

350

ROBERT G. SPIRO

I. INTRODUCTION One of the major modifications that a protein may undergo after synthesis of its peptide chains is the attachment of sugar residues. A large number of proteins of diverse origin and biological function are known to contain such covalently linked carbohydrate and are designated as glycoproteins. In the fifteen years that have elapsed since the last review of the carbohydrate-containing proteins was written for this series (Bettelheim-Jevons, 1958), remarkable progress has been made in an understanding of the basic structural features that characterize this group of compounds. This knowledge pertains not only to the nature of the covalent bonds that serve to link saccharides to the peptide chain, but also to the structure, number, and distribution of the carbohydrate units attached to the protein in this manner. Close on the heels of this enlightenment in regard to the structural chemistry of glycoproteins have come numerous significant studies describing the enzymatic machinery involved in the biosynthesis and degradation of these carbohydrate-containing molecules. More recently, attention has been focused on a clarification of the biological role which the carbohydrate may play in fulfilling the diverse functions of glycoproteins and in regulating their intracellular migration and export. While it is the purpose of this review to deal primarily with the structural aspects of the glycoproteins, whenever possible an attempt will be made to show how these are reflected in important biological properties of the molecules. Since the article by Bettelheim-Jevons (1958), a number of other general reviews on this subject have appeared, including those of Spiro (1963, 1969a, 1970a), Marshall and Neuberger (1970), Montgomery (1970), and the comprehensive compilations edited by Gottschalk (1966, 1972). 11. DEFINITION As already indicated, glycoproteins can be simply defined as proteins to which carbohydrate is covalently attached. This classification makes no distinction among carbohydrate complexes on the basis of the number or size of the saccharide units, and therefore also includes those compounds often referred to as mucopolysaccharides or proteoglycans. Biosynthetic studies on a number of glycoproteins from diverse sources have indicated that these proteins have in common not only covalently linked carbohydrate units, but a similar type of enzymatic machinery to attach the sugar residues to the completed peptide chain. 111. DISTRIBUTION AND BIOLOGICAL PROPERTIES It is evident from Table I that glycoproteins are widely distributed in nature, occurring not only in vertebrate and invertebrate animals but

351

GLYCOPROTEINS

TABLE I Distrihtion and Function of Some Glycoproteins ~~

Source. Vertebrates Plasma

Function Transport

Unknown Clotting Immunoprotective Freezing pointdepressing Enzyme

Urine Milk Saliva Pituitary Pancreas Liver

~~

Examplesb Transferrin (l), ceruloplasmin (a), thyroxine-binding globulin (3), corticosteroidbinding globulin (4), &lipoprotein (5), vitamin Blz-binding glycoprotein (6) Fetuin (7), aI-acid glycoprotein (8), a2macroglobulin (9) Fibrinogen (lo), prothrombin (11) Immunoglobulins; IgG (12), IgA (13), IgM (14); Clq complement (15) Antarctic fish glycoprotein (16)

Cholinesterase (17), atropinesterase (18), amine oxidase (19) Unknown Tamm and Horsfall glycoprotein (20) Hormone Chorionic gonadotropin (21) Transport Lactotransferrin (22) Lactose synthetase (A protein) (23) Enzyme Nutrition K-Casein (24) Enzyme a-Amylase (25) Hormone Follicle-stimulating hormone (20), luteinizing hormone (27), thyroid-stimulating hormone (27) Enzyme ltibonuclease (28, 29), deoxyribonuclease (30), lipase (31),kallikrein (32). Enzyme 8-N-Acetylglucosaminidase(33), 8-glucuronidase (34) Enzyme Hyaluronidase (35) Unknown Soluble glycoproteins (36) Hormone storage Thyroglobulin (37) Lubrication, protection Submaxillary glycoproteins (38, 39)

Testes Aorta Thyroid Submaxillary gland Gastrointestinal Lubrication, protection tract Stomach Enzyme Transport Kidney Enzyme Filter, cytoskeleton Lung Anticoagulant Skin Structural Cartilage Structural Lens Support, filter Retina Sensory process Cell membranes Receptors, cell adhesion Bird nest Cementing

Glycoproteins of mucous secretions (40,41) Pepsinogen and pepsin (42) Intrinsic factor (43) 7-Glutamyl transpeptidase (44) Glomerular basement membrane (45) Heparin (46) Collagen (47, 48) Proteoglycan (49, 50) Lens capsule (51) Visual pigment (52) Red cell membrane glycoproteh (63) Colloculia mucoid glycoprotein (54) (Continued)

ROBERT G . SPIRO

TABLE I (Continued) Sourcea

Function

Egg white

Nutrition, protective

Egg Yolk Snake venom Invertebrates

Nutrition Enzymes Protective Structural Transport Fertilization process Protective Lubrication

Green plank

Cell aggregation Enzymes

Fungi

Unknown Structural Enzymes

Bacteria

Structural Enzyme Unknown

Virus

Unknown

Exampled Ovalbumin (55), ovomucoid (56), avidin (57 ) Phosvitin (58) Proteinases (59) Annelid cuticle collagens (60, 61) Collagens (62) Snail hemocyanin (63) Sea urchin jelly coat glycoprotein (64) Silk fibroin from Bombyx mori cocoons (65) Glycoprotein from mucous secretions of whelk (66) Glycoproteins of sponge cells (67) Pineapple stem bromelain (68, 69), horseradish peroxidase (70) Phytohemagglutinins (71) Cell wall glycoproteins (72) Taka-amylase (73), glucose oxidase (74), glucoamylase (75), diphosphopyridine nucleosidase (76), chloroperoxidase (77), lipase (78), a-galactosidase (79), invertase (80) Glycoproteins of yeast cell wall (81) Bacillus subtilis diphosphopyridine nucleosidase (76); Micrococcus sodonensis nuclease (82) Escherichia coli cell membrane glycoprotein (83) Membrane glycoproteins (84)

Refers to source of isolation. numbers in parentheses: (1) Jamieson (196513); (2) Jamieson (1965a); (3) Giorgio and Tabachnick (1968); (4) Muldoon and Westphal (1967); (5) Marshall and Kummerow (1962); (6) Kidroni and Grossowicz (1969); (7) Spiro (1960); (8) Yamashina (1956); (9) Dunn and Spiro (1967a); (10) Bray and Laki (1968); (11) Lanchantin et al. (1968); (12) Rosevear and Smith (1961); (13) Dawson and Clamp (1968); (14) Miller and Metzger (1965); (15) Calcott and Muller-Eberhard (1972); (16) DeVries (1971); (17) Svensmark and Heilbronn (1964); (18) Margolis and Feigelson (1964); (19) Watanabe and Yasunobu (1970); (20) Fletcher et al. (1970); (21) Bahl (1969a); (22) Castellino et al. (1970); (23) Trayer and Hill (1971); (24) Mackinlay and Wake (1965); (25) Keller et al. (1971); (26) Cahill et al. (1968); (27) Liao and Pierce (1970); (28) Plummer and Hirs (1964); (29) Reinhold et al. (1968); (30) Cately et al. (1969); (31) Garner and Smith (1972); (32) Fritz et al. (1967); (33) Robinson and Stirling (1968); (34) Plapp and Cole (1967); (35) Borders and Raftery (1968); (36) Radhakrishnamurthy and Berenson (1966); (37) Spiro and Spiro (1965a); (38) Graham and Gottschalk (1960); (39) Carlson (1968); (40) Pamer et al. (1968); (41) Inoue and Yosizawa (1966); (42) Bohak (1969); (43) Grlisbeck et al. (1966); (44) Szewczuk and Connel (1964); (45) Spiro (1967a); (46) Lindahl and R o d h (1965); (47) Butler and Cunningham a

* References given by

GLYCOPROTEINS

353

also in plants, unicellular organisms, and even viruses. Indeed it is already clear that a considerable portion of the polymerized carbohydrate of higher animals is covalently conjugated to protein, and it is quite likely that a similar situation may prevail in the sugar polymers of less evolved animals, as well as in plants. While attention was directed initially to the isolation and characterization of the numerous glycoproteins present in body fluids, such as plasma, saliva, and gastrointestinal secretions, and to those which can be readily extracted in soluble form from various glandular and supportive tissues, increasing emphasis is being focused a t the present time on the study of the insoluble glycoproteins which are components of complex structures, such as plasma membranes, basement membranes, and plant cell walls. The known or presumed functions of glycoproteins are diverse, spanning a wide range of vital biological activities (Table I). Almost all the proteins of plasma, with the notable exception of albumin, contain carbohydrate and fulfill such varied roles as transport, clotting, and antibody activity. Gonadotropins from both pituitary and placental origin are glycoproteins, as are thyroid-stimulating hormone and thyroglobulin, the thyroid hormone storage protein. A rapidly increasing number of proteins with enzyme activity, including various hydrolases, oxidases, and transferases, are being reported to contain covalently bound carbohydrate. These originate from a large variety of tissues and from organisms throughout the phylogenetic scale. The protective and lubricating roles of the glycoproteins from epithelial secretions are well known. It is evident now that the members of the large collagen family are glycoproteins and that they along with the proteoglycans and various soluble glycoproteins make up the bulk of the intercellular matrix which provides structural support to multicellular organisms. Extracellular structures,

(1966); (48) Spiro (1969b); (49) Luscombe and Phelps (1967a); (50) Hascall and Sajdera (1970); (51) Fukushi and Spiro (1969); (52) Heller (1968); (53) Winzler (1969); (54) Kathan and Weeks (1969); (55) Johansen et al. (1961); (56) Montgomery and Wu (1963); (57) Huang and DeLange (1971); (58) Shainkin &ndPerlmann (1971a); (59) Oshima et al. (1968); (60) Muir and Lee (1970); (61) Spiro and Bhoyroo (1971); (62) Spiro (1972a); (63) Dijk et al. (1970); (64) Hotta et al. (1970); (65) Sinohara et al. (1971); (66) Hunt and Jevons (1965); (67) Margoliash el al. (1965); (68) Scocca and Lee (1969); (69) Yasuda et al. (1970); (70) Shannon et at. (1966); (71) Sharon and Lis (1972); (72) Lamport (1969); (73) Anai et al. (1966); (74) Pazur et al. (1965); (75) Pazur et al. (1963); (76) Everse and Kaplan (1968); (77) Morris and Hager (1966); (78) S6mBriva et al. (1969); (79) Suzuki et al. (1970); (80) Meachum et al. (1971); (81) Sentandreau and Northcote (1968); (82) Berry et al. (1970); (83) Okuda and Weinbaum (1968); (84) Strauss et al. (1970).

354

ROBERT G. SPIRO

such as basement membranes and cell walls, serve as supportive elements and may also function in the capacity of coarse sieves. Perhaps most intriguing of all is the biological function which the glycoprotein components of the plasma membranes of cells may have. These membranes, which separate the cell from its external environment, appear to play a crucial role in active transport of molecules, to serve as receptors for viruses, hormones, and antibodies, and to take part in intercellular recognition and adhesion. While the physiological function of many glycoproteins seems to be well established, the role which the carbohydrate plays in helping these proteins carry out their activities is in many cases much less clear and will be discussed in a subsequent section of these review. IV. ISOLATION Since glycoproteins span almost the entire spectrum of protein types and contain anywhere from less than 1% to 80% carbohydrate by weight, no uniform procedure can be employed in their isolation. I n general the techniques used are the classical methods of protein chemistry, involving precipitation, ion exchange chromatography, gel filtration, and zone electrophoresis and do not differ substantially from those employed for the purification of carbohydrate-free proteins. I n glycoproteins which contain only a small portion of their total weight as carbohydrate, the properties of the peptide portion will be the primary determinant of the behavior of the protein during the various isolation procedures. I n those proteins, however, in which the carbohydrate makes up a considerable portion of the total, such as some proteins of plasma, the proteins from mucous secretions, and the proteoglycans of connective tissue, i t will contribute greatly to the physical properties of the molecule. Such glycoproteins tend to be more soluble in aqueous solutions by virtue of their polar sugar constituents. For example, during the Cohn low-temperature ethanol fractionation procedure, the most soluble fraction (Fraction VI) contains glycoproteins rich in carbohydrate, such as fetuin from calf serum and the a,-acid glycoprotein and the zinc and barium a2glycoproteins from human serum. The high negative charge imparted to some glycoproteins by a large number of sialic acid or uronic acid and sulfate residues can be used to advantage in separation procedures employing anion exchange chromatography or zone electrophoresis. Moreover, such polyanionic proteins can be selectively precipitated by complex formation with quaternary ammonium salts, such as cetylpyridinium chloride, after adjustment to specific pH values and ionic strength. Glycoproteins of insoluble structures, such as plasma membranes can

GLYCOPROTEINS

355

usually be fractionated only after extraction with aqueous phenol or pyridine, sodium dodecyl sulfate, or similar reagents. The purification of glycoproteins is complicated by the characteristic heterogeneity of the carbohydrate units (Section XII) which can lead to the occurrence of multiple components which have identical peptide chains and differ only in their saccharide moiety. This phenomenon is well illustrated by ion exchange chromatography of ribonuclease from bovine (Plummer and Hirs, 1963; Plummer, 1968) and porcine (Reinhold et al., 1968) pancreatic juice by which several members of a family of each enzyme were obtained and shown to differ only in their carbohydrate composition. In the case of the porcine enzyme a t least eight major electrophoretically distinguishable components were shown to be present in the pancreatic juice.

V. COMPOSITION AND METHODS OF ANALYSIS As may be noted from Table 11, glycoproteins do not have a n amino acid composition distinct from that of other proteins. Moreover, they may differ strikingly from each other, as a comparison of the amino acid composition of collagen or basement membrane with that of a plasma protein such as fetuin will indicate. The sugars which have been found in the carbohydrate portion of glycoproteins include D-galactose, n-mannose, n-glucose, L-fucose, Dxylose, L-arabinose, D-glucosamine, D-galactosamine, D-glucuronic acid, L-iduronic acid, and the N - and O-acetyl and N-glycolyl derivatives of neuraminic acid (sialic acids). The hexosamines appear in the N-acetyl form except in the case of heparin and heparan sulfate, in which substitution of the amino groups with sulfate is found. The occurrence of O-sulfate groups has been reported in a number of proteins, which include in addition to the sulfated proteoglycans. glycoproteins from mucous secretions (Section XII1,F) , egg white (Section XIII,E), brain (DiBenedetta et al., 1969; Margolis and Margolis, 1970), and the jelly coat of the sea urchin egg (Hotta et al., 1970). Details of the methodology useful in the analysis of the saccharides of glycoproteins have been reviewed (Spiro, 1966, 1973). After mineral acid hydrolysis a t low protein concentration, neutral sugars can best be quantitated by automated borate-complex anion exchange chromatography or by gas-liquid chromatography of their alditol acetates. Hexosamines can be resolved and determined on the amino acid analyzer using either a conventional gradient or a t pH 5.0. Sensitive colorimetric methods are available for the determination of sialic acids (Warren, 1959), hexuronic acids (Dische, 1947), sulfate esters (Dodgson and Spencer, 1953), and acetyl groups (Ludowieg and Dorfman, 1960).

356

ROBERT G. SPIRO

TABLE

Composition of Several

Component

Fetuin

a2-Macroglobulin (human)

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine His tidine Arginine Tryptophan Hydroxyproline Hydroxylysine Amide

33 25 26 34 34 24 33 40 12 15 27 7 11 16 10 12 2 24

516 444 504 800 356 398 403 578 126 107 236 586 239 276 414 181 224 75 494

Galactose Mannose Glucose Fucose Glucosamine Galactosamine Sialic acids Xylose

12 8 15 3 14 -

62 94 13 146 48 -

6 2

3 1 2

-

1

(48)

(820)

(14.7)

(36)

(670)

(1)

(2)

(3, 4)

(5, 6)

(71

(MOL wt. Ref erencec

x

10-3)

Ribonuclease B (bovine)

Bromelain (pineapple stem)

Thyroglobulin (human)

15 9 14 12 4 3 12 9 7 4 2 2 5 3 10 4 4 -

400 283 484 697 347 394 383 310 244 62 155 476 110 259 195 80 275 117 -

17

29 14 28 23 14 35 35 22 10 5 21 10 21 9 23 2 12 8 42

-

-

-

_.

549

50 130

-

21 114 13 23

-

Expressed as moles per mole of glycoprotein except where indicated. Expressed as residues per 1000 total amino acid residues. References: (1) Spiro and Spiro (1962); (2) Dunn and Spiro (1967a); (3) Plummer and Hirs (1964); (4) Smyth el al. (1963); (5) Ota et al. (1964); (6) Yasuda et al. (1970); a

b

357

GLYCOPROTEINS

I1 Representative Glycoproteinsa

ThyroidSubmaxilstimulating lary gland hormone glycoprotein (bovine) (ovine) 14 18 10 14 12 7 12 9

1s

8 8 6 15 8 20 6 7

-

-

-

228 592 707 329 383 694 508 318 39 17 131 231 51 103 98 31 187

-

Ovalbumin (hen) 32 16 36 32 14 19 35 28

7

16 25 32 9 21 20

7

15 3 -

26

-

-

5

0.3 9 1 10 4 -

822 841 -

(27)

(1 x 103)

(8)

(9, 10)

-

-

31

Erythrocyte membrane Glomerular glycoTropobasement protein collagen membrane (human) (calf skin)b (bovine)b 6 14 14 10

7 7 7

8

5 5

4 4 4 4

4

-

-

23 4

-

-

3 -

-

9 18 28

(45)

(31)

-

2

-

45 18 36 72 138 320 112 20

__

4 11 25 3 13 27 5 50

-

94 7 46 2

-

1

-

68 37 55 96 69 208 61 38 31 14 29 59 18 28 27 16 49 6 68 22 68 20 5 16 2 10 1 4

Cuticle collagen (earthworm)b 60 50 87 84 7 318 98 17 1 1 16 29 2 6 18 1 25

-

179 1 96 60 2

-

-

-

3 1 1 3

2

2

d

114, 15)

(16)

(17)

(7) M. J. Spiro (1970); (8) Liao and Pierce (1970); (9) Bhargava and Gottschalk (1966); (10) Graham et al. (1963); (11) Tristram and Smith (1963); (12) Johansen et al. (1960); (13) Winzler (1969); (14) Piez and Gross (1960); (15) Spiro (1969b); (16) Spiro (1967a); (17) Spiro (1970b, 1972a).

358

ROBERT G. SPIRO

Amide analysis on glycoproteins containing sialic acid should be performed after selective removal of this sugar, either by mild acid hydrolysis or neuraminidase treatment. This is necessary, as approximately 25% of the amino groups of the sialic acid are released as ammonia under the conditions used for liberating the amide group ( 2 N HC1, 3 hours, 100°C). If the protein contains hexosamines, distillation of the ammonia after hydrolysis should be performed a t low temperature with 5 M K,CO, to prevent deamination of these sugars by the alkali (Spiro and Spiro, 1962)VI. GLYCOPEPTIDES A study of the carbohydrate portion of glycoproteins should focus on a number of major problems, which include a determination of the number, size, and structure of the units among which the sugar residues of the protein are apportioned, the nature of the sugar-amino acid linkage (s) which attach these units to the peptide chain (s), the distribution of such units among the chains of the protein, and the amino acid sequence on either side of the linkage site. Since many glycoproteins contain carbohydrate units and glycopeptide bonds of more than one type, an elucidation of these structural features usually requires extensive proteolytic digestion in order to obtain glycopeptides with only one linkage site. After digestion with proteases of broad specificity such as Pronase, papain, or subtilisin it is often possible to obtain the carbohydrate units with a minimum number of amino acids attached. Alternatively, proteolysis has been achieved with a combination of two or more specific proteases such as trypsin, chymotrypsin, collagenase, and pepsin and further shortening of the peptide brought about with exopeptidases. The glycopeptides in such digests arc usually of higher molecular weight than the other peptides due to the additional size imparted to them by the sugar components and due to the fact that steric hindrances imposed by the carbohydrate may have prevented them from being degraded as extensively by the proteases. They can therefore readily be resolved from noncarbohydrated peptides by gel filtration on columns of lowporosity in volatile buffers (Fletcher e t al., 1963; Arima et al., 1972). Gel filtration will also permit separation of glycopeptides that contain carbohydrate units that differ significantly in size, such as are present in digests of bovine glomerular basement membrane (Spiro, 1967b) (Fig. 1) and porcine ribonuclease (Jackson and Hirs, 1970). Further fractionation of glycopeptides is usually carried out by various forms of ion exchange chromatography which not only permits the separation of carbohydrate units of different types but also makes possible the resolution of

359

GLYCOPROTEINS

2.4

1

4240

TUBE NUMBER

FIG.1. Separation of glycopeptides by gel filtration of proteolytic digest. The collagenase-Pronase digest of 400 mg of bovine glomerular basement membrane was placed on a column (2.1 X 82 cm) of Sephadex G-25. Elution was performed with 0.1 M pyridine acetate buffer, pH 5.0, and fractions of 5.2 ml were collected. The two glycopeptide peaks emerge prior to the large amount of peptide material. Hexose by anthrone (-)

; peptide by ninhydrin (- - -). From Spiro (196713).

a given type into the variants which almost always exist (Section XII). DEAE-cellulose chromatography has been effectively used for the separation of the sialic acid-containing glycopeptides from such proteins as fetuin (Spiro, 1962a), a,-acid glycoprotein (Satake et al., 1965; Yamauchi and Yamashina, 1969), human and calf thyroglobulins (Spiro, 1965a; Arima et al., 1972), ceruloplasmin (Jamieson, 1965a), transferrin (Jamieson, 1965b), bovine glomerular basement membrane (Spiro, 1967b), a,-macroglobulin (Dunn and Spiro, 1967b), and phosvitin (Shainkin and Perlmann, 1971b). Dowex 1-X2 has been used for the resolution of the trypsin glycopeptides from bovine (Plummer and Hirs, 1964), and porcine (Kabasawa and Hirs, 1972), ribonuclease as well as from human chorionic gonadotropin (Bahl, 1969b). This exchanger has also been employed for the fractionation of the glucuronic acid-containing glycopeptides of the clamworm cuticle (Spiro and Bhoyroo, 1971) and of the chondroitin sulfate proteoglycan (Gregory et al., 1964). The hydroxylysine-containing glycopeptides of glomerular basement membrane have been chromatographed on Dowex 50-X2 (Spiro, 1967b), while similar glycopeptides from skin collagen have been separated on Dowex 50-X8 columns (Morgan et al., 1970). Chromatography on Dowex 50X 2 a t an acidic pH and low ionic strength has been a very useful procedure for resolving glycopeptides containing the neutral carbohydrate units from ovalbumin (Cunningham, 1968; Huang e t al., 1970) thyro-

360

ROBERT

G.

SPIRO

globulin (Arima et al., 1972), &-amylase (McKelvy and Lee, 1969) , and earthworm cuticle collagen (Spiro, 1970b). Other techniques which have been used to separate glycopeptides include zone (Rosevear and Smith, 1961; Lee and Montgomery, 1962; Montgomery and Wu, 1963) and paper electrophoresis (Dawson and Clamp, 1968; Wagh et al., 1969; Scocca and Lee, 1969), as well as charcoal :Celite (Spiro, 1965a) and paper (Clamp and Putnam, 1964) chromatography. While most glycoproteins are readily degraded by proteolytic enzymes into glycopeptides containing only a single carbohydrate unit, in some proteins the proximity, charge, or size of the saccharides can interfere with action of the enzymes and prevent complete scission of the peptide chains between the sites of carbohydrate attachment. This has been noted particularly in various glycoproteins from mucous secretions (Gottschalk, 1960) and in the blood group active glycoproteins from ovarian cysts (Morgan, 1968). Proteolysis of ovine submaxillary glycoprotein which contains very closely spaced carbohydrate units is substantially facilitated after removal of the terminal sialic acid residues (Gottschalk, 1960). Chondroitin sulfate proteoglycan, which contains bulky carbohydrate units segregated in small sections of the peptide chains, also shows a resistance to proteolysis (Section XII1,H), but prior removal of most of the sugar residues by hyaluronidase leads to more effective proteolytic action (Gregory et al., 1964). VII. CARBOHYDRATE-PEPTIDE LINKAGES The covalent sugar-amino acid bond which attaches the carbohydrate units to the peptide chains is of course the unique structural feature of glycoproteins. This linkage always involves C-1 of the most internal sugar residue and a functional group of an amino acid within the peptide chain. Three major types of glycopeptide bonds have been described, and the structures of these are shown in Fig. 2. The linkage to the amide group of asparagine is an N-glycoside (glycosylamine bond) and always involves N-acetylglucosamine. I n contrast, 0-glycosidic bonds exist between the sugar residue and serine (threonine) or hydroxylysine. The monosaccharide involved in the serine (threonine) bond can be Nacetylgalactosamine, galactose, mannose or xylose, while only galactose has been found as the linkage sugar to hydroxylysine (Table 111). More recently an additional linkage has been found in the cell wall of plants in which either arabinose (Lamport, 1969) or galactose (Miller et al., 1972) is linked 0-glycosidically to hydroxyproline. It is of interest that only a limited number of amino acids are involved

361

GLYCOPROTEINS CH20H

o Glycosylamine ; ; : : - ) O : HO (

{HZ

H

CH3CNH

COOH

8

CH20H

O-Glycoside serine to (or threonine)

H H ( r : P N H z t H H

CH3CNH

O-FHp

COOH

0

O-Glycoside to hydroxylysine H

COOH

F I ~2.. Structure of the three major glycopeptide bonds.

in the carbohydrate-peptide attachment and that such components as lysine, glutamine, aspartic acid, glutamic acid, histidine, and arginine have so far not been implicated in this process. Of the sugar components common to glycoproteins, only the sialic and uronic acids, fucose, and glucose remain uninvolved in the formation of glycopeptide bonds. The major types of glycopeptide bonds can readily be distinguished by their different susceptibility to alkaline and enzymatic cleavage, and a number of techniques for their identification have been developed (Spiro, 1 9 7 2 ~ ) . Since several proteins have now been shown to contain more than one type of glycopeptide bond (Section X ) , it is essential to account quantitatively for the linkage of all the carbohydrate present. If there is the suspicion that diverse linkages exist in a given protein, glycopeptides containing these must be prepared and studied separately.

A . Glycosylamine Bond Involving Asparagine The N-glycosidic linkage between N-acetylglucosamine and asparagine (2-acetamido- 1 - N - ( 4 ' - ~- aspartyl) - 2-deoxy-~-~-g~ucopyranosy~amine) was the first glycopeptide bond to be described (Johansen et al., 1961;

362

ROBDRT G. SPIRO

TABLE I11 Occurrence of Carbohydrate-Peptide Bonds i n Glywproteim Carbohydrate-peptide bond Amino acid5

Sugar

Am

GlcNAc

Ser, Thr

GalNAc

Ser, Thr Ser, Thr

Gal Man

Ser

XYl

HYl HYP HYP

Gal Ara Gal

Glycoproteins" Plasma proteins, hormones, immunoglobulins, enzymes, egg white proteins, basement membranes, plasma membranes, corneal keratan sulfate Glycoproteins from mucous secretions and ovarian cysts, immunoglobulins, fetuin, plasma membranes, chorionic gonadotropin, cartilage keratan sulfate, Antarctic fish freezing point-depressing glycoprotein Annelid cuticle collagen Clamwonn cuticle collagen, yeast cell wall and invertase, Aspergillus niger glucoamylase Proteoglycans (protein complexes of chondroitin sulfateti, dermatan sulfate, heparin and heparan sulfate) Basement membranes, collagens Cell walls of green plants Cell walls of volvocalean green algae

References are given with discussion in text. S-Glycosidic bonds between cysteine and galactose and between cysteine and glucose have been found in glycopeptides from urine and red cell, respectively (see text). a

b

Nuenke and Cunningham, 1961). The structure of this sugar-amino acid complex was determined after its isolation from hen ovalbumin and was verified by comparison with the chemically synthesized compound (Marks et al., 1963; Marshall and Neuberger, 1964). Since that time evidence for the occurrence of this linkage in a large variety of proteins has been brought forth (Table 111). This linkage compound (GlcNAcAsn) has also been isolated from a number of other glycoproteins including bovine ribonuclease (Plummer et al., 1968) , al-acid glycoprotein (Yamashina e t al., 1965a; Wagh et al., 1969), corneal keratan sulfate proteoglycan (Baker et al., 1969; Stuhlsatz et al., 1971) and calf and human thyroglobulin (Arima and Spiro, 1972). I n other proteins its occurrence has been clearly demonstrated through a study of glycopeptides in which i t is found (Sections VIII, IX, and X I I I ) . I n order to obtain glycopeptides containing asparagine as the only amino acid, extensive digestion with Pronase has been employed, often using an elevated temperature (45*-5OoC) and repeating the incubation with fresh enzyme several times (Montgomery et al., 1965a; Cunningham, 1968; Arima and Spiro, 1972). Release of the external sugars to yield N-acetylglucosaminylasparagine has been accomplished by means of

GLYCOPROTEINS

363

partial acid hydrolysis (Marshall and Neuberger, 1964; Yamashina et al., 1965a), incubation with glycosidases (Arima and Spiro, 1972), sequential application of the Smith periodate oxidation procedure (Makino and Yamashina, 1966), or a combination of these methods (Tarentino e t al., 1970). The GlcNAc-Asn can be identified on the amino acid analyzer where it has a n elution time of 0.27 (Technicon system) relative to aspartic acid (Spiro, 1973). On paper chromatography in l-butanol-acetic acidwater (4:1:5) it migrates with an RASPof 0.54 and yields a brown color with ninhydrin stains (Marks et at., 1963; Spiro, 1 9 7 2 ~ ) . The compound is readily split by glycosyl asparaginase to yield aspartic acid plus l-amino-N-acetylglucosamine (which rapidly decomposes to N-acetylcosamine and NH,) but is resistant to the action of P-N-acetylglucosaminidase (Makino et al., 1968). On acid hydrolysis GlcNAc-Asn yields 1 mole each of aspartic acid, glucosamine, and NH,. The compound is stable to mild alkaline treatment such as splits the O-glycosidic bond to serine or threonine, but can be cleaved by stronger alkaline conditions (Marks e t al., 1963). When treated a t 80°C for 16 hours with 2 N NaOH containing 2 M sodium borohydride, hydrolysis of the N-acetylglucosaminylasparagine bond takes place (as well as deacetylation of the N-acetylglucosamine) with the formation of glucosaminitol in about 75% yield (Spiro, 1 9 7 2 ~ ) .

B. O-Glycosidic Bond to Serine or Threonine The occurrence of glycosidic linkages to the hydroxyl groups of serine and threonine residues in peptide chains was first recognized to occur in glycoproteins from mucous secretions and proteoglycans (Anderson et al., 1964; Bhavanandan et al., 1964; Tanaka and Figman, 1965; Harbon et al., 1968). Subsequently such bonds were also found in a number of proteins from plasma, annelid cuticle collagens, plasma membranes, and fungal glycoproteins (Table 111). While in most proteins containing this type of linkage both serine and threonine residues are believed to be involved, in some cases only one or the other of these amino acids takes part in the sugar attachments. In proteoglycans and human chorionic gonadotropin, the linkage is solely to serine residues, while in the Antarctic fish freexing-point-depressing glycoproteins, clamworm cuticle collagen, and the hinge peptide region of the IgG immunoglobulin, only threonine has been found to be so substituted. When glycoproteins or glycopeptides containing carbohydrate units linked to these a-amino-P-hydroxyacids are treated with mild alkaline conditions (e.g., 0.1 N NaOH, 37"C, 48 hours) the glycopeptide bond is split by the process of p-elimination with the release of a reducing sac-

364

ROBERT G. SPIRQ

charide and the formation in the peptide of an unsaturated amino acid (Fig. 3). The linkage sugar can be identified if the alkaline treatment is performed in the presence of sodium borohydride by measuring, after acid hydrolysis, the sugar alcohol formed, while the amino acid involved in the glycopeptide bond can be determined after reduction or sulfite treatment of its unsaturated product (Fig. 3 ) . If the unsaturated amino acid is not so treated, it will be destroyed during acid hydrolysis and an assessment of the linkage amino acid can be made from such a loss. Even in the presence of palladium catalyst, the conversion of 2-aminoacrylic acid to alanine and 2-aminocrotonic acid to 2-aminobutyric acid, however, may be far from complete. Conversion to the sulfonyl derivatives is a more quantitative process, yielding cysteic acid or 2-amino-3sulfonylbutyric acid which can be separated by Dowex 1 chromatography employing the amino acid analyzer (Spiro, 1972c) or by gas-liquid chromatography of the trimethylsilyl derivatives (Simpson et al., 1972). While many studies on glycoproteins containing carbohydrate units linked to the p-hydroxyamino acids have relied on the p-elimination mechanism for linkage analysis, whenever possible isolation of the sugar amino acid linkage complex should be attempted. It must be kept in H O

I

G-O-CH2

CH,

II

-C-CI NH seryl-0-glycoside

I

I

l

I

[

:; J piI

II

[ G-0-1

I1J

Y 3

+ CH = C - C -

I

2-aminoacrylyl

NH

EHS (Pdy H O

I

II

CH3- C-C-

I

NH I

olonyl

SO,-CH,

H O

H O

- c-c-

C HJ-C H2-C-C-

I II I

NH

I

cysteiyl

t hreonyl-0- g Iycoside

6 - 0 1 -CH3 k ic-c-

I ).

o NH

0 I l l

NH

I [ G - O j + CH,=C-C-

H

G-O-CH-C-C-

I

I II I

NH

I

2-aminobutyryl

2-aminocrotonyl

\SO& SOiH 0

I

I l l

CHS-CH -C-CI

NH I

2-omino3-sulfonyl bu tyryl

FIG. 3. Mechanism of cleavage by p-elimination of 0-glycosidic bonds to or-amino-/3-hydroxyacids. As shown, the unsaturated amino acids formed can be converted into stable products by reduction or sulfite treatment. G = glycosyl.

GLYCOPROTEINS

365

mind that serine or threonine residues may be substituted with other than glycosyl groups, such as phosphoryl or possibly even fatty acyl groups, which could also be eliminated by alkaline treatment and give rise to the same unsaturated amino acid residues. Experiments with model compounds (Riley et al., 1957; Montreuil et al., 1967; Derevitskaya et al., 1967) have indicated that substitution of the carboxyl and amino group of the O-glycosidated amino acid greatly facilitates the elimination reaction. It has been shown that the electron attracting effect of a substituted carboxyl group is important in making possible the electron shift which occurs in the /3-elimination reaction. An unsubstituted amino group would also tend to increase the electron density on the carbon involved in the elimination and thereby slow the reaction. Alkali-resistant O-glycosides can therefore be encountered if the substituted serine or threonine residues are in C- or N-terminal positions. Substitution of such terminal groups would be expected to speed up the elimination reaction, and this has been shown to occur after acetylation of N-terminal residue (Bella and Danishefsky, 1968) of the dermatan sulfate-protein linkage region. Studies employing a-N-acetylgalactosaminidase have indicated that the glycosidic linkage of N-acetylgalactosamine to serine or threonine in the ovine submaxillary glycoprotein (Buddecke et al., 1969), hog stomach blood group A active glycoprotein (Weissmann and Hinrichsen, 1969), and fetuin (Spiro, 1972b) is a in anomeric configuration. Xylosylserine has been obtained after partial acid hydrolysis of the protein complexes of chondroitin sulfate and heparin and shown by optical rotatory dispersion studies and comparison with the two synthetic anomers to be j3 in configuration (Lindahl and Rod&, 1966; Lindahl, 1966). O-P-xylopyranosyl-L-serine can be separated by paper chromatography and electrophoresis, as well as on the amino acid analyzer (Lindahl and R o d h , 1966).

C. 0-Glycosidic Bond to Hydrosylysine Involvement of hydroxylysine in the linkage of carbohydrate was first noted in studies of guinea pig skin tropocollagen (Butler and Cunningham, 1966) and bovine renal glomerular basement membrane (Spiro, 1967b). Since that time the galactosylhydroxylysine bond has been observed in a large number of fibrillar collagens from both vertebrate and invertebrate sources, as well as in various basement membranes (Spiro, 1969b, 1972a). Indeed, this linkage has been detected in all proteins which contain hydroxylysine so far examined, with the exception of the silk collagen from Nematus ribesii (gooseberry sawfly) (Spiro et al., 1971).

366

ROBERT G . SPIRO

I n sharp contrast to the O-glycosidic bonds to the a-amino-P-hydroxyacids, the linkage to the 6-hydroxyl group of hydroxylysine is stable to strong alkali. After alkaline hydrolysis of sufficient strength (2 N NaOH, 105"C, 24 hours) to split, all the peptide bonds of a protein, the hydroxylysine-linked carbohydrate units, Gal-Hyl (5-O-/3-~-galactopyranosylhydroxylysinej and Glc-Gal-Hyl (2-O-~-~-glucopyranosyl-5-0-/3-~galactopyranosylhydroxylysine) can be identified and quantitated on the amino acid analyzer (Spiro, 1969b). Although these components can be resolved from amino acids with the usual buffers (Technicon), they can best be separated with a special pH 5.0 gradient, where they appear in the extended region between the phenylalanine and the basic amino acids and are well separated from tryptophan which emerges between Glc-GalHyl and Gal-Hyl (Spiro, 1 9 7 2 ~ ) . Glc-Gal-Hyl, which is the major form of hydroxylysine-linked carbohydrate, can be isolated from alkaline hydrolyzates by gel filtration on Sephadex G-15 (Spiro, 1 9 6 7 ~ ) . It is readily converted to Gal-Hyl by mild acid hydrolysis (0.1 N H,SO, a t 100°C for 28 hours) owing to the fact that the glycosidic linkage of galactose to hydroxylysine, which is vicinal to a positively charged amino group, is considerably more stable to acid than the more conventional glycosidic bond linking glucose to the galactose. After substitution of the amino group by N-acetylation, the rate of release of galactose by acid hydrolysis is very much increased and it approaches that of the liberation of the glucose. Gal-Hyl can be separated from amino acids by paper chromatography in l-butanol-acetic acid-water (4: 1:5) where it has a migration of 0.69 of hydroxylysine (Glc-Gal-Hyl has an RHylof 0.52). Upon paper electrophoresis a t pH 3.5, Gal-Hyl moves toward the cathode a t 0.73 the speed of hydroxylysine, while Glc-Gal-Hyl moves a t 0.60 the rate of the hydroxylysine. The Gal-Hyl in the glomerular basement membrane, lens capsule, ichthyocol, and several invertebrate collagens was shown to be /3 in configuration (Spiro, 1967c, 1972a). Galactose was split from the linkage compound or glycopeptides containing it by the action of P- but not a-galactosidase. Prior blockage of the amino groups of the substrate by N-acetylation was necessary for this enzymatic cleavage to take place. Hydroxylysine residues which are involved in glycosidic linkages are resistant to periodate oxidation in contrast to the unsubstituted form of this amino acid. The c-amino group of this O-glycosidically substituted amino acid reacts readily with dinitrofluorobenzene (Spiro, 1967b) .

D. Other Glycopeptide Bonds Hydroxyproline-linked oligosaccharides in which the linkage sugar is arabinose have been found in the cell wall of plants covering a wide

GLYCOPROTEINS

367

phylogenetic range (Lamport, 1969). More recently oligosaccharides linked to hydroxyproline through a galactose residue have been found in chlamydomonas, a primitive green alga (Miller et al., 1972). This glycopeptide bond, like that to hydroxylysine, is resistant to alkaline hydrolysis, and the hydroxyproline-linked carbohydrate units have been isolated after such treatment by chromatography on a cation exchanger. Recent reports describing the occurrence of digalactosylcysteine in an N-terminal position in a glycopeptide from human urine (Lote and Weiss, 1971) and triglucosylcysteine in a similar position in a glycopeptide from human erythrocytes (Weiss et al., 1971) suggest that S-glycosidic bonds may also occur in glycoproteins. The association of small amounts of L-arabinose with hyaluronic acid isolated from brain (Wardi et al., 1966) has raised the possibility that this sugar might be involved in the linkage of this polymer to protein in analogy with the role which xylose plays in attaching the carbohydrate units of many of the sulfated proteoglycans. VIII. SEQUENCE OF AMINOACIDSAROUND GLYCOPEPTIDE BONDS The location of the carbohydrate units on the peptide chain(s) must be established in order to understand their relationship to the threedimensional structure of the molecule. The total amino acid sequence of a numbcr of glycoproteins has now been elucidated and the position of attachment of their carbohydrate units determined. These proteins include bovine ribonuclease B (Hirs et al., 1960; Plummer and Hirs, 1964), porcine ribonuclease (Jackson and Hirs, 1970), IgG (Eu) myeloma immunoglobulin (Edelman et al., 1969), bovine thyroid-stimulating hormone (Liao and Pierce, 1971), ovine luteinizing hormone (Papkoff et al., 1971; Liu et al., 1972a,b), human chorionic gonadotropin (Bahl et al., 1972), hen egg avidin (DeLange and Huang, 1971) and the a,-acid glycoprotein of human plasma (Schmid et al., 1971). All of these fully sequenced proteins contain asparagine-linked carbohydrate units, while the chorionic gonadotropin has in addition carbohydrate units linked t o serine residues. Amino acid sequences around the carbohydrate linkage site have been established for a number of other proteins on the basis of investigations of their glycopeptides. A survey of the amino acid constellations around the various glycopeptide bonds promises to give insights into the specificity of the enzymatic glycosylation and the possible determinants for this process which may be required in the peptide chain.

A . Bond Involving Asparagine An inspection of the amino acids around asparagine-linked carbohydrate units (Table IV) indicates that the sequence Asn-X-Ser (Thr),

TABLEIV Amino Acid Sequences around Asparagine-Linked Carbohydrate Units of Sevmal Glycoproteins Protein

Amino acids around linkage site.

Nature of unitb

8

References

CHO I

a,-Acid glycoproteinC

IgG immunoglobulin, H chain (human myeloma (Eu)) IgG immunoglobulin, H chain (rabbit) IgG immunoglobulin, L chain (mouse plasma cell tumor) Bence-Jones protein, K chain (HBJ 4) Bence-Jones protein, A chain (Ful) IgG immunoglobulin, H chain variable region (human myeloma (Cor)) IgM immunoglobulin, H chain (human macroglobulin (Ou)). Fibrinogen, y chain (human) Ribonuclease B (bovine) Ribonuclease (procine)’

J

Pro-Ile -Thr -Am-Ala -Thr-Leu Glu-Glu-Tyr -Am-Lys -Ser-Val Phe-Thr-Pro -Am-Lys -Thr-Glu Cys-Ile -Tyr -Asin-Thr -Thr-Tyr Gln-Arg-Glu -Am-Gly -Thr-Ile Gln-Gln-Tyr -Am-Ser -Thr-Tyr (297) Gln-Gln-Phe -An-Ser -Thr-Ile

C C C C C C

Schmid et al. (1971)

C

Ala-Ser -Gln -Am-Ile -Ser -Am

C

Nolan and Smith (1962); Hill et al. (1967) Melchers (1969)

Ala -Ser -Glx -Am-Val -Ser -Asx (28) Cys-Ser -Gly -Am-Ser -Ser (26) Lys-Tyr-Tyr -Am-Thr -Ser -Leu (62) Phe-Gln-Glx -Am-Ala 8 e r -Ser Leu-Tyr -Am-Val -Ser -Leu Gln-Val -Glu -Am-Lys -Thr-Ser (52) Lys-Ser -Arg -Am-Leu -Thr-Lys (34) Ser -Ser S e r -Am-Ser S e r -Asn (21 1 Ser -Arg-Arg -Am-Met-Thr-Gln

C

Sox and Hood (1970)

C

Sox and Hood (1970)

-d

Press and Hogg (1970)

C S C

Shimizu et al. (1971)

S

Plummer and Hirs (1964); Him et al. (1960) Jackson and Hirs (1970)

,*A\

C

S

Edelman et al. (1969)

Iwanaga et al. (1968)

G,

Deoxyribonuclease (bovine) Stem bromelain (pineapple) Thyroid-stimulating hormone (bovine) cz subunith

6 subunit

Luteinizing hormone (ovine) j3 subunit Chorionic gonadotropin (human)’ a-subunit

p subunit

Ovalbumin (hen) Ovotransferrin (hen) Avidin (hen) Phosvitin (hen) Thyroglobulin (human)i Visual pigment (bovine)

Tyr-Gln-Ser -Am-Ser -Thr-Met (76) Ser -Am-Ala -Thr Pro-Arg-Asn -Am-Glu -Ser -Ser

C

S

S@

Catley et al. (1969) Yasuda et al. (1970)

Val -Pro-Lys -Am-Ile -Thr-Ser (56 ) Arg-Val-Glx -Am-His -Thr-Glu (82 ) Leu-Thr-Ile -Asn-Thr -Thr-Val (231

C

Liao and Pierce (1971)

Gln-Pro-Ile -Am-Ala -Thr-Leu (13)

C

Liu et al. (1972b); Papkoff et al. (1971)

Val -Glx-Lys -Am-Val -Thr-Ser (52) Lye-Val -Glx -Am-His -Thr-Ala (78) Arg-Pro-Ile -Am-Ala -Thr-Leu (13) Val-Cys-Ile -Am-Val -Thr-Thr (28) Glu-Lys-Tyr -Am-Leu -Thr-Ser

C

Bahl et al. (1972)

Leu-Ile -His -Am-Arg -Thr-Gly Leu-Gly-Ser -Am-MetrThr-Ile (17) Ser -Asn-Ser -Gly-Psr Ah-Leu-Glu -Am-Ala -Thr-Arg MetrAsn-Gly-Thr-Glu

C C

i?

2

0

E

C

x

G

C

rn

C

S S S

C

S

S

Johansen et al. (1961); Nuenke and Cunningham (1961); Lee and Montgomery (1962) Williams (1968) Delange and Huang (1971)

Shainkin andPerlmann (1971b) Rawitch et al. (1968) Heller and Lawrence (1970)

(Continued)

%

W

FOOTNOTES TO TABLE IV Arrow indicates the asparagine residue to which carbohydrate units attached by glycosylamine bond. Numbers in parentheses below asparagine residues are given for those proteins in which the residue number of this amino acid from the N terminus of the chain has been established. b S refers to simple carbohydrate unit consisting only of mannose and N-acetylglucosamine residues while C refers to complex units which contain sialic acid and galactose and sometimes fucose in addition to the mannose and N-acetylglucosamine (see Section IX,A). c Sequences around the attachment of the five carbohydrate units of al acid glycoprotein are given. d Nature of unit not known. Sequences shown for two of the five units known to be present on each H chain. f Sequences around the at,tachment of the three carbohydrate units of porcine ribonuclease are given. 0 Unit contains a single fucose and xylose residue in addition to mannose and N-acetylglucosamine. * Sequences around the attachment of the two units of the a subunit shown; same sequences were found in a-subunit of ovine lutehizing hormone (Liu et al., 1972a). i Sequences shown for the two attachments in the a and p subunits. i Each human thyroglobulin molecule contains 7-8 simple aspmagine linked units (Arima et al., 1972).

-a

0

0

E m

0

m Fl

g

GLYCOPROTEINS

371

with X being any amino acid (Neuberger and Marshall, 1969) pertains in every case except phosvitin. This protein, however, is unusual in having a large number of phosphoserine residues in the vicinity of the glycopeptide bond. Despite the characteristic amino acid sequence on the C-terminal side of the asparagine-carbohydrate linkage no identifiable pattern of amino acids can be recognized on the N-terminal side of the glycopeptide bond. Although i t was suggested on the basis of a limited number of amino acid sequences that the nature of X might determine the type of carbohydrate unit attached to the asparagine residue (Jackson and Hirs, 1970), it has now become evident that this amino acid itself does not play such a role, as no consistent difference in this component can be noted between simple and complex carbohydrate units (Table IV) . It is nevertheless likely that the general conformation of the protein in the vicinity of the attachment site helps to determine whether the carbohydrate units are available to glycosyltransferases which can add the sialic acid-galactose-N-acetylglucosamine side chains to the mannose-N-acetylglucosamine core to form the more complex carbohydrate units (Section IX,A) . A variety of factors aside from the amino acid on the C-terminal side of the carbohydrate-linked asparagine may help to determine this conformation. From X-ray crystallography data of bovine ribonuclease A, it has been inferred that the single carbohydrate unit of bovine ribonuclease B (Plummer and Hirs, 1964) and the three carbohydrate units of porcine ribonuclease (Jackson and Hirs, 1970) are located on the surface of the molecule and project into the solution environment. It would not be surprising to find that the rather bulky, hydrophilic carbohydrate groups of glycoproteins will generally be located in exterior positions on the molecule. While the Asn-X-Ser (Thr) sequence may be a necessary determinant for the glycosylation of the asparagine residue, it has become apparent that such a sequence alone is not sufficient to ensure the attachment of carbohydrate on a protein. A survey of tripeptide sequences from 264 proteins, the complete or partial primary structure of which had been elucidated a t that time, indicated that in the 101 Asn-X-Ser(Thr) sequences observed, only 20 had carbohydrate attached to them (Hunt and Dayhoff, 1970). While some of the nonglycosylated proteins may be the product of cells in which the necessary enzymatic machinery for carbohydrate unit assembly is missing, in other cases steric hindrances imposed by the peptide chain may prevent the attachment of carbohydrate. The latter possibility is illustrated by hen ovalbumin which, although it contains a Carbohydrate unit attached to asparagine in an Asn-Leu-Thr sequence (Table I V ) has another appropriate sequence (Asn-Leu-Ser) which is not glycosylated (Wiseman et al., 1972). The

372

ROBERT G . SPIRO

occurrence in bovine pancreatic juice of a ribonuclease (ribonuclease A) which contains no carbohydrate attached at residue 34,while other molecules of this enzyme (ribonucleases B, C, and D) do have carbohydrate attached at this position (Plummer and Hirs, 1964; Plummer, 1968) is more difficult to explain. Aside from the possibility that the carbohydrate is cleaved from some of the protein after secretion from the cell, the occurrence of a carbohydrate-free molecule would imply some cellular compartmentalization or inefficiency of the carbohydration step. While the distribution of the carbohydrate units of the al-acid glycoprotein have been shown to be extremely asymmetrical with all the units occurring in the N-terminal half of the peptide chain (Schmid et al., 1971), this does not appear to be a general phenomenon, as more even distributions have been found in the peptide chains of other proteins containing multiple carbohydrate units.

B. Bond Involving Serine or Threonine Only a limited amount of information is available as yet in regard to the amino acid residues surrounding O-glycosidic linkages to serine or threonine residues. Furthermore, the sequence information obtained so far is restricted to proteins in which N-acetylgalactosamine is the linkage sugar (Table V ) . The determination of the complete amino acid sequence of human chorionic gonadotropin has revealed t h a t the 8 subunit contains three serine-linked carbohydrate units which are located closely spaced within a six-residue segment of the peptide chain not far from its C-terminal end (Bahl et aE., 1972). This segment of the chain is very rich in proline, and there is a residue of this amino acid on the N-terminal side of each serine residue involved in the glycopeptide band (Table V) . Similarly, the threonine-linked carbohydrate unit present in the hinge region of the heavy chain of rabbit IgG immunoglobulin (Smyth and Utsumi, 1967) is located in a segment of the peptide chain that contains several proline residues and, like the gonadotropin, has this amino acid positioned on the N-terminal side of the glycosylated residue. A study of short glycopeptides containing the three serine (threonine) linked carbohydrate units of fetuin has indicated that they contain three proline residues in the immediate vicinity of the glycopeptide bond (Spiro, 1970d). The possible role of proline in helping to determine the glycosylation by N-acetylgalactosamine of serine and threonine residues is also suggested by the high content of this amino acid in the glycoproteins from mucous secretions (Hashimoto and Pigman, 1962), which contain numerous closely spaced carbohydrate units linked to the p-hydroxyamino acids. Further evidence of the possible role of proline in shaping the receptor region for serine and threonine glycosylation W R S

TABLE V Amino Acid Sequences around Several Serine (Threonine)-Linked Carbohydrate Units Protein IgG immunoglobulin, hinge region (rabbit)

Amino acids around linkage sites

1

Ser -Lys-Pro-Thr-Cys-Pro-Pro-Pro-Glu Smyth and Utsumi (1967)

I

1

1

Freezing point-depressing glycoprotein (Antarctic fish) Thr-Ala-Ala-Thr-Ala -Ah-Thr Submaxillary glycoprotein (bovine) Chorionic gonadotropin, 0 subunit" (human) Chondroitin sulfate proteoglycan (porcine cartilage) Myelin A1 protein (boviney

References

1

DeVries et al. (1971)

1

Ser -Thr-Gly-Ser

1

1

1

Pro-Pro-Pro-Ser -Leu-Pro-Ser -Pro-Ser -Arg (123) (118) (121) J.

Glu-Gly-Ser -Ah -Gly .1

Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser (981

Ozeki and Yosizawa (1971) Bahl et al. (1972) Katsura and Davidson (1966) Hagopian el al. (1971)

Arrows indicate threonine and serine residues to which carbohydrate units are attached by 0-glycosidic bonds; all attachments here shown involve N-acetylgalactosamine as the linking sugar except the one for chondroitin sulfate proteoglycan, which has a xylosylserine bond. Numbers in parentheses refer to position of amino acid from N terminus of peptide chain. This protein does not contain carbohydrate in the native state, but can act as acceptor at the indicated site for a N-acetylgalactosaminyltransferase.

374

ROBERT G. SPIRO

afforded by the finding that a N-acetylgalactosaminyltransferase isolated from bovine submaxillary glands acts upon a specific threoninc in the A1 protein from bovine myelin, which does not naturally contain any carbohydrate (Hagopian et al., 1971). This particular threonine residue was located in a proline-rich region of the peptide chain, similar in many ways to the hinge glycopeptide of the rabbit IgG immunoglobulin (Table V ). I n contrast, the amino acids surrounding the numerous threoninelinked carbohydrate units of the freezing point-depressing glycoprotein from the sera of Antarctic fish are all alanine, as indeed only alanine and threonine residues occur in this protein (DeVries et al., 1971).

C . Bond Involving Hydroxylysine A number of amino acid sequences have been determined in the vicinity of the hydroxylysine-linked carbohydrate units (Table VI) . All these units have been located within a glycine-containing collagen triplet, and all have been found to contain arginine a t a distance of three residues on the C-terminal side of the hydroxylysine. The general pattern, Gly-XHyl (carbohydrate)-Gly-Y-Arg, may apply, in which X and Y are quite diverse. Analysis of a number of glycopeptides from the renal glomerular basement membrane suggests that Glx and Asx may occupy positions X and Y in the vicinity of the numerous hydroxylysine-linked carbohydrate units of this protein (Spiro, 1967b). Furthermore, evidence has been obtained to suggest that hydroxylysine-linked carbohydrate units occur TABLE VI Amino Acid Sequenccs around Hydroxylysine-Linked Carbohydrate Units of Several Collagens Source Human and guinea pig skin; carp swim b1addel.b Human skin Carp swim bladder Cuttlefish skin

Amino acids around linkage sitea

Ref erences

Glc-Gal

1

G:y-Met-Hyl-Gly-His -Arg Gly-Phe -Hyl-Gly-Ile -Arg Gly-Ile -Hyl-Gly-His -Arg Gly-Ala -Hyl-Gly-Asp-Arg Hyp-Gly-Glu -Hyl-Gly-Ala-Arg Hyp-Gly-Gln -H yl-Gly-Ah -Arg Ser -Gly-Pro -Hyl-Gly-Ala -Arg

Morgan et al. (1970) Morgan et al. (1970) Morgan et al. (1970) Isemura et al. (1972)

Arrow indicates hydroxylysine residues to which glucosylgalactose disaccharide unite are linked by 0-glycosidic bonds. This sequence is found in the a1 chain in which the glycosylated hydroxylysine represents residue 103 from the N terminus of this chain (Traub and Piee, 1971;Butler, 1970).

GLYCOPROTEIN S

375

in locations outside of the helical portion of the peptide chain in collagenase-resistant regions (Spiro, 1 9 7 0 ~ ) . Some information in regard to the determinants involved in the glycosylation of hydroxylysine residues has been obtained from studies with a kidney galactosyltransferase (M. J. Spiro and Spiro, 1971). This enzyme functions specifically to transfer galactose from UDP-galactose to the hydroxyl group of hydroxylysine residues located in high molecular weight compounds, as long as the 6-amino groups of this amino acid is unsubstituted. Native collagens from both vertebrate and invertebrate sources are good acceptors and were found to have comparable activity per micromole of unsubstituted hydroxylysine. Furthermore, after selective removal of the hydroxylysine-linked carbohydrate units, the newly exposed hydroxylysine residues, which can be presumed to be the natural acceptors, were not more effective than those which had remained unsubstituted in the native molecule. This suggests that either incomplete glycosylation takes place on hydroxylysine residues in the proper amino acid constellation, or the requirement of amino acids around the acceptor site is not very stringent. That glycosylation of hydroxylysine may depend on the availability of the necessary glycosyltransferase was illustrated by studies on the silk collagen of the gooseberry sawfly (Nematus ribesii) (Spiro et al., 1971) , an unusual protein which contains 37 hydroxylysine residues per 1000 total amino acid residues, none of which are substituted by carbohydrate units. The kidney UDP-galactose :hydroxylysine galactosyltransferase can effectively use this protein as an acceptor with the formation of galactosylhydroxylysine. Presumably the silk glands of the sawfly larva are not equipped with the necessary enzymes to make possible this O-glycosylation so that potential carbohydration sites remain unoccupied.

IX. NATUREOF THE CARBOHYDRATE UNITS One logical method of classifying the carbohydrate units of glycoprotein is based on the amino acid and sugar involved in the glycopeptide bond (Spiro, 1970a). A . Aspara g i n e - Link e d Units

On the basis of their composition it is evident that asparagine-linked carbohydrate units occur as two distinct types (Table VII). One type consists only of mannose and N-acetylglucosamine residues, while the other is more complex and contains in addition other sugar components such as sialic acid, galactose, fucose, and in one case (bromelain), xylose. The structure of the simpler mannose-N-acetylglucosamine type of

w

4 Q,

TABLE VII Composition and Number of Some AsparagineLinked Carbohydrade Units Residues/carbohydrate unita Protein Fetuin crl-Acid glycoprotein Haptoglobin, 2-1 (human) a*-Macroglobulin (human) Transferrin (human) IgG immunoglobulin (human) IgM immunoglobulin, monomer (humany IgA immunoglobulin* Ribonuclease B (bovine) Deoxyribonuclease A (bovine)

Gal 3 4 3 2 2 2 2 2 -

-

-

Sialic Man GlcNAc acids 3 3 3 3 4 3 3 7 3

4

6 6

5 5 4 5 4 4 4 2 3 3 2 2

3 3 2 2 2 1 1

Fuc

1

1

1

1 1

-

-

-

-

2

-

1

-

-

Xyl I

-

-

-

-

Number of units Per molecule 3 5 13 31 2 2 6 4 3 1 1 1

m

8

! ?

References Spiro (1962a, 1970d) Satake ei al. (1965) Gerbeck et al. (1967) Dunn and Spiro (1967b) Jamieson (1965b) Clamp and Putnam (1964) Shmieu et al. (1971); Hickman et al. (1972) Dawson and Clamp (1968) Plummer and Him (1964) Salnikow et al. (1970)

z

u,

Stem bromelain (pineapple)” Taka-amylase (Aspergillus oryzae) Thyroglobulin (calf)b Thyroglobulin (human)h Ovalbumin (hen) Avidin, subunit (hen) Phosvitin (hen) Glomerular basement membrane (bovine) Erythrocyte membrane glycoprotein (human) Visual pigment (bovine)

-

-

Yasuda et al. (1970) Yamaguchi et al. (1969) Spiro (1965a); Arima et al. (1972)

4 2 3 4

3 6 3 9 3 9 5 5 3 3

2 2 5 2 4 2 3 4 5

5

2 1 2 3

3

3

4

1

Thomas and Winder (1971)

-

3

3

-

Heller and Lawrence (1970)

Arima et al. (1972) Johansen et al. (1961) Huang and DeLange (1971) Shainkin and Perlmann (1971a) Spiro (1967a)

0

0 Since variation exists in the composition of the units (Section XII), the average compositions are given expressed as full integers; in proteins in which sialic acid or fucose residues are present over 0-1 range a full residue is listed in each case. * These proteins contain both simple and complex types of asparagine-linked carbohydrate units. c Scocca and Lee (1969) only found two mannose residues in the bromelain carbohydrate unit. d The membrane contains 1.3 asparagine-linked units/lW molecular weight. e Number of units not known.

5

U

BY

E

2

378

ROBHRT G . SPIRO

unit, which ranges in molecular weight from 1200 to 2000, has been investigated in ovalbumin, calf and human thyroglobulin, bovine ribonuclease B, porcine ribonuclease, and Taka-amylase (Section XIII) . In each of these units studied so far, two N-acetylglucosamine residues linked to each other by a p(1- 4) glycosidic bond have been found to make up the most internal portion of the unit with one of the N-acetylglucosamines participating in the glycosylamine linkage to the asparagine. Mannose oligosaccharides of varying lengths have been found attached to the other of the two N-acetylglucosamine residues. In the case of ovalbumin, peripheral N-acetylglucosamine residues attached to the mannose chains also occur. The structure of the more complex units, which generally fall into the 2000-3000 molecular weight range, has been studied in a number of glycoproteins, including fetuin, a,-acid glycoprotein, transferrin, thyroglobulin, bromelain, IgG immunoglobulins, @,-macroglobulin, and erythrocyte membrane glycoprotein (Section XIII) . A variety of structures have been proposed, but in general, they consist of a mannose-N-acetylglucosamine core to which mono- or oligosaccharides are attached. One of the most common of these attached oligosaccharide chains has the sequence sialic acid-galactose-N-acetylglycosamine. Shorter versions of this chain or single fucose or xylose residues also may be found. In some of these units, such as in fetuin, IgG immunoglobulin, and bromelain, di-N-acetylchitobiose is attached to the peptide chain, and the remainder of the carbohydrate unit is attached to the outer of these two residues. I n porcine ribonuclease, attachment of the mannose to the outer as well as the inner of the two N-acetylglucosamine residues has been proposed, while in the a,-acid glycoprotein and transferrin, apparently only a single N-acetylglucosamine residue forms the bridge between the mannose and the asparagine on the peptide chain (Section XIII) . The number of asparagine-linked carbohydrate units varies widely among proteins (Table VII) , ranging from a single unit in hen ovalbumin to 31 in the human a,-macroglobulin. Several proteins, including calf and human thyroglobulin, porcine ribonuclease, and the IgM and IgA immunoglobulins have been found to contain both the simple and complex types of carbohydrate units.

B. Serine (Threonine)-Linked Units The carbohydrate units linked to the /3-hydroxyamino acids occur in various forms (Table VIII). Small units, often branched, linked to the peptide chain through N-acetylgalactosamine and containing one or more other sugar components such as sialic acid, fucose, or galactose, have been found in immunoglobulins, red blood cell membrane glycoprotein,

379

GLYCOPROTEINS

fetuin, Antarctic fish freezing point-depressing glycoproteins, and various proteins from mucous secretions. Some units which are attached to the peptide by N-acetylgalactosamine also contain N-acetylglucosamine residues. These are found in the blood group active glycoproteins of ovarian cyst, the keratan sulfate of cartilage proteoglycans, and in the recently described carbohydrate of canine submaxillary glycoprotein (Section XIII) , I n the latter two proteins moreover, the N-acetylglucosamine occurs in 0-sulfated form. Carbohydrate units linked to serine and threonine by galactose have so far been found only in annelid cuticle collagens. These units are quite simple, consisting of one to three galactose residues linked to each other by (1 + 2) glycosidic bonds (Section XIII,G) . The threonine-linked glucuronic acid-mannose (6-0-a-~-glucuronosyID-mannose) unit which has been found in the clamworm cuticle collagen has clearly indicated that mannose can be involved in carbohydratepeptide bonds to hydroxyamino acids and has further shown that uronic acids may be components of small carbohydrate units as well as of the larger ones characteristic of proteoglycans (Spiro and Bhoyroo, 1971). The nature of other mannose-linked carbohydrate units such as are believed to occur in yeast cell wall (Sentandreau and Northcote, 1968) and invertase (Greiling et al., 1969) as well as in A . niger glucoamylase (Lineback, 1968) remains to be elucidated. The carbohydrate units of a number of proteoglycans, including the chondroitin sulfates, dermatan sulfate, heparin, and heparan sulfate are linked to the peptide through P-xylosylserine bonds. These units are considerably larger than those of other glycoproteins, falling in the molecular weight range of 13,000 to 29,000. The basic structure of the xylose-linked carbohydrate is a repeating disaccharide in which hexuronic acid and N-acetylhexosamine residues alternate and in which variable degrees of 0- and N-sulfation of the sugar residues occurs (Section XII1,H). Studies on chondroitin sulfates, dermatan sulfate, and heparin have shown that they have identical carbohydrate-protein linkage regions ( R o d h , 1968) : (Y

61-3

GlcUA-

81-3

Gal-

Gal-

81-4

p1-3

Xyl-

Ser

The serine (threonine)-linked carbohydrate units of the glycoproteins from mucous secretions as well as of the proteoglycans are numerous and closely spaced. The density of such carbohydrate units on the peptide chain significantly exceeds that achieved by the asparagine-linked units. I n the ovine submaxillary glycoprotein, for example, there are an average of six amino acids between disaccharide units while in the cartilage proteoglycan an average of about 10-20 amino acids are interspersed be-

TABLE VIII Composition and Number of Some Serine(Threonine)-Linked Carbohydrate Units Residues/carbohydrate unitb Protein Submaxillary glycoprotein (ovine) Submaxillary glycoprok i n , A+ (porcine) Fetuin IgG immunoglobulin (rabbit)$ IgA immunoglobulin (human) Chorionic gonadotropin (human) Erythrocyte membrane glycoprotein (human)

Gal

GalNAc

Sialic acids

Fuc

Man

-

1

1

-

-

-

-

-

1

2

1

1

-

-

-

-

1 1

1 1

2 2

-

-

-

-

3

3

1

-

1

1

1

1

1

2

GlcUA GlcNAc

Xyl

Number of units per molecule

Sugar involved in glycopeptide bond

Referenceso

800

GalNAc

(1)

496c

GalNAc

(2)

Tim m T: 'd

-

-

-

-

3 1

GalNAc GalNAc

(3)

-

-

-

-

1

GalNAc

(5)

-

-

-

-

-

3

GalNAc

(6)

-

-

-

-

-

1st

GalNAc

(7, 8)

-

B ;

(4 1

B

Freezing poinbdepressing glycoprotein 5 (Antarctic fish) Cuticle collagen (Lumbricus ) Cuticle collagen (Nereisp Cartilage proteoglycank (bovine)

1

1

-

-

-

-

-

-

31

GalNAc

1-3

-

-

-

-

-

-

-

231

Gal

1-3 2 6

50 1

-

-

-

-

1

-

-

-

2

-

-

-

1 50 -

6

1

-

4'

2f 90 60'

Gal Man Xyl GalNAc

(9) (1%11) (12) (12) (13) (14)

a References: (1) Graham and Gottschalk (1960); (2) Carlson (1968); (3) Spiro (197Od); (4) Fanger and Smyth (1972a,b); (5) Dawson and Clamp (1968); (6) Bahl(1969b); (7) Thomas and Winzler (1969); ( 8 ) Adamany and Kathan (1969); (9) DeVries et al. (1971); (10) Muir and Lee (1969); (11) Spiro (1970b); (12) Spiro and Bhoyroo (1971); (13) Hascall and Sajdera (1970); (14) Hascall and Riolo (1972). b Some of the units vary in their composition (Section XII); for these the composition of the most complete unit is given. c Calculated from galactose content of protein; since incomplete units without galactose also occur this represents a minimal value. d Only 30% of the molecules contain this unit attached to the hinge region of one of i b H chains, * Calculated from the N-acetylgalactosamine content of the protein (Winzler, 1969). f Expressed as carbohydrate unib/lOOO total amino acid residues (Spiro, 1972a). 0 This collagen contaim two distinct types of serine(threonine)-liked carbohydrate units. The proteoglycan contains chondroitin sulfate and keratan sulfate units. Calculated on the basis of one N-acetylgalactosamine residue per carbohydrate unit.

382

ROBERT G . SPIRO

tween carbohydrate attachments. The highest concentration of serine(threonine)-linked carbohydrate units has been found in the freezing point-depressing glycoprotein, in which only two amino acids intervene between successive glycopeptide bonds, and in human chorionic gonadotropin, which has all three of its serine-linked carbohydrate units on the p subunit attached within a six-amino acid segment of the peptide chain.

C . H ydrox yl ysine-Linked Units Only the disaccharide 2-O-~-~-glucopyranosyl-~-galactose and the monosaccharide galactose have been found linked glycosidically t o hydroxylysine residues. These two units occur in variable proportions and amounts in basement membranes and collagens from both vertebrate and invertebrate sources (Table IX) . Recent studies moreover indicate that such hydroxylysine-linked units may also be present in the Clq complement protein of human serum (Yonemasu et al., 1971 ; Calcott and Muller-Eberhard, 1972). The highest densities of hydroxylysine-linked units are found in basement membranes and in collagens with a low degree of morphological organization, while the lowest density is observed in collagens with highly organized fibrils (Spiro, 196913) (Table IX) . The basement membranes have over 80% of their hydroxylysine residues substituted with carbohydrate, which occurs almost exclusively as the disaccharide unit. Fibrillar collagens in general have a smaller percentage of their hydroxylysine residues involved in glycosidic linkage, and while invertebrate collagens contain almost all of their units in the form of disaccharides, most vertebrate collagens have a substantial portion of their hydroxylysine-linked carbohydrate as single galactose residues.

X. PROTEINS WITH MORETHANONE TYPEOF CARBOHYDRATE UNIT As the number of glycoproteins under structural investigation has increased, it has become apparent that the occurrence of more than one distinct type of unit in a given protein is not uncommon (Table X). A number of proteins including thyroglobulin, porcine ribonuclease, and the IgM and IgA immunoglobulins contain both the simple and complex asparagine-linked carbohydrate units. Studies on porcine ribonuclease, moreover, have indicated that the two complex units and one simple unit of this protein are attached to specific asparagine residues and are not interchangeable (Jackson and Hirs, 1970). The occurrence of serine(threonine)-linked units in addition to units attached to asparagine has been observed in immunoglobulins, fetuin, human chorionic gonadotropin, corneal proteoglycan, and erythrocyte membrane glycoprotein.

TABLE IX HydroxylysineLinked Carbohydrate Units of Several Basement Membranes and Collagens

Proteins Membranes Bovine anterior lens capsule Bovine posterior lens capsule Bovine glomerular basement membrane Human glomerular basement membrane Collagens Bovine vitrosin Loligo, cephalic cartilage, citrate soluble Lumbricus, body wall, citrate-insoluble* Chick cartilage, a, (11) Loligo, fins, citrate-soluble Mytilus, foot, citrate-insolubleb Busywtypus, body wall, citrat&nsolubleb Rabbit cornea, citrate-insoluble* Calf cornea, citrate-insolubleb Calf skin, citrate-soluble Bovine, Achilles tendon Carp swim bladder, citrate-soluble Rat skin, citrate-soluble Rabbit sclera, citrate-insolubleb Gooseberry sawfly, silk collagen

Total Spacing of number of units linked Hyl-linked to Hyl GleGal-Hyl Gal-Hyl units (units/1000 (units/1000 (units/1000 (amino acid amino acid amino acid amino acid residues/ residues) residues) residues) unit) 37 32

1s 17

15 11 10 9.0 8.4 7.0 6.5 5.8 5.0 2.3 1.8 1.4 1.3 1.0 0

35 31 17 16

2.2 1.3 0.5 1.0

27 31 57 59

11

3.8

0

1.5 3.7 0.9 0.7 0.8 2.3 1.3 1.1 0.6 0.2 0.6 0.4 0

68 93 96 111 119 143 154 173 200 435 556 715 769 1000 -

9.7 8.9 5.3 7.5 6.3 5.7 3.5 3.7 1.2 1.2 1.2 0.7 0.6

1.1

HYl involved Total Hyl in linkage (residues/ of carbo1000 hydrate amino acid units (yo) Referencesa residues) 45 39 22 21

83 83 80 81

(1) (1) (2) (31

20 16 17 23 16 12 12 10 9.6 8.7 13 11 7.6 7.3 37

74 67 61 39 53 58 54 58 52 26 14 13 17 14 0

(4 )

5

n

(4)

(4) (5) (4) (4)

% gc3

E

5

(4 )

(6 1 (6) (6) (6) (6) (6) (6)

(7)

5 References: (1) Spiro and Fukushi (1969); (2) Spiro (1967~); (3) Beisswenger and Spiro (1970); (4) Spiro (1972a); (5) Miller (1971); (6) Spiro (1969b); (7) Spiro et al. (1971). Analyses performed on gelatins of collagens remaining insoluble after citrate buffer extraction.

*

0

w

384

ROBERT G. SPIRO

TABLE X Some Glycoproteins with More Than One Type of Carbohydrate Unit. Asn-linked Protein Thyroglobulin (calf) Thyroglobulin (human) Ribonuclease (porcine) IgM immunoglobulin (human) IgA immunoglobulin (human) IgG immunoglobulin (rabbit) Fetuin Chorionic gonadotropin (human) Erythrocyte membrane glycoprotein (human) Glomerular basement membrane (bovine) Lens capsule Corneal proteoglycan

Simple

+

+ +

+ +

Complex

+ + + + + + +

-1-

+

+ + +

Ser(Thr)linkedb

Hyllinked

Spiro (1965a) Arima et al. (1972) Jackson and Him (1970) Shimiau et al. (1971)

+

+ + + + + +

References

Dawson and Clamp (1968) Fanger and Smyth (19724 Spiro (1962a, 1970d) Bahl (1969b) Winxler (1969)

+ +

Spiro (1967b) Spiro and Fukushi (1969) Stuhlsats el al. (1971)

Units indicated by (+) have been identified in the listed proteins. Cuticle collagens from Nereis and cartilage proteoglycan contain two distinct types of ser(thr)-linked units (see Table VIII). a

Both hydroxylysine- and asparagine-linked units occur in glomerular basement membrane and lens capsule. Recent studies have indicated, moreover, that these two distinct types of units can occur on the same subunit of the glomerular basement membrane (Sato and Spiro, 1972), and that skin procollagen probably also contains both asparagine- and hydroxylysine-linked carbohydrate units (Spiro and Martin, 1972) (Section XII1,G). Clamworm cuticle collagen has been shown to have both serine (threonine) -linked galactose units and threonine-linked glucuronic acid-mannose units (Spiro and Bhoyroo, 1971). While the bovine corneal proteoglycan contains asparagine-linked keratan sulfate units as well as serine-linked chondroitin sulfate chains (Table X) the proteoglycan from bovine nasal cartilage has in addition to its chondroitin sulfate linked by xylosylserine bonds, keratan sulfate units which are attached to the peptide core by N-acetylgalactosaminylserine (threonine) linkages (Bray et aZ., 1967).

GLYCOPROTEINS

385

XI. METHODSFOR STRUCTURAL ANALYSISOF CARBOHYDRATE UNITS A number of techniques have been employed effectively to determine the sequence and linkages, both in terms of position and anomeric configuration, of the saccharide components of carbohydrate units. The details of these methods, which include enzymatic degradation with glycosidases, graded acid hydrolysis, isolation of oligosaccharides after alkaline-borohydride treatment or partial acid hydrolysis, periodate oxidation, and methylation have been reviewed (Spiro, 1966, 1 9 7 2 ~ ) . Owing to the multiplicity and heterogeneity (Section XII) of the carbohydrate units in glycoproteins, it is essential to resolve units of different types and variants of a given type prior to carrying out structural studies. This can usually best be accomplished by extensive proteolytic digestion of the glycoprotein followed by fractionation of the carbohydrate in the form of short glycopeptides. Such glycopeptides moreover are more accessible to degradation by glycosidases than the intact protein and make studies employing other techniques easier to perform and interpret. Treatment of glycopeptides with specific glycosidases has been instrumental in establishing the sequence and anomeric configuration of the saccharide components of the carbohydrate units of a variety of glycoproteins. A large number of such enzymes with different specifications have been purified and characterized from a wide variety of sources (Table XI). These enzymes are exoglycosidases which act by removing only the terminal nonreducing sugar from the polymer. If the complete sequence of a carbohydrate unit is to be established, sequential digestion by a number of enzymes is required. After each digestion a new sugar becomes uncovered and can in turn be released with the appropriate glycosidase. Sequential digestion of a carbohydrate unit in this manner in conjunction with methylation and periodate oxidation at various stages of enzymatic degradation permits the elucidation of the complete structure of a carbohydrate unit. Enzymatic degradation of the large carbohydrate units of proteoglycans with their repeating disaccharide sequence has been achieved with the use of endoglycosidases (endohexosaminidases) , such as testicular hyaluronidase (Meyer et al., 1960) and bacterial chondroitinase (Yamagata et al., 1968), to yield saturated oligosaccharides and unsaturated disaccharides, respectively. Methylation of glycopeptides can yield information in regard to linkages between sugar residues and the extent of branching. The method of Hakamori (1964) employs the methylsulfinyl carbanion in dimethyl sulfoxide to produce the carbohydrate alkoxide, which then is reacted

386

ROBERT G . SPIRO

TABLE XI Sources of Some Glycosidases Useful in the Study of the Carbohydrate Units of Glycoproteins Enzyme Neuraminidase

8-Galactosidase

Source Vibrio cholerae Clostridium perfringens Diplococcus pneumoniae Influenza virus Escherichia coli Jack bean Phaseolus vulgaris Aspergillus niger Clostridium perfringens Diplococcus pneumoniae

a-Galactosidase

8-N-Acetylglucosaminidase

Aspergillus niger Coffee bean Jack bean Pig epididymis Phaseolus vulgaris Aspergillus niger Diplococcus pneumoniae

a-Mannosidase

Jack bean Turbo cornutus

0-Mannosidase

Hen oviduct Turbo cornutus

a-Fucosidase

Pineapple Busycot ypus Clostridium perfringens

Aspergillus niger Itat epididymis a-N-Acetylgalactosaminidase Porcine liver Clostridium perfringen j 8-Xylosidase @-Glucuronidase

Lumbricus terrestris Charonia lumpas Bovine liver

a-Glucuronidase

Limpet

References* Ada et al. (1961) Cassidy et al. (1965) Hughes and Jeanloz (1964a) Rafelson et al. (1966) Hu et al. (1959) Li and Li (1968) Agrawal and Bahl (1968) Bahl and Agrawal (1969) Chipowsky and McGuire (1969) Hughes and Jeanloz (1964a) Muir and Lee (1969) Courtois and Petek (1966) Li and Li (1970) Findlay and Levvy (1960) Agrawal and Bahl (1968) Bahl and Agrawal (1969) Hughes and Jeanloz (1964b) Li (1967) Muramatsu and Egami (1967) Sukeno et al. (1971) Muramatsu and Egami (1967) Li and Lee (1972) Kabasawa and Hirs (1972) Aminoff and Furukawa (1970) Bahl (1970) Carlsen and Pierce (1972) Weissmann and Hinrichsen (1969) Chipowsky and McGuire (1969) Buddecke et al. (1969) Fakuda et al. (1969) Fishman and Bernfeld (1955) Marsh and Levvy (1958)

For examples of glycoproteins studied with these enzymes, see Spiro (1972~).

GLYCOPROTEINS

387

with methyl iodide; it has proved to be very effective for the methylation of glycopeptides (Spiro, 1972c) . Hexoses located in terminal nonreducing positions yield tetra-0-methyl ethers upon methylation, while from singly substituted internally located hexoses, tri-O-methyl ethers are obtained, and di-0-methyl derivatives indicate those serving as branch points (Table XII) . The hexosamines yield O-methyl ethers of N-methylamino sugars, with the formation of a tri-0-methyl ether in the case of a terminal sugar, and a di-O-methyl and mono-O-methyl ether for a singly or doubly substituted residue, respectively (Table XII) . Analysis of the O-methyl ethers of the neutral sugars can be achieved by gas-liquid chromatography while that of the O-methyl ethers of N-methylamino sugars can be performed by ion exchange chromatography with the use of the amino acid analyzer (Spiro, 1 9 7 2 ~ ) . Examination of the products of periodate oxidation of glycoproteins or glycopeptides can also provide valuable information in regard to the linkages which exist between sugar residues in the carbohydrate units. After borohydride reduction of the oxidized polymer the alcohols released upon acid hydrolysis are identified (Table XII). Terminal hexoses and those substituted a t C-2 and/or 6 give rise to glycerol, while hcxoses substituted only a t C-4 or a t C-4 and 6 yield either erythritol (from mannose and glucose) or threitol (from galactose). If the hexoses are substituted a t C-3 or a t C-2 and 4, no free vicinal hydroxyl groups are available, oxidation will not take place, and the undegraded hexose will be obtained upon hydrolysis. N-Acetylhexosamines will be oxidized only when located in terminal positions or when substituted solely a t C-6, yielding glycerol as the alcohol upon acid hydrolysis of the reduced periodate oxidation product. Fucose, when located in terminal positions, as is usually the case, yields 1,2-propylene glycol upon hydrolysis of the reduced oxidation product. Terminal sialic acid residues are susceptible to destruction by periodate, but this sugar is spared if singly O-substituted a t C-8 or if doubly O-substituted in any two positions. The product obtained by periodate oxidation of glycosidically bound sialic acid continues to be negatively charged and to react with the various colorimetric assays employed in the measurement of the unoxidized sugar (Spiro, 1964). Periodate oxidation can also be employed to determine the position on which N-acetylhexosamines involved in various glycopeptide bonds are substituted (Thomas and Winzler, 1969; Arima and Spiro, 1972). For this purpose reduced oligosaccharides obtained through p-elimination by alkaline borohydride treatment of glycoproteins or glycopeptides containing the N-acetylgalactosamine-serine (threonine) bond or produced by enzymatic cleavage of the N-acetylglucosamine-asparagine bond, fol-

TABLEXI1 Products of Methylation and Periodate Oxidation Obtained f r o m Various Sugars Occurring i n the Carbohydrate Units of Glywproteinsa Periodate products

Linkage to sugar Terminal 1-2 1-3 1-4 1-6 1 2 plus 1 3 1-+ 2 plus 1 4 1 + 2 plus 1 + 6 1-+ 3 plus 1-+ 4 1 3 plus 1 6 1-+4plus1-6

- ---f

Methylation productsb (0-methyl ethers)

3,63,4 2,6-t 2,4-t 2,3-t

Glucose or mannosec

Galactose"

Glycerol Glycerol Hexose Erythritol Glycerol Hexose Hexose Glycerol Hexose Hexose Erythritol

Glycerol Glycerol Galactose Threitol Glycerol Galactose Galactose Glycerol Galactose Galactose Threitol

N - Acetylglucosamine or N-acetylgalactosamine" Glycerol Hexosamine Hexosamine Glycerol

-

Hexosamine Hexosamine Hexosamine

N-Awtylglucosaminitold

-

L-Threosaminitol Xylosaminitol Serinol -

-

X ylosaminitol L-Threosaminitol Glucosaminitol

N-Acetylgalactmaminitold -

L-Threosaminitol L-Arabinosaminitol Serinol L- Arabinosamini to1 L-Threosaminitol Galactosaminitol

All saccharides are in the D-configuration except where otherwise indicated. 0-Methyl ethers of glucose, mannose, or galactose; glucosamine and galactosamine yield products indicated by t with an N-methyl instead of a 2-0-methyl substitution when the method of Hakamori is used. Sugar and sugar alcohols obtained after oxidation of specified sugar followed sodium borohydride reduction of product and acid hydrolysis. Amino polyols obtained from oligosaccharides terminating in specified N-acetylhexosaminitol after periodate oxidation and borohydride reduction followed by acid hydrolysis. a

s Ld

389

GLYCOPROTEINS

lowed by borohydride reduction, are submitted to oxidation. After reduction and acid hydrolysis of the products of such periodate oxidation, an amino alcohol is obtained which is indicative of the position of substitution of the terminal hexosamine (Table X I I ) . The amino alcohols (aminohexitols, aminopentitols, aminotetritols, and serinol) can be resolved and quantitated on the amino acid analyzer (Spiro, 1 9 7 2 ~ ) . The sequential application of the Smith technique of periodate oxidation, borohydride reduction and mild acid hydrolysis can serve to degrade the carbohydrate units of glycoproteins in a stepwise manner (Spiro, 1966). In this procedure the acetal bonds linking the oxidized and subsequently reduced sugars can be split under conditions of acid hydrolysis so mild as to leave intact the glycosidic bonds of the unoxidized sugars (Fig. 4 ) . Saccharides located in terminal nonreducing positions are oxidized and the reduced oxidation fragments are released by the mild acid hydrolysis. If oxidizable sugars are located in positions internal to resistant sugar residues, the mild hydrolysis will release the reduced oxidation products of the internal sugar with the more peripheral, nonoxidized portion of the sugar chain still attached. When only the sugars in nonreducing terminal positions are destroyed by periodate, the repeated application of the Smith degradation can then serve as a n alternate tool to stepwise removal of sugars by glycosidase action. The N-acetylneuraminyl- (2 + 3) -P-~-galactopyranosyI-(1 3 4) -N-acetyl-Dglucosamine chains of the ssparagine-linked units of fetuin (Spiro, 1964) and the N-acetylneuraminyl- (2 + 3)-P-D-galactopyranosyl- (1 + 3) -Nacetyl-D-galactosamine chains of the serine (threonine) -linked units (Spiro, 1970d) of this protein have been sequentially degraded by this technique. CH,OH

CH,OH

-.+ H*

p>,R CH,OH CH20H

CH,OH CHOH

I

CH,OH

+

CHO I

+ROH

CH20H

FIG. 4. Application of the Smith degradation technique involving periodate oxidation, sodium borohydride reduction, and mild acid hydrolysis to a terminal galactose residue. R = inner portion of the carbohydrate unit which is presumed to be unaffected by the oxidation step.

390

ROBERT G . SPIRO

Because of the difference in the stability to acid of the glycosidic bonds of the various sugars (which are presumed to occur in glycoproteins as pyranosides), it is often possible to obtain information in regard to sequence of saccharide residues by hydrolysis with dilute mineral acid for varying periods of time. The sialic acids, which are usually present in terminal positions, can be released under conditions (0.025-0.1 N sulfuric acid, 80°C, GO minutes) which leave other glycosidic bonds intact. Similarly, fucose when present in terminal positions can be released more rapidly than other sugars. Graded acid hydrolysis of fetuin glycopeptides, in which there are sialic acid-galactose-N-acetylglucosamine chains attached to a mannose-N-acetylglucosamine core, results in the release of sugars in the order: sialic acid > galactose > glucosamine > mannose when hydrolyzed with dilute sulfuric acid (0.05N , 100°C) (Spiro, 19G2b). When glycopeptides containing 2-O-c~-~-glucopyranosyl-5-O-/3-~galactopyranosylhydroxylysine are hydrolyzed in 0.1 N sulfuric acid a t 100°C, the glucose residue is released about 10 times more rapidly than the galactose (Spiro, 19G7c), even though methyl a-D-glucopyranoside is actually substantially more stable than methyl P-D-galactopyranoside to acid hydrolysis (Marshall and Neuberger, 1972). In this case the increased stability of the P-galactosyl bond results from the positive charge on the aglycon, i.e., on the c-amino group of the hydroxylysine which is vicinal to the glycosidic attachment. Blockage of this amino group by N-acetylation facilitates by as much as 35-fold (Fig. 5) the w

e 1.0 w

0

la

HOURS

FIG. 5. Differential releaw of glucose ( G ) and galactose (Gal) from the hydroxylysine-linked disaccharide unit during hydrolysis of native (Nat) and N-aeetylated (Ac) basement membrane glycopeptides with 0.1 N sulfuric acid a t 100°C. From Spiro (1967~).

GLYCOPROTEINS

391

hydrolysis of this bond and makes possible the isolation of the 2-0-a-Dglucosylgalactose disaccharide due to an equalization of the stability of the glucosidic and galactosidic bonds. When acid-labile bonds are located in internal positions of the carbohydrate units, oligosaccharides of varying size may be obtained from partial acid hydrolyzates, which may contribute important structural information. A number of oligosaccharides terminating in N-acetylglucosamine have been isolated from acid hydrolyzates of glycoproteins, with 4-O-/3-~-galactopyranosyl-N-acetyl-~-glucosamine (N-acetyllactosamine) being the most frequently obtained component (Eylar and Jeanloz, 1962 ; Spiro, 1962b). While the neutral N-acetyl-j3-n-glucosaminidic bond is fairly easily split by acid, if deacetylation should occur prior to its scission, the resulting glucosaminide with its positive charge close to the glycosidic linkage would become highly resistant (Moggridge and Neuberger, 1938). Carbohydrate units containing hexuronic acids tend to produce on acid hydrolysis high yields of disaccharides (aldobiuronic acids) containing the uronic acid linked to a more internal sugar due to the greater stability of the glucuronidic linkage. Chondrosine (3-O-&?-~-glucuronosyl-~galactosamine) from the chondroitin sulfate unit of proteoglycans (Davidson and Meyer, 1954), 3-O-/3-~-glucuronosyl-~-galactosefrom the carbohydrate-protein linkage region of a number of proteoglycans (Rod& and Armand, 1966) and 6-O-c~-~-glucuronosyl-~-mannose from the clamworm cuticle collagen (Spiro and Bhoyroo, 1971) are examples of such disaccharides obtained by acid hydrolysis. I n order to obtain sialic acid-containing oligosaccharides, acetolysis has been employed (Hickey et al., 1972). Preferential release of fucose from bromelain glycopeptide has been accomplished by hydrolysis in 1N acetic acid a t 100°C (Scocca and Lee, 1969) or trifluoroacetic acid a t 20°C (Yasuda et al., 1970). When carbohydrate units are linked to the peptide chain by O-glycosidic bonds involving the p-hydroxyamino acids, they can readily be obtained in the form of reduced oligosaccharides by mild alkaline treatment (0.1 N NaOH, 37°C) in the presence of sodium borohydride (Carlson, 1968; Mayo and Carlson, 1970). High concentrations of borohydride (1.0M) must be used during the alkaline treatment to ensure rapid reduction of the terminal sugar of the released oligosaccharide and to prevent alkaline degradation by a peeling reaction which is particularly likely to take place if this sugar is substituted at C-3. Using this approach the serine(thre0nine) -linked carbohydrate units of a number of glycoproteins have been obtained for structural studies, including those of several ovarian cyst and submaxillary gland glycoproteins, earthworm,

w

TABLE XI11 Ezamples of Heterogeneity in the Carbohydrate Units of Glycoproteins Protein

Type of unit

W

w

Type of variation

arMacroglobulina (human) (NAN-Gal-GlcNAc)n-(MamGlcNAc)-GlcNAc-Asn n = 0 4 ; extent of completion of chains attached to ManGlcNAc core n = 5-11 Thyroglobulin (calf and (Man),-GlcNAc-GlcNAc-Am human) (GlcNAc),-(Man),-GlcNAc-GlcNAc-Asn n = 0-3; m = 5-6 Ovalbumin* (hen) Collagensc (skin,tendon)

Glc-Gal-Hyl

Also occurs as Gal-Hyl

Cuticle collagens (Lumbricus, Nereis)

(Gal),,-Thr(Ser)

n = 1-3

Submaxillary glycoprotein (porcine)

GalNAc-Gal-GalNAc-Ser(Thr)

Fetuind

NAN-Gal-GalN Ac-Ser(Thr)

I

I

Fu NAN

I

NAN ~

~~~~

In various stages of completion ranging from GalNAc to pen tasaccharide Also occurs without the NAN attached to GalINAc ~

References D u n and Spiro (1967b)

Arima et al. (1972) Cunningham (1968); Huang et al. (1970) Cunningham and Ford (1968); Spiro (1969b) Muir and Lee (1969); Spiro (1970b); Spiro and Bhoyroo (1971) Carbon (1968)

Spiro (1970d)

~~~

Similar heterogeneity occurs in other plasma glycoproteins, glomerular basement membrane, and complex units of thyroglobulin and porcine ribonuclease. * Man and outer GlcNAc distributed between two chains (see Section XII1,E). c Also occurs in collagens from a large number of other sources (Spiro, 1972a). d Similar heterogeneity found in erythrocyte membrane glycoprotein (Winzler, 1969). 0

L-d

E Y

D

m

w

8

GLYCOPROTEINS

393

and clamworm cuticle collagens, fetuin, and red blood cell membrane glycoproteins (Section XIII) . Sugars such as sialic acid or fucose, easily cleaved by acid hydrolysis, can be obtained still linked to the carbohydrate units obtained by the alkaline-borohydride treatment.

XII.

HETEROGENEITY OF CARBOHYDRATE UNITS

One of the most characteristic features of the carbohydrate units of glycoproteins is their structural heterogeneity. While each type of carbohydrate unit has a distinct pattern, it usually occurs as a family of variants which appear to represent various stages of completion of this structural plan (Table XIII). This type of diversity, often termed microheterogeneity, must be differentiated from the presence of more than one distinct type of unit which also occurs in many glycoproteins (Section X) . The heterogeneity may occur in glycoproteins containing only a single carbohydrate unit, as well as in those containing multiple units. While in some glycoproteins the heterogeneity of the carbohydrate units can be recognized in the intact molecule due to differences in charge, if sialic acid is a variant, or to differences in size, if the carbohydrate makes up a substantial part of the total weight, in most cases, the diversity has become apparent only from a study of glycopeptides or oligosaccharides derived from the protein after enzymatic or chemical removal of the bulk of the peptide material. Charge differences in the carbohydrate can then become apparent and separation of glycopeptides or oligosaccharides containing varying numbers of sialic acid residues by anion exchange chromatography or electrophoresis can be achieved. Ion exchange chromatography on Dowex 50 a t an acid p H and low ionic strength has proved to be an effective tool for separating glycopeptides containing variants of the asparagine-linked neutral carbohydrate units (Fig. 6) (Section V1) . After strong alkaline hydrolysis, the disaccharide and monosaccharide forms of the hydroxylysine-linked carbohydrate units can be recognized and quantitated on the amino acid analyzer (Spiro, 1969b). No clear evidence for heterogeneity based on the substitution of one sugar for another has as yet been obtained, and this would be consistent with the high specificity of the glycosyltransferases involved in glycoprotein biosynthesis. While iduronic acid (Cifonelli and Dorfman, 1962) has been found in addition to glucuronic acid in the heparin proteoglycan, these sugars may occupy distinct positions in the chains rather than being randomly placed. Positional isomerism has so far been detected only in the a,-acid glycoprotein in which the terminal sialic acid residues can be linked to C-3, -4,

394

ROBERT G. SPIRO

FIG.6. Separation of variants of the asparagine-linked mannose-N-acetylglucosamine unit (unit A) of human thyroglobulin by chromatography on Dowex 50-X2 a t pH 3.0. The glycopeptides (105 mg hexose) from a Pronase digest of the thyroglobulin were applied to a column (1.9 X 120 cm) in 1 mM pyridine formate, pH 3.0, followed by elution with this buffer. A linear gradient of pyridine formatr buffer at this pH was started (arrow) from 1 mM (tube 171) to 0.155 M (tube 320). The molar ratio of mannose to 2 glucosamine residues is given for peaks 7-17 which contain the unit A of thyroglobulin. Peaks 1-6 represent primarily the unit B, which contains sialic acid, fucose, and galactose, in addition to mannos? and N-acetylglucosamine. From Arima et al. (1972).

or -6 of the galactose (Jeanloz, 1966). It is not yet known, however, whether this isomerism is random or is a specific structural phenomenon with a given linkage occurring only on certain oligosaccharide chains. The finding of heterogeneity in the single carbohydrate unit of ovalbumin (Cunningham, 1968; Huang e t al., 1970) clearly indicates that such diversity does not result exclusively from the presence of different units a t specified sites on the peptide chain such as might prevail in proteins with multiple carbohydrate units. Similarly it has been shown that each of the two complex heteropolysaccharide units of porcine ribonuclease has its own family of variants (Jackson and Hirs, 1970). While it is known from a study of glycoproteins with blood group activity that the structure of carbohydrate units can be genetically

GLYCOPROTEINS

395

determined through the inheritance of specific glycosyltransferases (Watkins, 19721, the microheterogeneity observed so widely in glycoproteins cannot be attributed to such a genetic phenomenon. The diversity is found in the carbohydrate units of glycoproteins from individuals of a given blood group type and has also been observed in the carbohydrate unit of ovalbumin isolated from the eggs of a single hen (Cunningham, 1968) and in IgG myeloma proteins which are believed to be synthesized by a single clone of cells (R. Kornfeld et al., 1971). Arguing further against a genetic determination of microheterogeneity are studies on the carbohydrate units of the a,-macroglobulin of human plasma which has 31 heteropolysaccharide units each of which may have from 0 to 4 sialic acid residues (Dunn and Spiro, 196713) (Table XIII). If, in this protein isolated from pooled plasma, molecules from a given individual would have uniform carbohydrate units with 4 residues of sialic acid each, 124 extra negative charges would be present compared to molecules from an individual whose carbohydrate units had no sialic acid. Molecules with such charge differences would clearly separate on electrophoresis instead of migrating in a uniform manner, as in fact they do. The underlying cause for the heterogeneity of glycoprotein carbohydrate units is most likely to be found in the mechanism operative in their biosynthesis (Section XV). This process is believed to be a postribosomal event which involves the stepwise attachment of sugar residues to the completed, membrane-bound peptide and is mediated by the action of glycosyltransferases. As the protein passes through the channels of the endoplasmic reticulum and Golgi apparatus where the glycosylation takes place, there may not always be the opportunity for all the transferases to act on a growing carbohydrate unit. If completion of the carbohydrate units is not required before the glycoprotein can leave the cell, molecules a t varying stages of synthesis would be exported and the degree of completion could depend on the rapidity with which the precursor protein passes through the membrane network. It also is possible that attachment of a branch point containing a charged sugar, such as sialic acid, prior to completion of the main saccharide chain could impose steric inhibition to the further action of glycosyltransferases. In the Carbohydrate unit of the submaxillary gland glycoproteins (Section XII1,F) , for example, such a premature attachment of a sialyl residue on the inner N-acetylgalactosamine could prevent the attachment of the outer sugar components. Similarly, early attachment of sialyl residues on the oligosaccharide chains of asparagine-linked carbohydrate units could sterically interfere with the completion of similar chains in the same unit. This, however, could happen only if the

TABLEXIV Carbohydrate Composition of Some Plasma Glywproteins

td

Sugar components (moles per mole of protein) Protein" Fetuin crl-Acid glycoprotein Haptoglobin, 2-1 Ceruloplasmin Transferrin arMacroglobulin Fibrinogen Prothrombin Hemopexin Corticosteroid-binding globulin Thyroxine-binding globulin

Molecular weight 48,000 44,000 200,000 160,000 90,000 820,000 330,000 70,000 70,000 52,000 58,000

Gal

Man

Fuc

GlcN

12 16 31 14 4 62 19

8 12 31 22 8 94 22

3 2 2 13

14 30 47 12

13

16d 32d

24d

16

5 -

8 146 19 10 29 26 12

GalN 3 -

-

3

Sialic acids 14 15 30 9 4

48 6 8 19 7 5

Glc

Totalb carbohydrate (yoof weight)

z8 cl

Referencesc

i z

Znas-Glycoprotein BaaTGlycoprotein 4s as-,%-Glycoprotein &-Glycoprotein 8-Lipoproteine High density lipoproteine Clq Protein Freezing point-depressing glycoprotein (Antarctic fish)’

41,000 40,000 60,000 31,000 100,000 75 000 390,000 21,500

7 8 14 13

)

56 38

15d 4.3

9 6 14 15 19

~~

~~

~~~~~~

All proteins except fetuin and the freezing pointrdepressing glycoprotein are from human plasma. * Calculated from sum of residue weights of sugar components, with the hexosamines considered to be in the N-acetyl form. c References: (1) Spiro (1960, 1962b) 1970d); (2) Yamashina (1956); (3) Gerbeck et al. (1967); (4) Jamieson (1965a); (5) Jamieson (1965b); (6) Dunn and Spiro (1967a); (7) Mester and Szabados (1968); (8) Lanchantin e6 al. (1968); (9) Hrkal and Muller-Eberhard (1971); (10) Muldoon and Westphal, 1967; (11) Giorgio and Tabachnick, 1968; (12) Burgi and Schmid, 1961; (13) Ishihara and Schmid (1967); (14) Iwasaki and Schmid (1970); (15) Labat et al. (1969); (16) Ayrault-Jarrier et al. (1961); (17) Scanu (1966); (18) Yonemasu et al. (1971); (19) DeVries et al. (1970). Determined as total hexose (galactose plus mannose). 6 Delipidated protein. Trematomus borchgrevinki. 5

398

ROBERT G . SPIRO

locations of the glycosyltransferases on the membranes are not effectively segregated, permitting their action on carbohydrate units a t varying stages of synthesis. The possibility that the catabolic machinery of glycoproteins is responsible for the heterogeneity of the carbohydrate units, however, cannot be discounted, in particular because of our sparse knowledge of this process. Glycosidases are widely distributed in tissues active in glycoprotein metabolism. While these enzymes have usually been believed to be entirely lysosomal in location, recent studies have indicated that they can also be found attached to the plasma membrane of cells (Section XVI) . Enzymes in the latter location could conceivably partially degrade the carbohydrate units of a glycoprotein in its passage in or out of the cell.

XIII. STRUCTURE OF CARBOHYDRATE UNITSOF SPECIFIC GLYCOPROTEINS With the preceding sections of this review serving as a background to

the structural concepts and methodology applicable to glycoproteins, one can now consider information available about the carbohydrate units of specific glycoproteins with diverse biological functions.

A . Plasma Glycoproteins All the numerous proteins of plasma, with the exception of albumin (Peters et al., 1971), prealbumin (Peterson, 1971), and some low molecular weight proteins (Nimberg and Schmid, 1972), contain covalently linked carbohydrate (Table XIV). These proteins serve various purposes, including those of metal, lipid, and hormone transport, clotting, freezing point-depression, and immunoprotection. The immunoglobulins serving the latter function will be considered separately because of their specialized structure and distinct site of synthesis (Section XII1,B). Most of the other major plasma glycoproteins are assembled in the liver (Spiro, 1965b) and are probably also degraded by the parenchymal cells of this organ (Ashwell and Morell, 1971). The most common carbohydrate units of plasma glycoproteins are asparagine-linked branched heteropolysaccharides, the structure of which has been studied in some detail in a number of proteins, including fetuin (Spiro, 1962a,b), a,-acid glycoprotein (Jeanloz, 1966; Wagh e t al., 1969), transferrin (Jamieson et al., 1971a) , and a2-macroglobulin (Dunn and Spiro, 1967b). There may be as few as 3 (fetuin) or as many as 31 (azmacroglobulin) of such units present in these proteins (Table VII). Carbohydrate linked to the a-amino-P-hydroxy acids also occurs in plasma proteins, having been found in fetuin and the freezing pointdepressing glycoproteins of Antarctic fishes (Table VIII) .

399

GLPCOPROTEINS

Studies on fetuin have revealed that its single peptide chain contains three asparagine-linked (Spiro, 1962a) and three serine (threonine) -linked (Spiro, 1970d) carbohydrate units (Fig. 7). The former units, which make up about 85% of the total carbohydrate and contain all of its N-acetylglucosamine and mannose, were investigated first through a study of glycopeptides obtained after papain or Nagarse digestion (Spiro, 1962a,b, 1964) and more recently by an investigation of glycopeptides isolated after proteolysis with Pronase (Spiro and Bhoyroo, 1972). The a-N-acetylneuraminyl- (2 + 3)-,B-D-galactopyranosyl- (1 + 4) -N-acetyl-D-glucosamine sequence of oligosaccharide chains attached to a mannose-N-acetylglucosamine core was established with the use of glycosidases, periodate oxidation including Smith degradation, and the isolation from partial hydrolysates of N-acetyllactosamine (4-0-,8-~galactopyranosyl-N-acetyl-D-glucosamine) . While the structure of the core portion has not yet been fully determined, Smith degradation, glycosidase studies and the isolation of oligosaccharides have indicated that it consists of 3 adjacent mannose residues attached to 2 or more internal N-acetylglucosamines. The anomeric configuration of the bonds between the mannose residues was found to be a, while a ,B linkage was shown to exist between the N-acetylglucosamines. Methylation and periodate oxidation of glycopeptides have shown that the two internal N-acetylglucosamine residues are attached to each other by a 1+ 4 glycosidic bond and that the inner two mannose residues serve as branch points. After extensive Pronase digestion of fetuin, glycopeptides containing only N-acetylgalactosamine, sialic acid, and galactose can be separated by gel filtration from the glycopeptides containing the asparagine-linked carbohydrate units (Spiro, 1970d). Studies on these glycopeptides further purified by DEAE-cellulose chromatography have shown that they contain carbohydrate units in which a-N-acetylneuraminyl- (2 + 3) -,B-D-galactopyranosyl- (1+ 4) -N-acetyl-D-galactosamine chains, with or without an additional sialyl residue attached to C-6 of the N-acetylNAN

NAN

NAN

NAN

la2-3 GY3 Gal al

Gal 1al-4

-

101-4

Gal

101-3

101-4

GLcNAc GlcNAc GlcNAc 181-2 p31-3(41 p ~ M a n al-2(61 Man-Man-GlcNAcal-3

*

S

)

NAN ~

PI-4

GlcNAc-Asn P

G

~

L

1" Ser(Thr)

N

FIG. 7. Structures proposed for the asparagine-linked and serine(threonine)linked carbohydrate units of fetuin. The latter units also occur in the form of a trisaccharide without N-acetylneuraminic acid linked to the N-acetylgalactosamine. Formulations based on the studies of Spiro (1962a,b, 1964, 1970d) and Spiro and Bhoyroo (1972).

A

~

400

ROBERT G . SPIRO

galactosamine are linked directly to serine (threonine) residues by aglycosidic bonds (Fig. 7). These carbohydrate units, contrary to the asparagine-linked carbohydrate, can be split from the peptide by alkaline borohydride treatment and obtained in the form of acidic tri- and tetrasaccharides with N-acetylgalactosaminitol in a terminal position. Because of the linkages prevailing in the sialic acid-galactose-N-acetylhexosamine chains of both the asparagine- and serine (threonine)-linked units, three sequential Smith periodate oxidations can degrade both types of chains in a stepwise manner. The a,-acid glycoprotein of human plasma consists of a single peptide chain to which five asparagine-linked heteropolysaccharide units are attached (Table VII) . Cyanogen bromide cleavage of this protein, followed by reduction and alkylation, yields an N-terminal fragment of 134 residues containing all the carbohydrate units (Schmid et al., 1971). Studies of glycopeptides obtained after Pronase digestion (Wagh et al., 1969) and oligosaccharides isolated after partial acid hydrolysis (Eylar and Jeanloz, 1962) or hydrazinolysis (Sato et al., 1967) have yielded information about the structure of the carbohydrate units of the a,-acid glycoprotein. Although these units demonstrate microheterogeneity (Yamauchi et al., 1968) their main structural feature (not unlike that of fetuin) consists of sialic acid-galactose-N-acetylglucosamine chains attached to a core portion consisting of mannose and additional N-acetylglucosamine residues. Methylation studies have indicated that the sialic acid residues are linked predominantly to C-4 and C-6 of the galactose residues, but that some bonds to C-3 also occur (Jeanloe, 1966). Thc location of the fucose residues of the a,-acid glycoprotein have not been clearly determined as yet although it has been proposed that they, like the sialic acid components, are located a t the end of the chains attached to galactose (Jeanloz, 1966). The structure of the inner portion of the heteropolysaccharide units of the a,-acid glycoprotein has not yet been established unequivocally although a full structural proposal has been formulated (Wagh et al., 1969) from studies of a glycopeptide with the use of glycosidases and periodate oxidation. In this protein, as in fetuin, 3 mannose and 2 N acetylglucosamine residues were found to make up the core portion, and two of these mannose residues were believed to be the points of attachment of four sialic acid-galactose-N-acetylglucosamine chains. The third mannose was considered to be part of a N-acetylglucosamine-mannose-N-acetylglucosamine sequence, with the innermost N-acetylglucosamine being involved in the linkage to asparagine, and both the inner and outer of the N-acetylglucosamines of this trisaccharide being substituted with one of the branched mannose residues. Such a structure would

GLYCOPROTEINS

401

therefore not include the di-N-acetylchitobiose-asparagine linkage region which has been found in several other glycoproteins, nor would i t contain any adjacent mannose residues. More recently methylation studies have confirmed the role which the two mannose residues play as branch points (Schwarzmann et al., 1972a). However, on the basis of glycosidase degradations it was found that the three mannose residues are contiguous and are indeed attached to a linkage region containing two N-acetylglucosamine residues (Schwarzmann e t al., 197210). Studies on glycopeptides from Pronase digests of the a,-macroglobulin of human plasma have revealed that the 31 carbohydrate units of this protein (Table VII) conform to a similar structural plan as described for fetuin (Dunn and Spiro, 1967b). Sialic acid (fucose)-galactose-Nacetylglucosamine chains linked to a core portion made up of three mannose and two N-acetylglucosamine residues appear to prevail. The isolation of N-acetyllactosamine, moreover, suggests a p (144) bond between galactose and N-acetylglucosamine in the chain, and the finding of a mannobiose in partial acid hydrolyzates indicates that at least two of the three mannose residues of the core are linked together. A high degree of microheterogeneity was observed in the units of the wmacroglobulin with the smallest being made up of only three residues of mannose and two of N-acetylglucosamine and the largest containing in addition four complete oligosaccharide chains (Table XIII) . Information about the two asparagine-linked carbohydrate units of human plasma transferrin has been obtained from a study of glycopeptides isolated after Pronase digestion of this protein (Jamieson et al., 1971a). Each unit was found to contain two sialyl- (2 + 6) -@-D-galactopyranosyl- (1+ 3 (4)) -N-acetylglucosamine chains which are attached to a core of four mannose and two N-acetylglucosamine residues. A structure was proposed in which the four mannose residues are placed contiguous to each other with the most internal of these serving as a branch point and being attached in turn to the N-acetylglucosamine component which is involved in the glycosylamine bond to asparagine. An entirely different type of carbohydrate unit has been found in the freezing point-depressing glycoproteins from the sera of the Antarctic fish Trernatornus borchgrevinki (Table VIII) (Komatsu e t al., 1970; DeVries et al., 1971). Studies of glycopeptides obtained after subtilisin or elastase digestion have indicated that these proteins have a very simple primary structure consisting of a repeating tripeptide with the sequence alanine-alanine-threonine (Table V) . P-Elimination studies under mild alkaline conditions indicated that all threonine residues are glycosidically substituted with disaccharide units consisting of galactose and N-acetylgalactosamine. Pcriodate oxidation demonstrated that galactose is in

TABLE XV Carbohydrate Composition of Human Immunoglobulins

Immunoglobulin IgG Ig-4 IgM

Heavy" chain type

Molecular weight

Y

150,000

a

170,000

p

(180,000)s ~~~~~~~~

Total carboSugar components (moles per mole of monomer) hydrateb (% of Sialic weight) Gal Man Fuc GlcN GalN acid 2.5 5.7 9.2

3 12 11

5

2

2

14 35 ~

6

~~~~

9 12 27

6

-

1 5 9

Total sugar residues Per monoPer mer molecule 20 51 88

20 51 440

~

The three classes of immunoglobulins may have either K or A light chains. Calculated from sum of residue weights of sugar components, with the hexosamines considered to be in the N-acetyl form. =References: (1) Rosevear and Smith (1961); (2) Dawson and Clamp (1968); (3) Johnson and Clamp (1971).

a

0

ReferenmC (11 (2) ( 31

8 ? u, 'd

GLYCOPROTEINS

403

the terminal position and is linked to C-3 or C-4 of N-acetylgalactosamine, which in turn is attached to the hydroxyl group of threonine. The occurrence of a somewhat larger number of galactose than N-acetylgalactosamine residues and the incomplete destruction of galactose with one periodate oxidation suggested that about 20% of the chains occur as galactose-galactose-N-acetylgalactosamine trisaccharides. Studies utilizing the nuclear magnetic resonance spectra have indicated that the disaccharide is 4-O-~-~-galactopyranosyl-N-acetyl-~-galactosamine and suggest that it is linked by an a-glycosidic bond to threonine residues (Shier et al., 1972). However, the recent finding that J3-elimination of the disaccharide results in the rapid formation of chromogen favors the existence of a 1 + 3 bond between the galactose and the N-acetylgalactosamine (Vandenheede et al., 1972). Human Clq protein, a component of complement, is so far the only protein of serum in which glucose has been found (Yonemasu et al., 1971; Calcott and Muller-Eberhard, 1972). It is most unusual, moreover, in containing substantial amounts of hydroxyproline and hydroxylysine, and i t appears that close to 60% of the residues of the latter amino acid may be substituted by glucosylgalactose disaccharide units such as are commonly found in basement membranes and collagens. The presence of mannose, fucose, sialic acid, and hexosamines in this protein indicates that the more typical asparagine-linked unit may also occur.

B. Immunoglobulins It has been clearly established that the immunoglobulins, from man and other species, contain covalently linked carbohydrate moieties. The amount of saccharide differs among the major classes of immunoglobulins, being the lowest in the IgG and highest in the IgM globulins (Table XV) . The nature and site of attachment of the carbohydrate units of the IgG globulins have been studied extensively after isolation of glycopeptides obtained from proteolytic digests of normal and myeloma proteins (Rosevear and Smith, 1961; Nolan and Smith, 1962; Clamp and Putnam, 1964; R. Kornfeld et al., 1971). Both human and rabbit IgG immunoglobulins uniformly contain an asparagine-linked carbohydrate unit on each of the two heavy chains (Fig. 8 ) . These units have been located in the Fc fragment and are therefore part of the constant portion of these peptide chains. Their exact site of attachment has been determined for a myeloma immunoglobulin through the complete sequence analysis of this protein (Edelman et al., 1969) (Table IV). A study of a large number of human IgG myeloma proteins has revealed that some contain

404

ROBERT G . SPIRO

-

-

Fob

( Fob = Fd + L)

s-s I t

s-5

L h ~ ~ v \ A s n mI

s-s

c‘,

s-s

HJ~vdAsn/vvr

\

H

I

I

COOH

S

I

4

P2 7

I Thr-Asn

mAsnI 1 1 s-s P

w s-s

COOH

3

sI

‘

I

s-s

7 I

s-s

I

COOH

S

/c4

Asn-

7 7-7 ‘c,

c3

s-s

L

,

-Fc-

I

s-s

1

COOH

FIG.8, Schematic portrayal of the distribution of carbohydrate units (C) on IgG immunoglobulin molecule. CI, present in both rabbit and human protein; C1, attached to hinge region of one heavy chain in most rabbit molecules; C3, found in a small percentage of both rabbit and human IgG; C,, reported to be present in a small percentage of human light chains. L = light (x or K) chains; H = heavy ( y ) chains. Papain cleavage of IgG yields Fab and Fc fragments. The variable portions of the chains are depicted by jagged lines, while the constant portion is shown by straight lines. See text for references.

additional asparagine-linked carbohydrate units attached to the variable portion of the F d fragments of the heavy chains (Spiegelberg et al., 1970). I n rabbit IgG immunoglobulins isolated from pooled sera, about 15% of the heavy chains contain such additional asparagine-linked carbohydrate units (Fanger and Smyth, 1972a). These carbohydrate units from the F d portion of the rabbit heavy chains contain glucosamine, mannose, and galactose like those attached to the Fc fragment, but, unlike the latter, they do not have sialic acid or fucose as sugar constituents. It has furthermore been shown that about 40% of the heavy chains of pooled rabbit IgG immunoglobulins contain a threonine-linked carbohydrate unit (Fanger and Smyth, 1972a) which is located in a prolinerich amino acid sequence in the hinge region of the molecule (Smyth and Utsumi, 1967) (Table V). This unit, which contains all of the galactosamine of the molecule (Table VIII) is present on only one heavy chain. Evidence for this asymmetric attachment of the threonine-linked unit in a t least 15% of the IgG molecules was obtained through the isolation of a glycopeptide containing this unit linked through a disulfide bridge to a second peptide of the same sequence but devoid of carbohydrate (Fanger and Smyth, 1972b). It was proposed that saccharide attachment to the second heavy chain is sterically prevented by the presence of this threonine-linked unit on the first. Such an explanation, however, could apply only if the enzymatic process of carbohydrate

GLYCOPROTEINS

405

attachment follows conjugation of the chains through disulfide bond formation. Galactosamine has not been detected in human normal or myeloma IgG globulins indicating that the occurrence of the threoninelinked unit is species dependent. Investigations of the carbohydrate of the light chains of the IgG immunoglobulins have been conducted on individual IgG myeloma and Bence-Jones proteins. The latter proteins are obtained from the urine of patients with multiple myeloma and are identical in amino acid sequence to the light chains of the corresponding myeloma globulins. These studies have shown that the light chains of some IgG globulins (15% of human myeloma proteins examined) contain asparagine-linked carbohydrate units which appear always to be attached in the variable portion of the chain (Edmundson et al., 1968; Melchers, 1969; Sox and Hood, 1970; Spiegelberg et al., 1970). Such units have been found in both K and X chains (Sox and Hood, 1970; Spiegelberg et al., 1970) and are attached t o asparagine residues in the characteristic Asn-X-Ser (Thr) sequence (Table IV) . Although the absence of such carbohydrate units on the variable portion of most light chains (as well as on the variable portion of most heavy chains) can be partly attributed to the lack of the necessary amino acid constellation next to an asparagine residue (Melchers, 1969), the finding of the Asn-X-Ser(Thr) sequence on some light chains which nevertheless do not contain carbohydrate suggest that steric hindrances or lack of availability of glycosyltransferases could also be responsible (Spiegelberg e t al., 1970; Sox and Hood, 1970). The carbohydrate units of the light chains, like those attached to the Fc portion of the heavy chains, contain galactose, mannose, glucosamine, fucose, and sialic acid as their saccharide constituents. The carbohydrate composition of the units on the Bence-Jones proteins is similar to that of the light chains isolated from the corresponding myeloma proteins, except for a higher sialic acid content (Spiegelberg e t al., 1970). I n vitro reconstitution experiments however have indicated that heavy chains combined as readily with light chains as with Bence-Jones proteins despite the higher sialic acid content of the latter (Spiegelberg et al., 1970). The structure of the carbohydrate units linked to the Fc portion of each of the heavy chains of human IgG immunoglobulins (Table VII) has received detailed attention from several laboratories (Rosevear and Smith, 1961; Rothfus and Smith, 1963; Clamp and Putnam, 1964; R. Kornfeld e t al., 1971). The most recent of these investigations was conducted on glycopeptides obtained by Pronase digestion of several myeloma globulins (R. Kornfeld e t al., 1971) in which all the carbohydrate units were characterized by the presence of sialic acid-galactoseN-acetylglucosamine chains attached to a mannose-N-acetylglucosamine

406

ROBERT G . SPIRO

core (Fig. 9). Two of such oligosaccharide chains occurred in each unit, but these were present in varying degrees of completion, often not having a full complement of sialic acid and galactose residues. Microheterogeneity was further evident in the fucose content of various carbohydrate units which ranged from 0.6 to 1.0 residues per unit. Since this microheterogeneity of peripheral residues was noted in myeloma proteins presumably produced by a single clone of cells, it attests to the nongenetic basis of this phenomenon. The core portion of the IgG carbohydrate units was found to be made up of 3 mannose and 2 or 3 N-acetylglucosamine residues. One mannose residue was found as a branch point in the glycopeptides of all myeloma proteins examined, and it was linked either to the outer (Fig. 9) or inner component of a di-N-acetylglucosamine, which in turn was attached to asparagine. Di-N-acetylchitobiose was isolated from the core of a glycopeptide in which the sequence mannose-N-acetylglucosamine-N-acetylglucosamine-asparagine occurred. The presence of two N-acetylglucosamine residues in the innermost portion of the IgG carbohydrate unit had previously been indicated by the periodate oxidation studies of Rothfus and Smith (1963). The core portion of the carbohydrate units of some IgG myeloma globulins contained a third N-acetylglucosamine residue the precise position and linkage of which has not yet been ascertained. Variations in the number and arrangement of the N-acetylglucosamine residues of the core portion was found only in proteins from different individuals and could therefore, contrary t o the microheterogeneity of the peripheral saccharides, have a genetic basis (R. Kornfeld et al., 1971). Most of the information available in regard to the carbohydrate units of the IgA protein comes from a study of glycopeptides isolated from proteolytic digests of an A myeloma globulin, K type (Dawson and Clamp, 1968). All the carbohydrate of this protein was found on the NAN a2-64

Gal Pl-6i

GlcNAc

P

GlcNAc

A

/PI-2

Man

a1 - 3\

Man

Man/ a 1- 6

{:2 al-3(4)

F U C L

p - 4

.1P

Asn

FIG.9. Proposed structure of carbohydrate unit of human IgG immunoglobulin. Based on studies of myeloma (M'M and Kel) proteins (R. Kornfeld et al., 1971).

GLYCOPROTEINS

407

heavy chains in the form of 3 complex and 1 simple asparagine-linked and 2 serine (threonine)-linked units per molecule (Tables VII and VIII) . The odd number of asparagine-linked units indicates an asymmetrical distribution of the carbohydrate units on the two heavy chains. The complex asparagine-linked units of this protein were shown to consist of three sialic acid-galactose-N-acetylglucosamine chains (occurring in various stages of completion) attached to an internal portion of 3 mannose residues and 1 additional N-acetylglucosamine. The location of the single fucose has not yet been clearly determined, even though in a tentative structural scheme its attachment to the N-acetylglucosamine residue involved in the glycopeptide bond was proposed. IgM globulins isolated from the sera of individual patients with Waldenstrom macroglobulinemia have been shown to contain all their carbohydrate on the two heavy ( p ) chains (Johnson and Clamp, 1971; Shimizu et al., 1971; Hickman et al., 1972). Each IgM monomer appears to have a total of 10 asparagine-linked carbohydrate units, of which 6 are of the complex type and 4 are of the simple form (Table V I I j (Shimizu et al., 1971 ; Hickman et al., 1972). Studies of glycopeptides have indicated that the carbohydrate is attached a t five sites in the constant region of each heavy chain (Shimizu et al., 1971). One complex unit is located in the Fd fragment, another in the hinge region, and the third in the Fc region. Both simple units are attached in the F c region of the heavy chain with one of these units being localized on the fourteenth residue from the C-terminus of the chain. The complex carbohydrate units demonstrate extensive microheterogeneity (Johnson and Clamp, 1971 ; Hickman et al., 1972) and appear to conform to a structural pattern similar to that of the carbohydrate units of human IgG immunoglobulins (Fig. 9 ) . The simpler carbohydrate units are also branched and consist of a variable number of mannose residues attached to two internally located N-acetylglucosamine residues (Hickman et al., 1972). (Y

C . Hormones All the gonadotropic hormones, thyroid-stimulating hormone (TSHj , and thyrogiobulin have been shown to be glycoproteins (Table XVI). While the latter protein is not a hormone itself, i t is the storage form of the thyroid hormones. The complete amino acid sequence of bovine TSH (Liao and Pierce, 1971), ovine (Liu et al., 1972a,b) and bovine (Maghuin-Rogister and Hennen, 1971) luteinizing hormone (LH), and human chorionic gonadotropin (HCG) (Bahl et al., 1972) have been reported, providing im-

TABLEXVI Carbohydrate Composition of Same H m n e Glyeoprateins Totala carbo-

Sugar components (moles per mole of protein) Protein Thyroid-stimulating hormone (bovine) a-subunit @-subunit buteinizing hormone (bovine) a-subunit &subunit Follicle-stimulating hormone (ovine) Chorionic gonadotropin (human) Thyroglobulin (calf) Thyroglabulin (human)

Molecular weight

Gal

Man

Fuc

GlcN

GalN

weight)

References*

s 13,300 13,600 12,000 13,700 32,000 27,000 670, OOO 670, OOO

0.2

-

0.1 0.1 6

9 50 50

6 3 7 3 6 9 86 130

0.3 0.9 0.4 0.7 1 1 17 21

6 3 5 4 6 11 97 114

3

2

2 1 3 3

13 ~~~

a

acids

-

-

21 12

(1) (1)

8 Ei I3 0

m

6

22 12 18

8 30 23

31 7.9 9.7

(1) (1) (2) (31 (4) (4, 5)

Calculated from s u m of residue weights of sugar components with the hexosamines considered to be in the N-acetyl form. References: (1) Liao and Pierce (1970); (2) Cahill ed al. (1968); (3) Bahl (1969a); (4) Spiro and Spiro (1965a); ( 5 ) Arima et al. (1972).

409

GLYCOPROTEINS

portant information in regard to the nature and site of attachment of the carbohydrate units of these proteins (Tables IV and V) . TSH, LH, and HCG are each made up of two noncovalently linked subunits designated as a and @ (Pierce, 1971). Through the work of Pierce and his collaborators (Liao and Pierce, 1970; Pierce et al., 1971a), it has been shown by recombination experiments that the a subunits of these three hormones are biologically interchangeable, while the p subunits are hormone specific. The amino acid sequence of the subunit of L H (ovine) (Liu et al., 1972a) has been shown to be identical to that of the a subunit from TSH (bovine) (Liao and Pierce, 1971), as had previously been anticipated from a comparison of tryptic and chymotryptic peptides from the bovine hormones (Pierce et al., 1971b). Although the a subunits of both hormones contain two asparagine-linked carbohydrate units situated at the same position in the chains, small differences in carbohydrate composition have been noted which if substantiated by structural studies could provide evidence for synthesis of the chains of the two hormones in different cell types or via distinct glycosyltransferase systems of the pituitary gland (Pierce et al., 1971b). The a subunit of HCG, although biologically interchangeable with bovine TSH-(Yand ovine LH-a, has about 25 amino acid substitutions (Bahl et al., 1972). The a subunit of HCG, moreover, differs markedly from that of TSH and LH in containing large amounts of galactose and siaIic acid and by the absence of galactosamine. Although the p subunits of TSH, LH, and HCG have quite different amino acid compositions, considerable homology exists among them (Pierce, 1971; Liu et al., 197213; Bahl et al., 1972). While TSH- and LH-/3 subunits have been reported to contain one carbohydrate unit each, HCG has been found to have 2 asparagine-linked and 3 serine-linked carbohydrate units on each J3 chain (Tables I V and V ) . The asparagine-linked units of HCG are believed to be similar to those already described for several plasma glycoproteins in consisting of sialic acid ( fueose) -galactose-N-acetylglucosamine chains attached to a mannose-N-acetylglucosamine core while the serine-linked carbohydrate units were found to consist of one residue each of sialic acid, galactose, and N-acetylgalactosamine, with the latter sugar taking part in the glycopeptide bond (Bahl, 1969b). The occurrence of N-acetylgalactosamine in the and p subunits of TSH and LH suggests the possibility that in addition to the already identified asparagine-linked units, serine (threonine) -linked units also occur in these hormones. The elucidation of the structure of these carbohydrate units will therefore be of importance, as asparagine-linked carbohydrate units containing galactosamine have so far not been described. (Y

(Y

(Y

410

ROBERT G. SPIRO

Thyroglobulin is an iodinated glycoprotein which contains about 10% of its weight in the form of carbohydrate (Spiro and Spiro, 1965a). Studies of this protein from calf (Spiro, 1965a), human (Spiro and Spiro, 1965b; Arima et al., 1972), and porcine (Fukuda and Egami, 1971) glands have shown that its saccharides are distributed among simple (unit A) and complex (unit B) forms of asparagine-linked carbohydrate units (Table VII). The human protein, which contrary to the other thyroglobulins, contains galactosamine, has in addition been found to contain a number of serine(threonine)-linked units (unit C) in which all of the residues of this sugar are found (Arima et al., 1972). A study employing glycosidase treatment, methylation, and periodate oxidation of glycopeptides obtained after Pronase digestion of the calf and human thyroglobulins has revealed that the simple asparaginelinked carbohydrate units consist of an extensively branched mannose portion linked to the outer residue of a N-acetylglucosamine disaccharide, which in turn is linked to asparagine on the peptide chain (Fig. 10) (Arima and Spiro, 1972). After enzymatic degradation, di-N-acetylchitobiose (GlcNAc-GlcNAc) , di-N-acetylchitobiose linked to asparagine by a P-glycosylamine bond (GlcNAc-GlcNAc-Asn) , and N-acetylglucosamine linked by this bond to asparagine (GlcNAc-Asn) were isolated from the glycopeptides. This unit exhibits extensive microheterogeneity (Table XIII) , and a study of its variants separated by Dowex 50 chromatography (Arima et al., 1972) indicated that in its smallest form it consists of 5 mannose residues linked to the di-N-acetylchitobiose (Fig. 10). Larger units with a maximum of 11 mannose residues were found to be made up of this core portion through the addition of extra residues of mannose primarily

- -- -----_ --_- -

I--I I

Man

- ---- - - -- - - - - - - - - --,

Man: -Man L

I

I

I

I

L

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ___--------__-_-

1

FIG.10. Proposed structure of carbohydrate unit A of calf and human thyroglobulin and its peptide attachment. The components within the box make up the smallest unit observed and constitute the core portion to which other mannose residues are attached to form the larger units. The anomeric configuration of the bond between the most internal mannose and N-acetylglucosamine is not designated, as there is still some uncertainty in regard to its nature. In (Man)., x = 0 to 6. Taken from Arima and Spiro (1972).

GLYCOPROTEINS

411

on to the termini of the 3 major chains. Pronase digestion of both human and calf thyroglobulin yielded in addition to the glycopeptides some neutral oligosaccharides which contained 7 to 8 mannose residues attached to di-N-acetylchitobiose located in a terminal reducing position. These oligosaccharides were presumed to have resulted from the cleavage by Pronase of the glycosylamine bond which attaches the mannose-Nacetylglucosamine units to the peptide chains (Arima et al., 1972). The complex asparagine-linked units of both calf (Spiro, 1965a ; Spiro and Spiro, 1965b) and porcine (Fukuda and Egami, 1971) thyroglobulin have been found to contain chains with the sialic acid-ga1actose-Nacetylglucosamine sequence attached to a core of 3 mannose and 2 N-acetylglucosamine residues. While the location of fucose in nonreducing terminal positions has been demonstrated, the sugar to which it is attached has not yet been clearly established. The complex carbohydrate units also exhibit extensive microheterogeneity which becomes most apparent upon fractionation of their glycopeptides on DEAE-cellulose (Spiro, 1965a ; Arima et al., 1972).

D . Enzymes An increasing number of enzymes from diverse sources are being characterized as glycoproteins (Tables I and XVII) . Of these enzymes the ribonucleases have been investigated most extensively in regard to the nature of their carbohydrate units. After the entire primary structure of bovine ribonuclease A, which contains no carbohydrate, had been elucidated (Anfinsen and Redfield, 1956; Hirs e t al., 1960) the isolation from bovine pancreatic juice of another ribonuclease (ribonuclease B) containing about 9% carbohydrate was reported (Plummer and Hirs, 1963). The carbohydrate-containing enzyme was shown to have the same activity toward ribonucleic acid as ribonuclease A, suggesting that the location of the sugar attachment was not near the active site. A study of peptides obtained after trypsin digestion of ribonuclease B indicated that this protein has the same primary structure as ribonuclease A except for the presence of a single carbohydrate unit on the asparagine residue in position 34 (Plummer and Hirs, 1964) (Table I V ) . This unit was obtained in the form of a glycopeptide which contained 6 mannose and 2 N-acetylglucosamine residues (Table VII) . Further fractionation of bovine pancreatic juice revealed the presence of 2 additional ribonuclease components, termed C and D, which contained sialic acid, fucose, and galactose residues in addition to mannose and N-acetylglucosamine (Plummer, 1968). These proteins had the same specific activity as ribonuclease A and B and on the basis of pep-

TABLEXVII

Carbohydrate Composition of Some Enzyme Glycoproteins

Totala carbohydrate

Sugar components (moles per mole of protein) Protein Ribonuclease B (bovine pancreas) Ribonuclease (porcine pancreas) Deoxyribonuclease A (bovine pancreas) Bromelain (pineapple stem) a-Amylase (Aspergillus oryzue)d a-Amylase (human parotid) Invertase (yeast) Invertase (Neurospora crassa) Glucose oxidase (Aspergillus niger)

Molecular weight

Gal

Man

Fuc

GlcN

GalN

Sialic acids

14,700 22,300 31,000 33,000 53,000 62,000 270,000 210,000 150,000

5

-

17 6

3 -

18 2

-

3 -

(% of

Xyl

weight)

-

9.4

-

38 4.5

1

3.5 2.6 3.7 48 13 17

-

-

Em!

M

Referencesb

5 3

& E

Lactose synthetase, A protein, (bovine milk) Hyaluronidase (bovine testicles) Amine oxidase (bovine plasma) Esterase (bovine plasma) Proteinase b (snake venom) Diphosphopyridine nucleotidase (Bacillus subtilis) Lipase (Rhiwpus arrhizus) Lipase (porcine pancreas)

42,000

6

2

3

3

61,000 170,000 82,000 95,000 26,000

9 16

19 16 8

-

40

-

7 12 15 34 13

43,000 50,000

-

14 4

-

2f 3

17

436

3

-

Calculated from the sum of residue weights of sugar components, with the hexosamines considered to be in the N-acetyl form. References: (1)Plummer and Hirs (1964); (2) Reinhold et al. (1968); (3) Salnikow et al. (1970); (4) Yasuda et al. (1970); (5) Yamaguchi et al. (1969); (6) Keller et al. (1971); (7) Neumann and Lampen (1967); (8) Meachum et al. (1971); (9) Pazur et al. (1965); (10) Trayer and Hill (1971); (11) Nagasawa et al. (1971); (12) Borders and Raftery (1968); (13) Watanabe and Yasunobu (1970); (14) Schoenmakers et al. (1965); (15) Oshima et al. (1968); (16) Everse and Kaplan (1968); (17) SBmBriva et al. (1969); (18) Garner and Smith (1972). c Only 2 moles of mannose were detected by Scocca and Lee (1969). The presence of 1 mole of galactose and small amounts of arabinose and xylose was reported by McKelvy and Lee (1969). c Determined as total hexose (galactose plus mannose). f Nature of hexosamine not determined. 5

b

414

ROBERT G. SPIRO

tide mapping after tryptic digestion also appeared to have the carbohydrate attached to the asparagine a t position 34. Fractionation of porcine pancreatic juice by a combination of ion exchange chromatography and gel filtration demonstrated the presence of a ribonuclease of high carbohydrate content and of pronounced heterogeneity (Reinhold et al., 1968), a t least 8 major components being distinguishable 'by acrylamide gel electrophoresis. Analysis of several enzyme fractions obtained by the column chromatography indicated identical amino acid compositions and enzyme-specific activities despite marked variation in the carbohydrate content, which varied from 18 to 32%. With the elucidation of the complete amino acid sequence of porcine ribonuclease, complex carbohydrate units were found to be attached to asparagine residues a t positions 21 and 76 while a simple unit was located on the asparagine a t residue 34 (Table IV) (Jackson and Hirs, 1970). On the assumption that the configuration of the peptide chains as revealed by X-ray diffraction studies (Kartha et al., 1967) is identical in bovine and porcine ribonuclease, the three attachment sites are all on residues that project into the environment and are a t quite some distance from the active site. The carbohydrate unit of bovine ribonuclease attached to its asparagine linkage site was obtained after extensive digestion of this protein with Pronase (Tarentino et al., 1970). Treatment of this glycopeptide with a-mannosidase released 5 of the 6 mannose residues in the carbohydrate unit yielding a fragment containing 1 mannose, 2 N-acetylglucosamine, and 1 asparagine residue. The structure of this asparagine-linked trisaccharide was shown to be B

B

Man --t GlcNAc 8'-f GlcNAc -+ Asn

through the removal of the mannose by the action of P-mannosidase (Sukeno et al., 1971) or a Smith periodate oxidation (Tarentino et al., 1970) and through the isolation of di-N-acetylchitobiose after cleavage of the glycosylamine bond by glycosyl asparaginase. The simple carbohydrate unit linked to asparagine 34 of porcine ribonuclease consists, like the analogous unit in bovine ribonuclease B of 6 mannose and 2 N-acetylglucosamine residues. Treatment of glycopeptides containing this unit with mannosidases indicated that 4 of these mannose residues are a-linked while 2 are attached in &configuration (Kabasawa and Hirs, 1972). The a- and P-linked mannose components could be removed independently by the appropriate mannosidase, suggesting that they are present in distinct side chains, which are attached

415

GLYCOPROTEINS

to an N-acetylglucosamine disaccharide which is in turn linked by a glycosylamine bond to the asparagine residue. A study of the complex carbohydrate units of porcine ribonuclease has indicated that they consist of a core made up of 3 mannose, 3 N-acetylglucosamine, and 1 fucose residue to which a number of side chains containing sialic acid, galactose, and variable amounts of N-acetylglucosamine are linked. These side chains could be removed from the core by the successive action of neuraminidase, p-galactosidase, and P - N acetylglucosaminidase. Periodate oxidation indicated that the N-acetylglucosamine residues in these chains are substituted a t C-6 (Kabasawa and Hirs, 1972). The core heptasaccharide could be degraded by the action of a-mannosidase and P-N-acetylglucosaminidase to an asparaginelinked tetrasaccharide with the partial structure : Fuc

- - 1-3(4)

GlcNAc

p1-3

Man

al-3(4)

GlcNAc -+ Asn

It was suggested that the inner N-acetylglucosamine residue of this sequence serves as a branch point in the intact unit to which both the a-linked mannose and a P-linked N-acetylglucosamine residue are attached. However, the occurrence of such an internal di-N-acetylglucosamine sequence which such a formulation would imply has not yet been directly demonstrated in these carbohydrate units. a-Amylase from Aspergillus oryzae (Taka-amylase) contains a single carbohydrate unit (Table VII) made up of an average of 6 mannose and 2 N-acetylglucosamine residues (Yamaguchi et al., 1969) although in some enzyme preparations additional galactose, xylose, and arabinose components have also been observed (McKelvy and Lee, 1969). Two sequential applications of the Smith periodate oxidation procedure remove all of the mannose leaving 2 N-acetylglucosamine residues attached to asparagine (Yamaguchi et al., 1969) and these 2 components have now been shown to be present in p (1 + 4) linkage (Lee and Scocca, 1972). Release of all but one of the mannose residues has been accomplished by a-mannosidase, after which the remaining residue of this sugar has been cleaved by the action of a &specific enzyme (Li and Lee, 1972; Sugahara et al., 1972). This has indicated that at least in respect to its internal portion, the carbohydrate of the a-amylase appears t o be very similar to that of bovine ribonuclease B. Glycopeptides containing only mannose (5 residues) and N-acetylglucosamine (2 residues) have also been obtained from Pronase digests of plasma amine oxidase (Watanabe and Yasunobu, 1970). The combined action of a-mannosidase and ,&?-N-acetylglucosaminidasewas reported to release all the saccharide components of this unit, which is linked to asparagine.

416

-

Man U l - 2

ROBDRT G. SPIRO

U1-2(6) Pl-4 PI-4 Man-Man-GLcNAc-GLcNAcNAsn

bl-6(21

Fuc

P

fP

XY

FIG. 11. Probable structure of carbohydrate unit of pineapple stem bromelain. For,mulation based on studies of Yasuda et a1. (1970) and Lee and Scocca (1972).

Pineapple stem bromelain contains an asparagine-linked carbohydrate unit which is composed of 1 residue each of xylose and fucose in addition to 2 to 3 residues of mannose and 2 of N-acetylglucosamine (Table VII) . Structural studies on glycopeptides obtained by Pronase digestion of this glycoprotein indicated that the xylose and fucose residues were both located in nonreducing terminal positions (Scocca and Lee, 1969; Yasuda et al., 1970). On the basis of periodate oxidation, glycosidase digestion, and the isolation of oligosaccharides (Man-Man-Man, Man-Man, and Man-GlcNAc) after partial acid hydrolysis, a structural formulation for this unit has been proposed (Fig. 11) (Yasuda et al., 1970). Characterization of the products obtained by Smith periodate degradation of bromelain glycopeptides indicated that di-N-acetylchitobiose makes up the linkage portion of the carbohydrate unit (Lee and Scocca, 1972). The most internal mannose residue, which was resistant to a-mannosidase treatment, was obtained as a mannosyl- (1+ 4) -N-acetylglucosamine disaccharide after partial acid hydrolysis of the peptide free carbohydrate unit from which the other mannose residues had been removed. This compound was cleaved by the action of a mannosidase with P-specificity.

E. Hen Egg Glycoproteins A large number of proteins from the egg white, as well as phosvitin, a phosphoprotein of egg yolk, have been shown to contain substantial amounts of carbohydrate (Table XVIII). The single carbohydrate unit of hen egg ovalbumin (Table I V and VII) has received detailed attention from a number of investigators and was indeed the first saccharide moiety of glycoproteins to be clearly defined (Neuberger, 1938). While this unit consists on the average of 5 mannose and 3 N-acetylglucosamine residues, fractionation on Dowex 50-X2 of asparaginyl oligosaccharides containing this unit obtained after extensive Pronase digestion indicated it to be quite heterogeneous, with the mannose residues varying from 5 to 6 and the N-acetylglucosamines ranging from 2 to 5 residues (Table X I I I ) . From intensive studies of ovalbumin glycopeptides, a 'branched structure for the carbohydrate unit was proposed in which the most internal portion of the unit was made up of 2 N-acetylglucosamine residues, with the innermost N-acetylglu-

TABLE XVIII Carbohydrate Composition of Some Hen Egg Glywproteins Sugar components (moles per mole of protein) Protein Ovalbumin Ovomucoid Avidin Ovotransferrin Ovoglycoprotein Ovoinhibitor (A) Phosvitin a-Ovomucind

Molecular weight

45,000 28,000 16,OOob 80,000 24,000 49,000 40,000 210,000

Gal

Man

5. 7 5 4 12

2 6 1o c 3 21

3 46

GlcN

3 23 4 8 19 14 5 63

GalN

Sialic acids

1

6

2 0.2 2 7

Total carbohydratea (% of weight)

References

3.2 23 10 2.8 31 9.2 6.4 13

Johansen et al. (1960) Chatterjee and Montgomery (1962) Huang and DeLange (1971) Williams (1968) Kletterer (1965) Davis et al. (1969) Shainkin and Perlmann (1971a) Robinson and Monsey (1971)

Calculated from the sum of residue weights of sugar components, with the hexosamines considered to be in the N-acetyl form.

* Weight of subunit; four such subunits are present in each avidin molecule. c

Determined as total hexose (galactose plus mannose). Also contains 15 moles of sulfate ester per mole of protein.

418

ROBERT G. SPIRO

cosamine being involved in the linkage to asparagine and also serving as a branch point (Montgomery et al., 1965a,b; Makino and Yamashina, 1966; Huang et al., 1970). The other N-acetylglucosamine residues were shown to be located on the periphery of the unit and separated from the inner N-acetylglucosamines by mannose residues (Fig. 12). More recently, however, it has been shown that the mannose portion of the unit is attached to the outer residue of the asparagine-linked N-acetylglucosamine disaccharide, as digestion of ovalbumin asparagine-linked oligosaccharides with a Streptomyces griseus endoglycosidase yielded GlcNAc-Asn and the remainder of the carbohydrate unit with N-acetylglucosamine on the reducing end (Tarentino et al., 1972). After removal from the ovalbumin unit of the outer N-acetylglucosamine residues with P-N-acetylglucosaminidase all but one mannose residue was found to be liberated by the action of a-mannosidase (Huang et al., 1970) suggesting that this residue was attached in a p-anomeric configuration. Treatment of the Man-GlcNAc-GlcNAc-Asn fragment obtained after such enzymatic degradation of ovalbumin with P-mannosidase did indeed release this remaining mannose residue (Sukeno et al., 1971; Sugahara et al., 1972; Li and Lee, 1972). This 8-mannosyl linkage to N-acetylglucosamine was shown to be a 1 - 4 bond through a characterization of the Man-GlcNAc disaccharide obtained after treatment of the Man-GlcNAc-GlcNAc-Asn fragment with the S. griseus endoglycosidase (Tarentino et al., 1972) or after the degradation of this fragment by partial acid hydrolysis (Lee and Scocca, 1972). That the two N-acetylglucosamine residues in this fragment are P(1- 4) linked (di-N-acetylchitobiose) was demonstrated by a study of the mannosyl-di-N-acetylchitobiotol formed after alkaline cleavage of the glycopeptide bond in the presence of sodium borohydride (Lee and Scocca, 1972). @

P

a

PI-4

01-3

( G L C N A C )M~a~n - 4 Man)3-GlcNAc-GlcNAc ta

(Man)o-l

P

-Asn

tPl-4

Man

tP

FIQ.12. Probable structure of carbohydrate unit of hen ovalbumin based on studies of Huang et al. (19701, Tarentino et al. (1972), Li and Lee (19721, Sugahara et al. (1972), and Lee and Scocca (1972). Although it is clear that the outer N acetylglucosamine residue of the asparagine-linked di-A'-acetylchitobiose is substituted with a p(1 -+4)-linked mannose residue, i t has not yet been clearly established whether it also serves as a point of attachment for an a-linked mannose. Moreover, it is not known whether the p-linked mannose is in chain A or B.

GLYCOPROTEINS

419

The asparagine-linked carbohydrate units of other egg glycoproteins, including ovomucoid (Montgomery and Wu, 1963) and phosvitin (Shainkin and Perlmann, 1971a) have also been investigated. The amino acid sequence of the subunits of avidin has been elucidated and a simple asparagine-linked carbohydrate unit has been located on position 17 of the chain (Table IV and VII) (DeLange and Huang, 1971).

F . Glycoproteins of Mucous Secretions Many proteins of mucous secretions contain large amounts of carbohydrate and they impart a high viscosity to these protective and lubricating fluids (Table X I X ) . The carbohydrate units of these proteins are characterized by the occurrence in them of sialic acid, fucose, galactose, N-acetylglucosamine, and N-acetylgalactosamine as sugar components, and by their linkage to the peptide chain by means of O-glycosidic bonds involving N-acetylgalactosamine and serine and/or threonine (Table VIII). Mannose does not usually occur as a saccharide component. These glycoproteins are often quite acidic in nature, owing to the presence of numerous sialic acid and/or sulfated sugar residues. The glycoprotein from the ovine submaxillary gland has been shown to contain its carbohydrate primarily in the form of about 800 closely spaced a-N-acetylneuraminyl- (2 + 6) -N-acetylgalactosamine disaccharide units (Graham and Gottschalk, 1960). This disaccharide is linked to serine and threonine residues (Harbon et al., 1964; Carubelli et al., 1965) by an O-glycosidic bond which is in the a-configuration (Buddecke et al., 1969). Alkaline borohydride treatment of the ovine glycoprotein has resulted in the release of the disaccharide by the process of p-elimination and its isolation in high yield (Murty and Horowitz, 1968). Alkaline borohydride treatment of the glycoprotein from bovine submaxillary gland has revealed that although the NAN- (2 + 6) -GalNAc disaccharide is the main serine (threonine) -linked carbohydrate unit, other units, including NAN- (2 + 6) -GalNAc- (1+ 4)-GalNAc, NAN- (2 + 6) GlcNAc- (1 + 6)-GalNAc ; GalNAc-GalNAc; GlcNAc-GalNAc ; FUC(Ga1,GlcNAc)-GalNAc; and NAN- (Ga1,GalNAc) -GalNAc are also present (Bertolini and Pigman, 1970). Detailed information in regard to the carbohydrate units of the porcine submaxillary glycoprotein has been obtained through a study of the oligosaccharides released after alkaline borohydride treatment of this protein (Carlson, 1968) . The most complete oligosaccharide isolated was a branched acidic pentasaccharide (Table VIII) (Fig. 13) with galactosaminitol in the terminal position. Less complete units obtained included the pentasaccharide without the nonreducing terminal N-acetylgalactosamine ; the pentasaccharide minus the N-glycolylneuraminic acid ;

TABLE XIX Carbohydrate Composition of Some Glywproteins from Mucous Secretions Components (moles/l06 g dry weight) Source

Molecular weight

Gal

Fuc

GlcN

GalN

3 9 64 90 148 250 163 93 120 119 153 192 94

5 8

41

52 71 46 76 85 25 68 52 31 100 110

-

112 82 106 75 74 51

Sialic acids

6

0

Total carbohydratea (% of Sulfate weight) References* ~~

Submaxillary gland (ovine) Submaxillary gland (bovine) Submaxillary gland, A+ (porcine) Submaxillary gland (canine) Gastric mucosa, Fr.111: B (canine) Gastric mucosa, Fr.IIb, (porcine) Gastric juice, gastroferrin (human) Colonic mucosa (ovine) Colonic mucosa, F3 (porcine) Gall bladder, A+ (porcine) Cervical mucin, oestrus (bovine) Ovarian cyst fluid, B+ (human) Ovarian cyst fluid, A+ (human)

1 x 106c 1.3 X lPc

830,000 -d -c

-f

260,000 212,000 -e

3

4

x

-d -d

106

62 74 121 144 90 108 86 104 98

1989

58 30 108 64 36 109

105 107 47 29 16 4 13 39 24 2 45 11 7

-

-

40 36 36 16 22 33

-

-

-

55 59 53 65 69 91 84 73 64 71 73 77 75

(11 (11 (21 (3) (4) (51 (6) (71 (8 ) (9) (10) (11, 12) (12, 13)

Calculated from sum of residue weights of sugar components and sulfate esters, with the hexosamines considered to be in the N-acetyl form. b References: (1) Tettamanti and Pigman (1968); (2) Carlson (1968); (3) Lombart and Winder (1972a); (4) Pamer et al. (1968); ( 5 ) Slomiany and Meyer (1972); (6) Multani et al. (1970); (7) Kent et al. (1967); (8)Inoue and Yosizawa (1966); (9) Neiderhiser et al. (1971); (10) Gibbons (1959); (11) Donald et al. (1969); (12) Watkins (1972); (13) Amiioff et al. (1950). After hydroxyapatite gel fractionation, molecular weights of about 4 X 106 were observed. Not reported. 8 Papain digestion used in isolation. f Trypsin digestion used in isolation. 0 Total hexosamines. h Pronase digestion used in isolation.

m

M

n m

+d

421

GLYCOPROTEINS al-3

01-3

GalNAc-Gal-GalNAc-Ser 1.1-2

Fuc

(Thr) ta2-6

NGN

FIG.13. Structure proposed for the carbohydrate unit of the porcine submaxillary glycoprotein with blood group A activity (Carlson, 1968). NGN denotes N glycolylneuraminic acid. Less complete forms of this unit also occur (see text).

the pentasaccharide without either the outer N-acetylgalactosamine or the N-glycolylneuraminic acid; the disaccharide N-glycolylneuraminic acid-(2 + 6) -N-acetylgalactosaminitol; and N-acetylgalactosaminitol itself. The high concentration of borohydride (1.0M) used in the preparation of these oligosaccharides prevented alkaline degradation by a “peeling” reaction, thereby assuring that the variants of the carbohydrate units represented genuine examples of microheterogeneity (Table XIII) . Oligosaccharides containing the N-acetylgalactosamine in a nonreducing terminal position were found only in glycoproteins isolated from porcine glands with blood group A activity, in agreement with the specifying role this sugar plays (Watkins, 1972). Many glycoproteins from mucous secretions contain antigens which in common with those on the erythrocyte cell surface belong to the ABH and Lewis blood group system (Watkins, 1966, 1972; Marcus, 1969). While these antigenic determinants on the red blood cell are believed to reside in the carbohydrate portion of glycosphingolipids, those present in the mucous secretions are located in the carbohydrate units of glycoproteins. Since the latter can readily be obtained in water-soluble form, intensive studies of their carbohydrate were undertaken in several laboratories to provide insights into the immunochemical determinants of the erythrocyte (Kabat, 1956; Watkins, 1966; Morgan, 1968). While the earlier investigations relied primarily on immunochemical means, more recently structural studies have been performed on oligosaccharides obtained by partial acid hydrolysis and alkaline borohydride treatment. Foremost among the blood group active glycoproteins examined have been those isolated from human ovarian cyst fluid (Painter e t al., 1965; Lloyd et al., 1968; Marr e t al., 1967; Morgan, 1968; Iyer and Carlson, 1971). From these investigations i t has become evident th a t blood group activity in the ABH and Lewis system is determined by terminal sugar substituents in the form of galactose, N-acetylgalactosamine, and fucose linked to core chains made up of additional galactose and hexosamine residues (Fig. 14). Although the size and structure of this core portion of the chain may vary, attachment to the peptide through an 0-glycosidic bond involving N-acetylgalactosamine and serine or threonine, seems t o be a uniform phenomenon. In blood group active glyco-

422

ROBERT G . SPIRO

H

Le

p1-3(4) Gal-GlcNACtal-2 Fuc

pl-3 GaL-GCcNAc-

tal-4 Fuc

pl-3

Leb

Got -GlcNActal-2 tal-4 Fuc Fuc

FIQ. 14. Structures of immunological determinants of ABH and Lewis blood group activities. Formulations derived primarily from work on human ovarian cyst glycoproteins (Watkins, 1966).

proteins from stomach, moreover, this linkage has been shown to be a in anomeric configuration (Weissmann and Hinrichsen, 1969). In the ovarian cyst glycoproteins, H, A, and B activities arise from the attachment of fucose, N-acetylgalactosamine, and galactose, respectively, to chains containing either /3( 1+ 3) or P (1+ 4) -linked ga1actose-Nacetylglucosamine, while Lewis activity seems to result from the attachment of one or two fucose residues to chains containing only the /3 (1 -+3) linkage (Fig. 14) (Watkins, 1966, 1972). The isolation of a pentasaccharide containing both &Gal- (1+ 3)-GlcNAc and P-Gal- (1 3 4) GlcNAc linked to a galactose 'branch point by P(1+ 3) and p ( l + 6) linkages, respectively, has suggested that in the ovarian cyst glycoprotein both types of chains may occur in a single carbohydrate unit consisting of 14 to 18 sugar residues (Lloyd et al., 1968). It is certain, however, that much smaller carbohydrate units can have blood group activity such as the penta- and tetrasaccharides terminating in N-acetylgalactosamine found in A+ porcine submaxillary glycoprotein (Carlson, 1968) (Fig. 13). While sialic acid substituents may occur on the carbohydrate units of glycoproteins with ABH and Lewis blood group activity, this sugar does not have an antigenically determining role, and indeed its removal often permits enhanced reactivity (Carlson, 1968; Baig and Aminoff, 1972). Glycoproteins manifesting ABH, Lewis blood group activity usually

GLYCOPROTEIN S

423

have a very high complement of P-hydroxyamino acids (40% in ovarian cyst glycoprotein) (Donald et al., 1969) to which the carbohydrate units are attached in a very closely spaced manner, and the high density of carbohydrate units in these proteins probably contributes to their relative resistance to proteolytic digestion. The genetically determined ABH and Lewis antigenic activity has been shown to be brought about through the inheritance of glycosyltransferases necessary for the attachment of the specific determinant sugar residues onto the core portion of the carbohydrate units (Watkins, 3 966, 1972). These enzymes include an N-acetylgalactosaminyltransferase, a galactosyltransferase, and two fucosyltransferases. The genetics of the ABH, Lewis system appear to affect the transferases of all tissues responsible for the assembly of carbohydrate units with blood group activity, whether these are the glycoproteins of mucins, glycolipids of erythrocyte membrane, or even the oligosaccharides of milk (Ginsburg et al., 1971). Like all glycoproteins, those with blood group activity demonstrate microheterogeneity of their carbohydrate units which are found in various stages of completion. This is reflected in multiple antigenic specificity, as for example the occurrence of A and H activity in the same molecule. A number of sulfated glycoproteins have been isolated from the mucous secretion of the digestive tract (Table X I X ) . These proteins, which are the product of epithelial cells, differ from the sulfated glycoproteins (proteoglycans) of connective tissue by the absence of uronic acid. Since these sulfated compounds are usually isolated after proteolytic digestion of the mucosa or glands in which they are found, they cannot be considered to represent discrete molecules, and only minimal molecular weights are available for many of them (Table XIX) . The carbohydrate units of these sulfated glycoproteins from dog (Pamer et al., 1968) and hog (Slomiany and Meyer, 1972) gastric mucosa show considerable heterogeneity as reflected in their saccharide composition and sulfate content. A study of the structure of several sulfated glycoprotein fractions isolated after trypsin digestion of hog gastric mucosa has given some insights into the structure of their carbohydrate units (Slomiany and Meyer, 1972). On the basis of sequential Smith periodate degradations, these units, which contain both A and H blood group activity, were visualized as branched structures consisting of 14 to 18 glycosyl residues attached to the peptide chain by an O-glycosidic linkage between N-acetylgalactosamine and serine or threonine. Two chains, one terminating in N-acetylgalactosamine (responsible for the A activity) and the other ending in fucosylgalactose (responsible for H activity), both linked to a galactose branch point, are believed to

424

ROBERT G . SPIRO

be present in each unit, although molecular weight measurements to verify this were not made. The core of the unit is made up of galactose and N-acetylglucosamine attached to the N-acetylgalactosamine residue which participates in the glycopeptide bond. N-Acetylglucosamine 6-sulfate was obtained as the only sulfated sugar after mild acid hydrolysis, and on the basis of the Smith degradations was assigned a single position remote from the immunodeterminant groups. Examination of other fractions indicated that more than one N-acetylglucosamine residue may be present in the sulfated form. That the occurrence of sulfate groups in proximity to the antigenic determinant sugar residues may interfere with immunological activity was suggested by the finding of decreasing activity of glycoprotein fractions with increasing sulfate content. Recently a sulfated glycoprotein has been obtained by water extraction of canine submaxillary glands (Lombart and Winzler, 1972a) (Table XIX) . A study of oligosaccharides isolated after alkalineborohydride treatment of this protein indicated that two distinct types of acidic carbohydrate units exist. One type of unit contains sialic acid, fucose, galactose, and N-acetylgalactosamine, while the other type is devoid of sialic acid but contains sulfated glucosamine in addition t o the fucose, galactose, and N-acetylgalactosamine (Lombart and Winzler, 1972b). Both kinds of units exist in varying stages of completion and both appear to be linked to the peptide through N-acetylgalactosamine attached to serine or threonine residues. These findings help to explain the observations of Dische (Dische et al., 1962; Dische, 1963), who found that the ratio of sialic acid to fucose in the submaxillary secretions of the dog varies greatly, although their sum remains constant, after chemical or electrical stimulation of the gland. This phenomenon could result from a change in the relative proportion of the two types of carbohydrate, as the unit containing sialic acid was found to contain less fucose than the one containing the glucosamine-sulfate, and could be mediated by a regulatory effect on key glycosyltransferases, such as the N-acetylglucosaminyl- or sialyltransferase. The change in the proportion of the carbohydrate groups would not, however, alter the net charge on the glycoprotein, as both the sulfate and carboxyl groups are dissociated at physiological pH.

G. Collagens and Basement Membranes The collagens represent one of the largest families of proteins found in the animal kingdom, being present in multicellular organisms ranging from sponges to the higher vertebrates. Included in this group are the various fibrillar collagens, as well as the amorphous extracellular base-

TABLE XX Carbohydrate Composition of Some Basement Membranes and Collagens Sugar components (moles/lOs g dry weight)

Protein Membranes Glomerular basement membrane (bovine) Glomerular basement membrane (human) Anterior lens capsule (calf) Posterior lens capsule (calf) Corneal Descemet’s membrane (ovine) Collagens Carp swim bladder, citrate-soluble R a t skin a, chain 02 chain Achilles tendon, buffer-insoluble Lumbricus cuticle, citrate-soluble Nereis cuticle, citrate-soluble Loligo cephalic cartilage, citrate-soluble Loligo fins, citrate-insoluble Lumbrim body wall, citrate-insoluble Meiridium body wall, pepsin-solubilized

Hexuronic acids

Totala carbo- Referhydrate encesb

Man

Fuc

GlcN

GalN

Sialic acids

17

4.3

1.3

8.4

1.2

3.7

-

8.9

13

14

3.4

1.0

5.9

0.8

2.1

-

7.0

23 18 19

26 21 21

4.1 3.9 11

1.5 1.5 3.7

8.2 12 7

0.7 0.9 1.8

1.0 2.0 2.3

1.7 6.7

11.3 11.5 11.2

-

-

0.6

-

0.6 1.7 2.1 0.5 -

0.3 0.6 1.4 9.3 2.3 3.0 2.9 2.5 7.5

Glc

Gal

14

1.2

1.5

0.3

-

0.7 1.1 1.3 0.3 0.2 6.8

1.0 2.2 2.5 49 8.6 7.5 8.2 7.1 18

0.9 1.7 1.9 0.8 0.3 1.2 1.9

-

-

0.2 2.1 0.3 0.3 0.8 1.8

2.5c 0.9 1.1 1.0 1.@ 0.8~ 0.3 2.2 0.6

8.0

5.2 21

0.5c

-

0.2

-

-

-

-

-

Xyl

Calculated from sum of residue weights of sugar components, with the hexosamines considered to be in the N-acetyl form. References: (1)Spiro (1967a); (2) Beisswenger and Spiro (1970); (3) Fukushi and Spiro (1969); (4) Kefalides and Denduchk (1969); (5) Spiro (1969b); (6) Spiro and Martin (1972); (7) Spiro (1972a); (8) Katzman and Oronsky (1971). Total hexosamines. 0

426

ROBERT G. SPIRO

ment membranes (Traub and Piee, 1971; Spiro, 1972a). The occurrence of covalently linked carbohydrate has now been demonstrated in a large number of these proteins (Table XX) and several distinct types of carbohydrate units and glycopeptide bonds have been elucidated (Table

XXI) .

The most characteristic carbohydrate units of the collagens and basement membranes are those which are linked to the peptide chain by a glycosidic bond involving the hydroxyl group of hydroxylysine (Table IX). Recognition of the involvement of this amino acid in a carbohydrate-peptide bond came through studies carried out on guinea pig skin tropocollagen (Butler and Cunningham, 1966) and bovine renal glomerular basement membrane (Spiro, 196713). After collagenase and Pronase digestion of the glomerular basement membrane, a number of short glycopeptides were isolated, each of which contained hydroxylysine, glucose, and galactose in a molar ratio of 1:l:l. Structural studies carried out on these glycopeptides indicated that they contained a ~-O-(Y-Dglucopyranosyl-D-galactose disaccharide linked by a /3-glycosidic bond to the hydroxyl group of hydroxylysine (Fig. 15) (Spiro, 1 9 6 7 ~ ) . The position of the linkage between the glucose and galactose was established by methylation and periodate oxidation, and its anomeric configuration was determined through the use of specific glucosidases. Isolation of the glucosylgalactose disaccharide could be achieved after partial acid hydrolysis of N-acetylated glycopeptides. Prior N-acetylation was required, as in the presence of an unsubstituted r-amino group on the hydroxylysine, the differential in the stability of the glucosylgalactose TABLE XXI Carbohydrate Units Found in Collagens and Basement Membranes Amino acid involved in glycopeptide Carbohydrate units bond Glc-Gal; Gal (Gal)r; (Ga1)z; Gal GlcUA-Man Heteropolysaccharide

Hyl Thr,Ser Thr Asn

Proteins

Referencesa

Vertebrate and invertebrate fibrillar collagens; basement membranes Annelid cuticle collagens Nereis cuticle collagen Basement membranesb

(1, 2) (3-5) (5 1

(6)

= References: (1) Butler and Cunningham (1966); (2) Spiro (1967c, 1969a, 1972a); (3) Muir and Lee (1969); (4) Spiro (1970b); ( 5 ) Spiro and Bhoyroo (1971); (6) Spiro (196713). b Mannose and glucosamine, in addition to galactose and glucose, have been found in rat skin procollagen suggesting the presence of Asn-linked unit in this protein (Spiro and Martin, 1972).

427

GLYCOPROTEIN S

and galactosylhydroxylysine bonds was so great as to preclude the release of the two sugars linked to each other (Fig. 5 ) . Because of the linkages involved, a single Smith periodate degradation can remove both glucose and galactose from the peptide chain. The galactosylhydroxylysine bond, while resistant to cu-galactosidase, was cleaved with a galactosidase of ,&specificity, but only if the r-amino group on the hydroxylysine was blocked by N-acetylation (Spiro, 1 9 6 7 ~ ) . The occurrence of a glycosidic linkage adjacent to a free amino group, as in the attachment of this unit to the peptide chain (Fig. 15), has so far not been detected in other compounds. Amino groups of hexosamines are always substituted by acetyl or sulfate, the amino group of sphingosine is acylated, and the a-amino groups of glycosylated serine and threonine residues are usually in peptide linkage. Hydroxylysine-linked disaccharide units of the same structure have been found in a number of other basement membranes (Spiro and Fukushi, 1969) and collagens from both vertebrate and invertebrate sources (Spiro, 1969b, 1972a; Spiro and Bhoyroo, 1971; Katzman et al., 1972). While these disaccharide units account for all the glucose present in collagens and basement membranes and along with hydroxylysine-linked galactose residues (Table IX) make up most if not all of the total carbohydrate present in vertebrate tropocollagens, a substantial number of sugar components not associated with hydroxylysine are present in other proteins of the collagen family (Table XX) , particularly basement membranes (Spiro, 1967a; Fukushi and Spiro, 1969) and insoluble collagens (Cunningham and Ford, 1968; Spiro, 1969b). The nature of these other units has been most clearly determined in the 'bovine glomerular basement membrane, which after collagenase and Pronase digestion yielded glycopeptides containing heteropolysaccharide units with an NH2

H

H

/

I y 2

H ~ cNH I COOH

OH

FIQ.15. Structure and peptide attachment of the disaccharide unit of basement membranes and collagens. From Spiro (1967~).

428

ROBERT G. SPIRO

average molecular weight of 3500 made up of sialic acid, fucose, galactose, mannose, and glucosainine residues and apparently linked to the peptide chain by glycosylamine bonds to asparagine (Spiro, 1967b). Fractionation of the reduced and alkylated peptide subunits of the glomerular basement membrane by gel filtration and ion exchange chromatography (Hudson and Spiro, 1972) and more recently by preparative polyacrylamide gel electrophoresis (Sato and Spiro, 1972) has indicated that hydroxylysine- and asparagine-linked carbohydrate units occur on the same peptide chain, Indeed it appears that all the hydroxylysinelinked carbohydrate of the glomerular basement membrane is present on chains t o which heteropolysaccharide units are also attached, although some chains containing only the latter type of unit may also occur (Sato and Spiro, 1972). While it is quite likely that heteropolysaccharide units similar to those found in glomerular basement membrane also occur in vertebrate insoluble fibrillar collagens, difficulties in solubilization and purification of these proteins have so far interfered with the resolution of this question. Isolation of a hydroxyproline-containing glycopeptide from a collagenase digest of pepsin-solubilised collagen from the sea anemone (Metridium dianthus) with carbohydrate units consisting of fucose, mannose, and N acetylglucosamine linked to asparagine may be considered further evidence for the existence of this type of glycopeptide linkage in collagens (Katzman and Oronsky, 1971). Recently, analysis of rat skin procollagen has shown the presence of mannose and glucosamine, in addition t o glucose and galactose (Spiro and Martin, 1972), suggesting the occurrence of a t least one asparaginelinked heteropolysaccharide unit per peptide chain in addition to the hydroxylysine-linked carbohydrate units. If such units are indeed present in procollagen despite their absence in the tropocollagen from this source (Table XX) their location must be on the peptide segment which is cleaved off by proteolytic action during the conversion of the precursor collagen (Lapikre e t al., 1971 ; Bornstein et al., 1972). The large number of heteropolysaccharides evident in basement membranes could be due to absence of such a protease in cells responsible for their synthesis. Carbohydrate units linked to the peptide chain of collagen by yet a third type of glycopeptide bond, namely the one involving a glycosidic linkage to the hydroxyl group of serine or threonine have been found in the annelid cuticles. These collagens arc essentially devoid of hydroxylysine (Table 11) and therefore do not have this amino acid to serve as a point for carbohydrate attachment (Spiro, 1972a). Lumbricus (earthworm) cuticle collagen contains most of its carbohydrate in the form of galactose residues (Table XX) which can be

429

GLYCOPROTEINS

released by alkaline borohydride treatment as reduced tri-, di-, and monosaccharides (Muir and Lee, 1969; Spiro, 1970b; Spiro and Bhoyroo, 1971) or as glycopeptides after collagenase digestion (Muir and Lee, 1970; R. G. Spiro, 1970b). Structural investigations of the reduced galactose oligosaccharides obtained after alkaline borohydride treatment or of the reducing oligosaccharides isolated after hydroxylamine treatment have indicated that the disaccharide is 2-O-a-~-galactopyranosyl-~galactose and the trisaccharide is 2-O-a-~-galactopyranosyl-2-O-a-~galactopyranosyl- galactose (Muir and Lee, 1969). On the basis of the destruction of threonine and serine, approximately 18 threonine and 5 serine residues per 1000 amino acid residues were found to be involved in the linkage of these galactose units (Spiro, 1970b). Analyses of the carbohydrate released after alkaline borohydride treatment indicated that 19 disaccharides, 5 trisaccharides, and 1 monosaccharide unit were present per lo00 amino acid residues (Spiro and Bhoyroo, 1971). The cuticle collagen of Nerels (clamworm) contains in addition to these serine (threonine) -linked galactose mono-, di-, and trisaccharides, another unusual type of unit which consists of a 6-O-a-D-glucuronosyl-~mannose linked to threonine (Spiro and Bhoyroo, 1971) (Fig. 16). This acidic disaccharide, which represents the only case so far described in which a uronic acid has been found in units other than the large polysaccharides of proteoglycans, was obtained in high yield after partial acid hydrolysis and as glucuronosylmannitol after alkaline borohydride treatment of the clamworm collagen. Small glycopeptides containing the disaccharide were isolated after collagenase and Pronase digestion of the protein and fractionated by Dowex 1 chromatography. The limited number of amino acids which these glycopeptides contained included a hydroxyproline residue as well as the threonine involved in the glycopeptide bond (Spiro and Bhoyroo, 1971). Methylation and periodate oxidation studies were used to determine the position of linkage of the glucuronosyl bond, while treatment of the disaccharide with glucuronidases of a- and 8- specificity established the anomeric configuration of this linkage.

H

OH

H

H

kOOH

FIG. 16. Structure and peptide attachment of the acidic disaccharide unit of Nereis (clamworm) cuticle collagen. Formulation based on studies of Spiro and Bhoyroo (1971).

430

ROBERT G . SPIRO

H . Proteoglycans The intercellular matrix contains a group of glycoproteins which by their high carbohydrate content and the large size of their saccharide units appeared to differ so significantly from other glycoproteins that they were for a long time considered as distinct entities and classified under the term of mucopolysaccharides. The pioneering investigations of K. Meyer and his colleagues (Meyer, 1970) indicated that the carbohydrate portion of these polymers is characterized by a basic structural pattern which consists of a repeating disaccharide sequence of a sulfated hexosamine alternating with a uronic acid or sulfated galactose residue (reviewed by Brimacombe and Webber, 1964) (Table X X II) . While the existence of a covalently linked peptide moiety was not originally recognized, occurrence of a carbohydrate-protein bond involving serine became established through the observations of Muir (1958) and Anderson et al. (1964), and these molecules were then more properly referred to as proteoglycans to specify their glycoprotein nature. The chondroitin sulfates, dermatan sulfate, and heparin are exclusively linked to serine residues in the peptide chain by means of a glycosidic bond involving xylose preceded by 2 galactose residues (~-O-@-Dgalactopyranosyl-4-0-~-~-galactopyranosyl-~-xylose)(Rodkn, 1968). The major portion of these polymers is believed to be a linear arrangement of alternating hexosamine and hexuronic acid residues. In the chondroitin sulfates, the hexosaminidic linkage in this array is /3 (1 + 4) while the uronidic bond is p(1+ 3) ; in dermatan sulfate these bonds are 8(1+ 4) and a ( l 3 3 ) , respectively; while in heparin, both hexosaminidic and uronidic linkages are of the a ( l 3 4) type. The repeating disaccharide units end in a glucuronic acid residue which is attached to the terminal galactose of the linkage region by a @(1+3) glycosidic bond (RodBn, 1968). The observation that glucuronic acid is involved in the linkage to the galactose even in dermatan sulfate where the major uronic acid is iduronic acid (Fransson, 1968) indicates that this most internal uronic acid occupies a special position in the molecule. Indeed, in the enzymatic assembly of chondroitin sulfate, transfer of glucuronic acid to the trisaccharide linkage region proceeds through the action of a glucuronosyltransferase distinct from that involved in the formation of the remainder of the polysaccharide chain (Helting and Rod&, 1969). O-Sulfate groups are present on C-4 or C-6 of the hexosamine residues of the proteoglycans (Table XXII). The position of the sulfate group on the N-acetylgalactosamine distinguishes two forms of chondroitin sulfate chains which are referred to as chondroitin 4-sulfate and chondroitin 6-sulfate, respectively. Heparin, in addition, has approximately

TABLE XXII Carbohydrate Units of Protwglycans~ Glycopeptide bond Polymer Chondroitin sulfates Dermatan sulfate Heparin Cartilage keratan sulfate Corneal keratan sulfate 0

Amino acid Ser Ser Ser Ser,Thr Asn

Sugar

Position of 0-sulfate groups

GalN; GlcUA GalN-PS; GalN-BS GalN; IdUA or GlcUA GalN-4-S GlcN ; GlcUA or IdUA GlcN-64, IdUA-243, GlcUA-’AS GlcN-63, Gal-6-S GalNAc GlcN; Gal GlcN-6-53, Gal-6-8 GlcNAc GlcN; Gal Xyl Xyl Xyl

See text for references.

* The iduronic acid is in the cconfiguration. c

Sugars in repeating disaccharide unitb

Other hexosamines are in the N-acetyl form.

Occurrence of N-sulfate groupC

-

+ -

-

Other sugar components Gal G a1 Gal NAN,Fuc,Man N AN,Fuc,Man

432

ROBERT G . SPIRO

half of its uronic acid residues substituted with a sulfate ester on C-2, and according to recent investigations, i t is the iduronic acid component which is primarily involved in this sulfation (Lindahl and Axelsson, 1971). The occurrence of N-sulfate groups on hexosamine residues further distinguishes heparin from the other proteoglycans, in which these amino groups are in the N-acetyl form. However, analyses of fragments of the polysaccharide chain of heparin have indicated that the glucosamine residues close to the carbohydrate-peptide linkage region are N acetylated (Lindahl, 1970). The keratan sulfates are distinguished from the carbohydrate units of other proteoglycans by the absence of uronic acids and the presence of sialic acid, fucose, and mannose residues (Table XXII). The predominant structural feature of the keratan sulfates is a core portion consisting of repeating galactose and N-acetylglucosamine residues in which the galactose is linked to the glucosamine by a 8- (1 + 4) bond and the glucosamine is attached to the galactose by a p- (1+ 3) linkage (Bhavanandan and Meyer, 1967, 1968). Sulfate groups are located on C-6 of many of the N-acetylglucosamine and galactose residues, and it is likely that short-chain substituents containing sialic acid, fucose, and additional galactose residues are attached to the repeating core portion of the polymer. While the carbohydrate-peptide linkage in cartilage keratan sulfate has been found to involve N-acetylgalactosamine attached to serine and threonine residues (Bray e t al., 1967), the corneal keratan sulfate carbohydrate units are attached to the peptide chain by glycosylamine bonds between N-acetylglucosamine and asparagine (Baker e t al., 1969; Stuhlsatz e t al., 1971). In the latter polymer mannose residues are believed to be located close to the linkage region. All of the proteoglycan carbohydrate units manifest heterogeneity in regard to chain length, degree of sulfation, and probably also, in the case of the keratan sulfates, in the monosaccharide substituents on the core chain. While both glucuronic acid and iduronic acid moieties occur in the repeating segments of heparin and dermatan sulfate, i t is not clear as yet whether these two sugars are randomly placed in the chain or whether they occupy specified positions therein. While a large amount of information is now available in regard to the nature and structure of the carbohydrate units associated with the connective tissue proteoglycans, less is known about the protein components with which they are associated in the native state. T o fill this void a number of investigators have concentrated on a study of these proteincarbohydrate complexes, as indicated in the volumes edited by Quintarelli (1968) and Balazs (1970). The most thoroughly studied of these complexes has been the pro-

GLYCOPROTEINS

433

teoglycan from bovine nasal cartilage, which is believed to contain both chondroitin sulfate and keratan sulfate carbohydrate units and which can be extracted with water (Malawista and Schubert, 1958), dilute buffer (Hoffman et al., 1967) or 4 M guanidinium chloride (Hascall and Sajdera, 1969). The latter method of extraction has been shown to disaggregate the protein-polysaccharide complex and to permit the separation by density gradient centrifugation of the proteoglycan subunits from a glycoprotein fraction. The latter, while only making up 3% of the weight of the complex, is necessary to achieve aggregation of the subunits. The proteoglycan subunit isolated in this manner consists by weight of 87% chondroitin sulfate, 6% keratan sulfate, and 7% protein (Hascall and Sajdera, 1970). Since the molecular weight of the proteoglycan subunit was determined to be 2.5 X lo6, the size of the peptide portion was calculated to be 180,000, to which about 90 chondroitin sulfate chains of molecular weight 25,000 are attached. Removal of the chondroitin sulfate chains has been achieved by digestion of the cartilage proteoglycan with bacterial chondroitinase with the formation of a core having an average molecular weight of 450,000 and containing the protein moiety to which the linkage regions of the chondroitin sulfate chains are still attached (Hascall and Riolo, 1972). Proteolysis of this core with papain resulted in the isolation of polydisperse glycopeptides containing the keratan sulfate which had an average molecular weight of 8500 and were made up of 13 sulfated repeating galactose-N-acetylglucosamine disaccharides, 1 mannose, 2 N-acetylgalactosamine, and 2 to 3 sialic acids, as well as a limited number of amino acid residues. Since alkaline treatment of these glycopeptides caused a significant decrease in their molecular weight, it was suggested that the keratan sulfate units occur in doublets separated by only a short papain resistant peptide chain and that this doublet structure was split by /3elimination of the glycopeptide bonds. Such an interpretation, which, however, could pertain only if no significant peeling degradation of the carbohydrate had taken place during the alkaline treatment, would leave carbohydrate units containing only a single N-acetylgalaetosamine residue, and would indicate the presence of about 60 keratan sulfate chains attached to the proteoglycan peptide core. A doublet structure has also been proposed for the chondroitin sulfate chains of cartilage proteoglycans on the basis of the observation that glycopeptides containing these carbohydrate units obtained after trypsin digestion could be halved by subsequent papain digestion or mild alkaline treatment (Luscombe and Phelps, 1967b; Mathews, 1971). On the basis of such studies the distribution of chondroitin sulfate units along the peptide chain was visualized as occurring in pairs consisting of units joined

434

ROBERT G. SPIRO

by fewer than ten amino acid residues and separated from each other by about 35 amino acid residues (Mathews, 1971). While molecular weight determinations for bovine nasal cartilage chondroitin sulfate units based on a study of glycopeptides obtained after papain digestion give values of about 25,000 (Partridge et al., 1961; Buddecke et al., 1963; Luscombe and Phelps, 1967b; Mathews, 1971), measurements of chondroitin sulfate chains released from porcine costal cartilage by alkaline-sulfite treatment have indicated an average molecular weight of only 14,000 (Hranisavljevic et al., 1972).

I . Glycoproteins of Plasma Membranes

It has become evident in recent years that glycoproteins are important constituents of the plasma membrane of animal cells, and along with glycolipids account for their carbohydrate content. The immense biological importance of these membranes which make up the discriminating barrier between the intracellular and extracellular environment has focused attention on the necessity of defining their subunit composition. While the presence of glycoprotein components has been demonstrated in plasma membranes from a number of cells (Glossmann and Neville, 1971), glycoproteins or glycopeptides suitable for structural investigations have been obtained only from a few (Table X X III). I n particular, the glycoproteins of the erythrocyte membrane have received detailed attention because of the ready availability of the red cell stroma. Solubilization of glycoproteins from this membrane has been achieved with the use of aqueous phenol (Kathan et al., 1961), aqueous pyridine (Blumenfeld, 1968), sodium dodecyl sulfate (Bakerman and Wasemiller, 1967; Rosenberg and Guidotti, 1%8), and lithium diiodosalicylate (Marchesi et al., 1972). Glycopeptides have been obtained by trypsin digestion of either the intact erythrocyte or its stroma (Winder et al., 1967; Jackson and Seaman, 1972). The release of glycopeptides containing over one-third of the protein-bound sialic acid of the erythrocyte by such proteolytic digestion of the intact cell indicates that glycoproteins are located on the exterior surface of the cell membrane. Indeed, incubation of intact erythrocytes with neuraminidase results in the liberation of all of its sialic acid, demonstrating that a t least this component of the glycoproteins is entirely located a t the outer surface of the plasma membrane (Eylar et ul.,1962). Approximately 80% of the sialic acid of the human erythrocyte membrane (Winzler et al., 1967) is to be found in a glycoprotein (Table XX I I I ) which in its disaggregated form has a molecular weight of about 31,000 and contains the M and N blood group antigens as well as the myxovirus receptor sites (Kathan et al., 1961 ; Kathan and Winder, 1963 ;

GLYCOPROTEINS

435

Morawiecki, 1964; Springer et al., 1966; Kathan and Adamany, 1967). The sialic acid-rich glycopeptides (Table XXIII) released by trypsin treatment of the intact erythrocyte are believed t o be derived in part or possibly entirely from this major glycoprotein. The sugar components of the erythrocyte membrane glycoprotein are distributed between serine (threonine) -linked units and asparagine-linked carbohydrate units (Winzler, 1969) (Table X) . The 0-glycosidically linked units were obtained as reduced oligosaccharides after alkaline borohydride treatment of the erythrocyte glycoprotein (Adamany and Kathan, 1969) or after such treatment of M and N active glycopeptides from trypsin digests of the erythrocyte membrane (Thomas and Winzler, 1969). The most complete oligosaceharide released by this treatment was a tetrasaccharide with the structure N-acetylneuraminyl- (2 -+ 3) -P-Dgslactopyranosyl- ( 1 + 3) - [ N-acetylneuraminyl- (2 + 6) ] -N-acetyl-Dgalactosaminitol. Approximately 25-30% of the carbohydrate present in the erythrocyte membrane glycopeptides was found in these alkalilabile carbohydrate units (Thomas and Winzler, 1969). Upon Pronase digestion of the peptides resistant to alkaline borohydride treatment, a number of glycopeptides were obtained (Thomas and Winzler, 1971). One of these which contained 1 fucose, 1 sialic acid, 3 galactose, 3 mannose, 4 N-acetylglucosamine, and 1 aspartic acid residue, in addition to small amounts of threonine, serine, and glutamic acid, was degraded with glycosidases and shown to consist of a branched structure in which two chains of sialic acid (fucose) -galactose-N-acetylglucosamine and one of galactose-N-acetylglucosamine are linked to a core consisting of the 3 mannose and the additional N-acetylglucosamine residues. From physicochemical studies of the erythrocyte membrane glycoprotein in detergents an asymmetric distribution of hydrophilic residues has been deduced and a model proposed in which the protein is anchored to the lipoidal plasma membrane by a region rich in hydrophobic amino acids, while the hydrophilic part of the molecule containing most of the carbohydrate extends outside of the lipid barrier and is in contact with the external aqueous environment (Morawiecki, 1964). The hydrophilic portion is believed to be available to the action of trypsin, influenza virus, and M and N antibodies and to contribute through its sialic acid residues to the high negative charge present on the erythrocyte surface (Winzler, 1969). Terminal analysis of the membrane glycoprotein, isolated by lithium diiodosalicylate extraction, has suggested that it is a single polypeptide chain, and a study of fragments obtained after cyanogen bromide cleavage has shown that the nonpolar amino acids are located predominantly in the C-terminal region while most of the carbohydrate is attached to the N-terminal half of the chain (Marchesi et al., 1972).

TABLEXXIII Carbohydrate Composition of Plasma Membranes and Glycoproteins or Glycopeptides Derwed Therefrom

Ld 0

Sugar components (g/100 ga)

Source Membranesf Erythrocyte (human) Erythrocyte (rat) Liver (rat) Kidney, brush border (rat) Kidney, brush border (rabbit) Ascites hepatoma (rat) Platelet (human) Adipose tissue (rabbit) Glycoproteins, glycopeptides: Erythrocyte glycoprotein (human)h Erythrocyte (human)<

Hexoseb Glc

Gal

-

-

2.7 6.4 4.5 3.7

-

-

-

-

-

0.2 0.2 3.0 0.7

-

2.5 0.8 5.5 1.4 13 20

Man

m

Totald Sialic carbo- ReferFuc GlcNAdGalNAa acids hydrate encese

-

-

-

1.6 1.0 5.1 1.7

0.9 0.3 0.8 0.4

3.50 3.30 1.00 3.10 5.0 1.2 4.6 3.1

2.6 3.6

1.2 1.8

6.5 6.3

1.3 0.4 1.0 0.2

13 11

2.6 2.0 0.8 1.1 2.3 0.6 1.7 0.9

28 37

8.8 11.7 6.3 7.9 13.8 4.5 21.7 8.4

(3) (4) (5) (6)

64.3 41.7

(7) (7)

(1) (2) (2) (2)

E R

L U

Erythrocyte (rabbit) Reticulocyte (rabbit)i Platelet, GPI (human) Spleen, H-2b glycopeptideL Mammary adenocarcinoma, TA3, ascites cell (mouse)i

5.4 -

1.6 3.3 -

11 5.7 4.9 27

6.3 2.7 5.3 0.5

1.8

0.8 1.3

-

14 5.6 14

-

5.59 9

19

2.3

0.7

2.4 1.2 13

37.0 18.8 27.9 12.1 68.5

(8) (8) (9) (10) (11)

Membranes expressed as g/100 g protein; glycoproteins and glycopeptides expressed as g/100 g dry weight. Determined as total hexose. Determined as free based but expressed as N-acetyl derivative. Sum of sugar components. References: (1) Rosenberg and Guidotti (1968); (2) Glossmann and Neville (1971); (3) Quirk and Robinson (1972); (4) Shimizu and Funakoshi (1970); (5) Barber and Jamieson (1970); (6) Kawai and Spiro (1972); (7) Winzler (1969); (8) Harris and Johnson (1969); (9) Pepper and Jamieson (1969); (10) Shimada and Nathenson (1969); (11) Codington ei al. (1972a). f All membranes except platelet membrane were delipidated prior to analysis. Total hexosamines. h Obtained by phenol-water extraction. Isolated after trypsin digestion of cells. j Obtained after trypsin-Pronase digestion. Obtained after papain digestion. 5

b

0

E

438

ROBERT G. SPIRO

The glycopeptides released by trypsin treatment of the intact erythrocyte were furthermore shown to come from the N-terminal half of this protein. While M and N blood group activity has clearly been attributed to the major erythrocyte membrane glycoprotein (Kathan e t al., 1961 ; Kathan and Adamany, 1967), the antigenic determinants have not as yet been established. No important differences have been demonstrated in the composition of the MM, NN, and MN active glycoproteins (Kathan and Adamany, 1967) or in the carbohydrate units derived therefrom, either alkali-labile (Thomas and Winzler, 1969), or asparagine-linked (Thomas and Winzler, 1971). Although the finding that removal of sialic acid from erythrocytes by neuraminidase destroys M and N blood group activity (Springer and Ansell, 1958), might implicate this sugar as a determinant group in the antigenic site, it is more likely that its absence causes conformational changes which interfere with antigen-antibody reaction. The lack of M and N activity which the oligosaccharides and the low activity which the glycopeptides from the erythrocyte membrane glycoprotein demonstrate suggests that the peptide portion of the glycoprotein a t least plays a major contributing role in the antigen-antibody reaction. Indeed blockage of the amino groups of the erythrocyte glycoprotein results in a loss of M and N blood group activity (Lisowska and Morawiecki, 1967). The myxovirus hemagglutination inhibition activity of the erythrocyte glycoprotein, like the M and N blood group activity, is destroyed by neuraminidase treatment (Springer and Ansell, 1958) , and this hemagglutination inhibition activity is also lost upon proteolytic digestion (Springer et al., 1966). The erythrocyte glycoprotein in its disaggregated form of molecular weight 31,000 appears to have considerably less of both hemagglutination inhibition and MN blood group activities than the high molecular weight aggregates in which it probably exists in the native membrane (Springer, 1967). Information in regard to the structures of the erythrocyte membrane phytohemagglutinin receptor sites have been obtained from studies of glycopeptides released by trypsin treatment of red cells (Kornfeld and Kornfeld, 1970; S. Kornfeld et al., 1971). After removal of the serine (threonine) -linked carbohydrate units from such glycopeptides by alkaline borohydride treatment and further digestion with Pronase, a number of fractions with Phaseolus vulgaris hemagglutinin activity were obtained. These fractions yielded a purified glycopeptide containing 1 sialic acid, 2 galactose, 2 mannose, and 3 N-acetylglucosamine residues as sugar components. On the basis of sequential cleavage with glycosidases , a branched structure for the carbohydrate unit of this glycopeptide was proposed in which a chain with the sequence sialic acid-galactost+-N-

GLYCOPROTEINS

439

acetylglucosamine and another chain with the sequence ga1actose-Nacetylglucosamine are linked to an internal portion consisting of 2 mannose and 1 N-acetylglucosamine residues (Kornfeld and Kornfeld, 1970). On the basis of the alkali stability and amino acid composition of the glycopeptide, this carbohydrate unit is believed to be attached to the peptide chain by a glycosylamine bond to asparagine. While removal of the sialic acid from the glycopeptide did not affect its phytohemagglutinin inhibition, release of the galactose caused a 90% loss in this activity. Since glycopeptides with a similar structure from other glycoproteins had inhibition activity, while oligosaccharides containing the galactose-N-acetylglucosamine sequence had negligible activity, it would appear that some particular feature of these carbohydrate units, such as the branched structure, the mannose-N-acetylglucosamine core, or the amino acids around the asparagine linkage site are also necessary determinants. That the activity of the carbohydrate unit is significantIy affected by the size of the peptide chain to which it is attached was shown by a marked loss in inhibition which occurred when glycoproteins obtained by direct chloroform-methanol extraction of erythrocyte stroma were treated with trypsin. The receptor site for the Lens culinaris phytohemagglutinin also seems to reside primarily in an asparagine-linked branched carbohydrate unit on the erythrocyte membrane. The major determinant sugar for this reaction, however, appears to be the N-acetylglucosamine of the oligosaccharide chains (S. Kornfeld et al., 1971). It is not unlikely that the phytohemagglutinin receptor sites reside on the same erythrocyte membrane glycoprotein which also contains the M and N antigens and myxovirus receptor sites, as this protein appears to account for most of the carbohydrate of the membrane. While the proposed structure of the asparagine-linked carbohydrate units from the M and N glycoprotein (Thomas and Winzler, 1971) differs somewhat from that of the units on glycopeptides with phytohemagglutinin activity (Kornfeld and Kornfeld, 1970) in 'both instances only a fraction of the carbohydrate-containing material was isolated and characterized. Differences observed could well be attributed to heterogeneity of these units. Similar asparagine-linked carbohydrate units consisting of sialic acid, fucose, galactose, mannose, and N-acetylglucosamine may exist in mouse spleen H-2 alloantigens (Table XXIII) (Muramatsu and Nathenson, 1970) and in the human platelet membrane (Pepper and Jamieson, 1969). Trypsin treatment of TA3 mammary adenocarcinoma ascites cells has resulted in the release of two high molecular weight glycopeptides in which sialic acid, galactose, N-acetylgalactosamine and N-acetylglucosamine were the primary sugar components (Table XXIII) (Coding-

440

ROBERT G . SPIRO

ton et al., 1972a). Alkaline-borohydride treatment of these proteins moreover has indicated that much of this carbohydrate is attached to serine and threonine residues through O-glycosidic bonds involving the N-acetylgalactosamine residues (Codington et al., 1972b). While the occurrence of glycoproteins in plasma membranes has now clearly been demonstrated, their presence in other cellular membranes, including those of the mitochondria (Patterson and Touster, 1962; Yamashina e t al., 1965b; DeBernard et al., 1971; Bosmann et al., 1972), microsomes (Patterson and Touster, 1962; Miyajima et al., 1969), and nuclei (Kashnig and Kasper, 1969) is suggested from the analytical data.

PROPERTIES XIV. PHYSICAL

It is not unlikely that the carbohydrate units of glycoproteins contribute to the physical properties of these molecules, which in turn form the basis of many of their biological functions (Section XVII). However, a t the present time only a limited amount of information in this regard is yet available. The presence of a substantial amount of carbohydrate tends to enhance the solubility of proteins, and this has formed the basis of procedures for the isolation of some glycoproteins (Section IV ). Glycoproteins such as the a,-acid glycoprotein (Schmid, 1953) and fetuin (Spiro, 1960), unlike most other proteins, are soluble in trichloracetic and perchloric acids as well as after boiling. The low partial specific volume of sugar residues (0.61 for an anhydrohexose) compared to that of amino acids (Cohn and Edsall, 1943) accounts for the fact that experimentally determined P values for carbohydrate-rich glycoproteins are significantly lower than those of other proteins. Values of 0.675 for a,-acid glycoprotein (Smith et al., 1950), 0.696 for fetuin (Spiro, 1960), 0.63 for bovine cervical glycoprotein (Gibbons and Glover, 1959), 0.685 for ovine submaxillary gland glycoprotein (Gottschalk and McKenzie, 1961), 0.68 for porcine ribonuclease (Reinhold et al., 1968), 0.63 for human ovarian cyst Lea blood group active glycoprotein (Pusztai and Morgan, 1961), 0.68 for the native (Luscombe and Phelps, 1967a) and 0.54 for the trypsin digested bovine nasal cartilage proteoglycan (Luscombe and Phelps, 1967b) can be compared to 0.733 for bovine serum albumin. The higher density of the carbohydrate-rich glycoproteins has been used to advantage for the separation of ovarian cyst blood group active glycoprotein (Creeth and Denborough, 1970) and nasal cartilage proteoglycan (Hascall and Sajdera, 1969) from other proteins in cesium chloride density gradients. The importance of the terminally located, negatively charged sialic acid residues in helping to determine the physical properties of glyco-

GLYCOPROTEINS

441

FIG. 17. Influence of sialic acid on glycoprotein electrophoretic mobility. Plot of mobility against pH for native fetuin on the left and sialic acid-free fetuin on the right. The dotted lines indicate the isoelectric points of the proteins. From Spiro (1960).

proteins is now generally recognized. The ease with which this sugar can be specifically removed from a protein by neuraminidase action or very mild acid treatment has made possible such an understanding. After selective removal of the 13 to 14 sialic acid residues from the fetuin molecule, a marked increase in the isoelectric point from 3.3 to 5.2 was noted (Spiro, 1960) (Fig. 17). The greater than expected increase in mobility of the sialic acid-free protein in the acid range was attributed to a more effective expression of positively charged groups of the protein with a change in the surface charge density. The isoionic point of the sialic acid-free fetuin was determined to be 5.33 compared to 4.03 for the untreated protein. The finding of a much smaller difference between the isoelectric and isoionic points of the sialic acid-free protein than of the native protein indicated a smaller degree of anion binding in the former. A comparison of the titration curves of native and sialic acid-free fetuin (Fig. 18) has shown that in the range above pH 4 the displacement of the two curves is about equal to the sialic acid content of the untreated protein (Spiro, 1960). A plot of the difference between the two curves below pH 4 gave a titration curve with a pK of about 2.4, which is close to the pK of 2.6 for free sialic acid. The negative charges on the numerous, closely spaced sialic acid residues of the ovine submaxillary gland glycoprotein have been shown to be responsible for the high viscosity which this protein demonstrates in solution (Gottschalk and Thomas, 1961) owing t o expansion and stiffen-

442

ROBERT G . SPIRO

a

,,,

+

z

B

I: -

r

0.0 0.6

i.2

2

3

4

5

6

7

8

9 1014

42

PH

FIQ. 18. Effect of sialic acid on titration of glycoprotein. Titration curves of deionized native fetuin ( X ) and deionized sialic acid-free fetuin ( 0 ) .The milliequivalents of acid or base added are expreHed per gram of dry weight of native fetuin. From Spiro (1960).

ing of the molecule caused by their mutual repulsion. When this sugar is removed by neuraminidase, or its charge reduced by acidification, a marked decrease in viscosity is observed. The lowering of viscosity achieved by titration occurs primarily between pH 4.3 and 1.8 and is completely reversible upon readjustment of the pH to a neutral value. The significant contribution of sialic acid to the net negative surface charge of the erythrocyte and probably of many other mammalian cells has already been mentioned (Section XII1,I) . Upon treatment of intact erythrocytes of most species with neuraminidase, an increase in isoelectric point from the range of pH 2-3 to that of p H 4.5-5.5 was noted (Eylar et al., 1962). A comparison of the physical properties of porcine ribonuclease with bovine ribonuclease A has indicated that the three carbohydrate units associated with the former (two of which contain a high complement of sialic acid, Section XII1,D) significantly affect its hydrodynamic properties (Reinhold et al., 1968). The porcine protein was found to have a much lower diffusion coefficient and higher viscosity than the carbohydrate-free bovine enzyme. The calculated friction ratio of 1.64 for porcine ribonuclease (fraction 11) compared to that of 1.05 for bovine ribonuclease A suggests that the carbohydrate units contribute to the

GLYCOPROTEINS

443

asymmetry of the molecule, probably owing to their extension into the solvent environment. One of the most interesting examples of the role which carbohydrate units play in determining the physical properties of glycoproteins is to be found in the freezing point-depressing glycoproteins of the Antarctic fish Trematomus borchgrevinki (DeVries and Wohlschlag, 1969; Komatsu et al., 1970; DeVries, 1971 ; Shier et al., 1972). The freezing point of the serum of this fish, which lives in the ice-laden waters of McMurdo Sound, is -2”C, in contrast to the value of -0.5 to -0.8”C of most other marine fish. The low freezing point is partly the result of glycoproteins with molecular weights ranging from 10,OOO to 21,000. On a molal basis, these proteins are about 200-500 times as effective as sodium chloride in lowering the freezing point of water, and therefore their interaction with that solvent must involve other factors than the usual colligative properties of solutes. While salt solutions have nearly identical freezing and melting points, the freezing and melting points of aqueous solutions of these glycoproteins were shown to be quite different and to vary with concentration (0.4”Cdifference a t 4 mg/ml; 023°C difference a t 12 mg/ml) . While the “antifreeze” property of the glycoproteins was shown to depend upon the complete integrity of the peptide chain, being lost by scission of as few as three bonds, experiments in which the numerous P-galactosyl-N-acetylgalactosamine disaccharide units of this protein (Section XII1,A) were modified indicated that the cis hydroxyl groups a t C-3 and C-4 of the galactose residues are specifically involved. The presence of borate, which complexes with these hydroxyl groups, caused a loss in antifreeze activity, but this property could be completely restored upon removal of the borate ions by dialysis. Periodate oxidation, which caused destruction of the galactose but spared the N-acetylgalactosamine, also resulted in a loss of antifreeze activity, while treatment with galactose oxidase, which reacts with C-6 of the galactose but leaves the rest of the molecule intact, did not affect this property. Acetylation of as few as 30% of the hydroxyl groups resulted in a loss of activity, which could be restored upon deacetylation with hydroxylamine. While the mechanism of the antifreeze effect exerted by the many terminal galactose residues (Table V) is not known, it has been suggested that the glycoprotcin might, by being adsorbed onto the surface of the ice crystals, prevent water molecules from settling into the lattice of the crystal until a much lower temperature is reached. Since the ice crystals melt a t the expected temperature, such molecules would then apparently not impede the movement of water molecules from the crystals into solution (DeVries, 1971).

444

ROBERT G . SPIRO

Proteins containing a substantial amount of carbohydrate have been shown to behave in an anomalous manner during gel filtration and gel electrophoresis. While a linear relationship exists for most globular proteins between their elution volumes on Sephadex gel columns and the log of their molecular weight, glycoproteins such as fetuin, ovomucoid, and thyroglobulin do not conform to this relationship (Andrews, 1965). These proteins emerge from the columns in a less retarded position than expected from their molecular weights, suggesting that due to a greater hydration in solution brought about by the carbohydrate units, they have a more expanded structure than proteins not containing carbohydrate. This overestimation therefore precludes the use of gel filtration for the purpose of molecular weight determination of glycoproteins. The finding that the migration of most proteins during acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) is directly related to the log of their molecular weight has served as the basis of a very effective tool for the characterization of a large number of proteins (Shapiro et al., 1967; Weber and Osborn, 1969). Glycoproteins with high carbohydrate contents, such as the human erythrocyte glycoprotein, as well as the trypsin peptide derived therefrom, and porcine ribonuclease, however, have been found to migrate in this type of electrophoresis a t rates slower than would be expected from their molecular mass (Segrest e t at., 1971). The low mobility of these carbohydrate-containing proteins was shown to be the result of their binding a smaller amount of SDS on a weight basis than standard proteins of the same mass, presumably due to the smaller peptide chains with the appropriate binding sites, Bovine serum albumin was found to bind 0.73 g of SDS per gram of protein ; the erythrocyte glycoprotein which contains about 60% carbohydrate only bound 0.38g while its glycopeptide which has 80% of its weight in the form of carbohydrate, bound only 0.023 g of SDS per gram. Ovalbumin, however, which contains only 3.5% carbohydrate, was found to bind 0.88 g of SDS per gram of protein. The anomalous migration of the glycoproteins in SDS gel electrophoresis was most pronounced a t low gel concentrations ( 5 % ) where the sieving function of the gel is least. At high acrylamide concentration (10-12.5%) migrations close to those expected from the molecular mass were observed (Segrest et al., 1971). Consistent with a decreased SDS binding, the apparent free electrophoretic mobility of the erythrocyte glycoprotein in SDS, determined from a plot of the log of its relative mobility against gel concentration, was found to be extremely low (Banker and Cotman, 1972). Since the free mobility of standard proteins in SDS varies only slightly, with a &fold increase in protein mo-

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lecular weight corresponding to only a 10% increase in free mobility of the corresponding protein-SDS complex, anomalous migration of a glycoprotein can be assessed from determination of this value (Neville, 1971; Banker and Cotman, 1972). OF GLYCOPROTEIN BIOSYNTHESIS XV. CONCEPTS

While no attempt will be made in this article to review the extensive literature (for reviews see Roseman, 1968; Spiro, 1969a, 1970a) pertaining to the biosynthesis of glycoproteins, a brief description of the mechanism by which this process is believed to take place can contribute to a fuller understanding of the structure and biological role of the carbohydratecontaining proteins. It has become apparent, primarily on the basis of studies employing puromycin (Spiro and Spiro, 1966) that synthesis of the peptide portion of glycoproteins precedes and occurs independently of the carbohydrate attachment. While the linear assembly of the polypeptide chain takes place on the polysomes by translation of the information in the RNA, the machinery operative in the synthesis of the carbohydrate units is not under such direct genetic control but involves the action of a series of glycosyltransferases. The enzymes function in concert to build up the carbohydrate units by transferring one sugar at a time from its nucleotide derivative to the protein acceptor, with the product of one enzymatic reaction becoming the substrate for and the determinant of the next reaction. The high specificity of the glycosyltransferases, which is directed toward both the sugar nucleotide and the acceptor, permits the synthesis of complex carbohydrate units with structures defined in regard to sequence and branching, as well as position and anomeric configuration of the linkages. While the major determinant on the acceptor is the nature of the terminal nonreducing sugar of the carbohydrate chain to which the new sugar is to be attached, the penultimate sugar, the linkage of the terminal sugar to the penultimate sugar, the nature of other sugar substituents, and the size and ionic charge of the peptide chain to which the carbohydrate unit is attached may play further specifying roles (M. J. Spiro and Spiro, 1968, 1971; R. G. Spiro and Spiro, 1971; Roseman, 1968; Helting and R o d h , 1969; Ginsburg et al., 1971). As already indicated (Section VIII) the enzymes involved in the formation of the glycopeptide bond may require a defined amino acid sequence around the residue to which the sugar is to be attached. While it is apparent that the glycosyltransferases are bound to cellular membranes, the precise nature of these membranes is still not completely clear. On the basis of both radioautographic (Neutra and Leblond,

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1966; Bennett and Leblond, 1970) and biochemical studies (Fleischer et al., 1969; Schachter et al., 1970) , the membranes of the Golgi apparatus appear to be the site of a number of glycosyltransferases, particularly those involved in the attachment of the peripheral sugars. However, other transferases, such as those involved in the assembly of the mannose-N-acetylglucosamine core portion of the asparagine-linked carbohydrate units, may be located on the membranes of the rough and smooth endoplasmic reticulum (Whur et al., 1969; Spiro and Spiro, 1972). Indeed it has been suggested that the most internal glucosamine residue of the asparagine-linked units is attached while the peptide chain is still bound to the polysomes (Molnar et al., 1965; Lawford and Schachter, 1966). On the basis of studies with intact neural retina cells, it has even been proposed that some glycosyltransferases may be located on the external surface of the plasma membrane (Roth et al., 1971). Although the enzymes involved in the assembly of a carbohydrate unit may be spatially separated on the membranes, this would not be absolutely necessary since their high acceptor specificity would appear to be sufficient to determine the sequence of the chains. The heterogeneity which is so commonly observed in the carbohydrate units of glycoproteins (Section X I I ) could be the result of the mechanism of stepwise attachment of sugar residues by the glycosyltransferases. As the protein moves through the membranous channels of the endoplasmic reticulum and Golgi apparatus, there may not always be opportunity for completion of the chains by the action of all the transferases due to rapidity of the passage or steric hindrances imposed by the peptide chain. Failure of an internal sugar to be added in time would preclude the attachment of all of the more externally located saccharide residues. Although no information is available as yet about the nature of the enzymatic reaction involved in the synthesis of the glycosylamine type of carbohydrate peptide bond, the glycosyltransferases responsible for the synthesis of O-glycosidic glycopeptide linkages, including the N-acetylgalactosaminylserine (threonine) (McGuire and Roseman, 1967) ; galactosylhydroxylysine (M. J. Spiro and Spiro, 1971) and xylosylserine (Stoolmiller et at., 1972) bonds have been described. Attachment of carbohydrate, as a postribosomal event, is controlled by enzyme specificity and environmental influences, such as substrate and cofactor availability. Along with other enzymatic modifications which a protein can undergo, including hydroxylation, N-methylation, oxidative deamination, iodination, and phosphorylation, the glycosylation steps stand out as potential points for rapid physiological regulation of their synthesis and perhaps release from the cell.

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XVI. CONCEPTSOF GLYCOPROTEIN CATABOLISM Although a large number of enzymes which can function in the hydrolysis of the carbohydrate units of glycoproteins have now been described in the tissues of higher animals, no clear picture as to the physiological sequence by which the degradation of glycoproteins proceeds has yet emerged. Glycosidases specific for almost every sugar and anomeric configuration occurring in glycoproteins, as well as a glycosyl asparaginase which can split the N-acetylglucosamine-asparagine bond, have been located in lysosomes (Pate1 and Tappel, 1971), and it has been shown that these enzymes can act in concert to break down the carbohydrate units of glycoproteins (Aronson and deDuve, 1968; Mahadevan e t al., 1969). More recently, a number of glycosidases located outside the confines of the lysosome have been found, including liver plasma membrane-bound (Schengrund et al., 1972), liver cytosol (Tulsiani and Carubelli, 1970), and brain synaptosomal membrane (Schengrund and Rosenberg, 1970) neuraminidases. Detailed studies carried out on the survival time of various radiolabeled glycoproteins of plasma by Ashwell and Morell (1971) and their collaborators (Morell et al., 1968, 1971; Gregoriadis et ab., 1970; Pricer and Ashwell, 1971) have given us valuable insights into the mechanism by which this group of glycoproteins is catabolized and into the crucial role which their carbohydrate units may play in this process. From these investigations it has become evident that selective removal of sialic acid residues from such proteins as ceruloplasmin, fetuin, cY,-acid glycoprotein, haptoglobin, @,-macroglobulin, human chorionic gonadotropin, and follicle-stimulating hormone leads to a remarkable increase in their rate of removal from the circulation through an uptake by the liver. Whereas 15 minutes after injection into rats 90% of native ceruloplasmin remained in the serum, less than 10% of the sialic acidfree ceruloplasmin was detected in the circulation a t that time. Essentially all the injected sialic acid-free glycoproteins were recovered in the liver, and on the basis of historadioautographic evidence and inhibition techniques, their uptake was clearly assigned to the parenchymal rather than the Kupffer cells of this organ. The removal from the circulation of the sialic acid-free proteins by the liver cells was shown to depend on their terminal galactose residues. If this sugar, which had become exposed by the initial neuraminidase treatment, was itself released by p-galactosidase digestion, an almost complete restoration of the prolonged survival time of the native protein was achieved. Similarly, treatment of the sialic acid-free protein with

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galactose oxidase to convert the primary hydroxyl group of the galactose to an aldehyde resulted in a marked extension of the survival time, indicating that intact terminal galactose residues for recognition by the liver cells. The plasma membranes of the liver have been implicated as the locus of the major binding sites for the circulating sialic acid-free glycoproteins. Effective binding of these glycoproteins by isolated liver plasma membranes was shown to depend on the presence of calcium ions and the sialic acid of the membrane itself. Competitive inhibition for membrane binding sites was noted between various sialic acid-free glycoproteins and glycopeptides, just as inhibition of uptake by liver in the intact animal of sialic acid-free glycoproteins was affected by the simultaneous injection of a variety of other desialylated glycoproteins or gly copeptides. The dual role which sialic acid appears to play (by its absence on the protein and its presence on the membranes) in the liver binding of plasma glycoproteins gives increasing importance to the aforementioned neuraminidase which has been found on the plasma membrane of that organ. This enzyme could function to cleave sialic acid residues from the plasma glycoproteins in preparation for their binding to the cell membrane and could also serve to remove sialic acid residues from the membrane itself. Indeed in vitro removal of sialic acid from the membrane results in release of the bound plasma glycoproteins (Pricer and Ashwell, 1971). After withdrawal of glycoproteins from plasma through a binding of their sialic acid-free derivatives on the liver cell membrane, they are transferred to the lysosomes (Gregoriadis e t al., 1970) where the full machinery for the degradation of the peptide and carbohydrate portion of glycoprotein resides. The importance of the carbohydrate in directing the uptake of plasma proteins by liver cells was emphasized by the work of Rogers and Kornfeld (1971), which showed that removal of albumin from the circulation by this organ was stimulated a t least 30-fold by coupling it with a fetuin glycopeptide from which the sialic acid was removed. As expected from the studies of Ashwell and Morell, no stimulation of hepatic uptake of albumin was brought about when the fetuin-albumin conjugate was injected without prior removal of the sialic acid residues. It is obvious, therefore, that the catabolism of serum albumin, which does not contain carbohydrate, must be directed by a mechanism quite different from that just described. Alternate paths of catabolism must indeed exist for some glycoproteins, as i t has been shown that the clearance from serum of transferrin is not enhanced by removal of its sialic acid and that sialic acid-free glycopeptides prepared from this protein do

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not inhibit the uptake by liver of other desialylated glycoproteins (Morel1 e t al., 1 9 7 1 ) . XVII. BIOLOGICAL ROLEOF OF

THE CARBOHYDRATE PORTION GLYCOPROTEINS

While the vital function of many glycoproteins has been clearly established (Section 111), the particular contribution of the carbohydrate units to this biological role is in many cases less evident and remains the subject of intense interest and speculation. For some glycoproteins, including the ovine submaxillary glycoprotein, Antarctic fish freezing-point depressing glycoproteins, various plasma proteins, as well as antigens and virus receptors of cell surfaces, a definite function has been attributed to the carbohydrate units (Table XXIV) and the basis for this assignment has already been discussed (Sections XIII, XIV, and XVI) . In many enzyme glycoproteins, on the other hand, the carbohydrate TABLEXXIV

Functions of the Carbohydrate of Glywproteins Function Demonstrated Increase viscosity of mucous secretions Lower freezing point of blood sera Regulate catabolism of circulating proteins by liver Cell surface antigens Virus receptor sites Postulated Mineralize connective tissue Regulate collagen fibril formation Determine porosity of basement membranes Modulate hormone effects on cell membranes Intercellular adhesion and recognition Regulate hemostasis Permit export of proteins from cells Regulate rate of protein synthesis Binding (noncovalent) of proteins to membranes

Examples" Ovine submaxillary glycoprotein Antarctic fish serum glycoproteins Plasma glycoproteins and hormones

M - and N-glycoprotein of erythrocytes Myxovirus hemagglutinin inhibition glycoprotein of erythrocyte

Pro teoglycans Hydroxylysine-linked carbohydrate of collagens Renal glomerular basement membrane Adipocyte cell surface glycoprotehs (insulin effectors) Glycoproteins of plasma membrane Platelet-collagen interaction Extracellular glycoproteins Action of membrane-bound glycosyltransferases General

See text for references and further discussion.

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has been shown not to be involved in the active site. Bovine pancreatic ribonuclease A, which contains no carbohydrate, has the same specific activity as ribonuclease B which has a carbohydrate unit attached to its peptide chain (Plummer and Hirs, 1963) (Section XII1,D) , while porcine ribonuclease fractions differing substantially in their carbohydrate content were found to manifest no significant differences in activity (Reinhold et al., 1968). Similarly, bovine @-glucuronidase has been resolved into multiple forms which differ primarily in their carbohydrate content and yet show no significant differences in specific activity (Plapp and Cole, 1967). Moreover, human parotid a-amylase isoenzymes which occur in both glycosylated and carbohydrate-free form show no difference in their specific activities (Keller e t al., 1971). Destruction of onethird of the sugar residues of A . niger glucoamylase by periodate oxidation led to no loss of enzyme activity (Pazur e t al., 1970), while oxidation of 80% of the sugar residues of pineapple stem bromelain resulted in only a 17% loss in enzyme activity (Yasuda et al., 1971). That the antibody activity of immunoglobulins is not associated with carbohydrate has been suggested by the fact that the F a b fragments, which have little if any carbohydrate associated with them, retain power to combine with antigen, while the Fc region of the molecule, which always contains the 2 asparagine-linked carbohydrate units, has little such activity (Porter, 1959; Fleischman e t al., 1963). Selective removal of sialic acid residues from follicle stimulating hormone (Gottschalk et al., 1960) and human chorionic gonadotropin (Goverde et al., 1968) leads to a loss of biological activity as assessed by in vivo assays of these hormones. The biological activity of the latter hormone has been shown, moreover, to be directly related to its sialic acid content, while its potency as determined by immunochemical means was unrelated to the content of this sugar (Sehuurs et al., 1968; Van Hall et al., 1971). From the studies of Morel1 et al. (1971) it would appear that the loss of activity of these hormones resulting from sialic acid removal is due to an uncovering of galactose residues, which, by their interaction with liver plasma membrane receptors, permits their rapid clearance from the circulation and thereby interferes with their action on target organs (Section XVI) . It has been suggested that the carbohydrate units of the proteoglycans may play a biological role, by virtue of their numerous anionic groups, in the mineralization of connective tissue and in the arrangement and stabilization of collagen fibers (Meyer, 1970) (Table XXIV). The former function would arise from the binding of calcium ions, whereas the second-mentioned activity would result from interaction between the cationic groups of collagen fibers with the anionic functions of the proteoglycans.

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45 1

The hydroxylysine-linked carbohydrate units of collagens and basement membranes may play a role in regulating the packing arrangement of the peptide chains (Table XXIV). The necessity of a hole region (Butler, 1970) into which the bulky carbohydrate substituents can fit suggests that the carbohydrate units can modify or interfere with the staggered array of collagen molecules which is presumed to be necessary for fibril formation. I n support of this thesis is the inverse correlation which has been noted between the number of hydroxylysine-linked carbohydrate units present in a collagen and the degree of morphological organization (Spiro, 1969b, 1972a). I n basement membranes where the density of hydroxylysine-linked carbohydrate units is greatest (Spiro, 1967c; Spiro and Fukushi, 1969) (Table I X ) , no fibril formation occurs, and these membranes are characterized by a lack of structure as viewed under the electron microscope. The porosity of these membranes, which function as coarse filters, may be influenced by the effect which the carbohydrate units have on the packing of the peptide chains. The increased permeability of the renal glomerular basement membrane in diabetes mellitus has been attributed to the presence of an increased number of subunits rich in the hydroxylysine-linked disaccharide units Beisswenger and Spiro, 1970,1973). A number of important biological functions which appear on the plasma membrane of the cell have been attributed to the carbohydrate portions of the glycoproteins which are known to be major components of this structure (Section XIII). Studies with mouse teratoma cells grown in completely synthetic media have suggested that the carbohydrate units of these surface glycoproteins are required for intercellular adhesion (Oppenheimer et al., 1969). Omission of L-glutamine from such growth media prevented aggregation of cells, and only two compounds, namely, D-glucosamine and D-mannosamine, were able to replace this amino acid. Moreover, since specific antagonists of L-glutamine, but not of the hexosamines, blocked the aggregating stimulation of the amino acid, it was thought likely that the reaction promoting the adhesiveness involved the transfer of the amide nitrogen from glutamine to fructose 6-phosphate to yield glucosamine 6-phosphate. The latter intermediate being the precursor of the hexosamines as well as the sialic acids would be crucial for the synthesis of nearly all the carbohydrate units of glycoproteins. Only glucosamine and mannosamine, which can be directly phosphorylated by hexokinase, could bypass a block a t the glucosamine 6-phosphate synthetase step. The “homing” property of lymphocytes, which determines the migration of these cells from the blood into the lymphoid tissue, is believed to be brought about by interaction of cell surface components on the lymphocytes with endothelial cells in the capillary walls (Gesner and

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Ginsburg, 1964). This interaction can be altered by glycosidase treatment of the cells, suggesting that sugars are part of this recognition site. The adhesion of platelets to collagen or basement membrane is the primary step in the process of hemostasis and represents an example of cell surface-glycoprotein interaction. The carbohydrate units of collagen have been implicated in this process on the basis of the observations that galactose oxidase treatment of this protein completely abolished its ability to promote platelet aggregation and that this activity could be restored upon subsequent reduction of the enzymatically modified protein (Chesney et al., 1972). Since galactose oxidase converts C-6 of the galactose in the hydroxylysine-linked glucosylgalactose and galactose units to an aldehydic function (Spiro, 1967c, 1969b) the integrity of this sugar appears to be necessary for the formation of the collagen-platelet membrane complex. However, since limited digestion with bacterial collagenase also abolished the ability of collagen to aggregate platelets, while removal of the telopeptides with trypsin had no such effect, the helical portion of the peptide chain onto which the hydroxylysine-linked carbohydrate units are believed to be attached must also play a crucial determining role (Chesney e t al., 1972). The occurrence of an enzyme on the platelet membrane (Jamieson et al., 1971b) with properties similar to the UDP-glucose :galactosylhydroxylysine-collagen glucosyltransferase (R. G. Spiro and Spiro, 1971) and the presence of hydroxylysine-linked galactose residues on many collagens (Spiro, 196913) has suggested to Jamieson that the basis of the platelet-collagen interaction is an enzyme-substrate interaction between the glucosyltransferase and the galactosylhydroxylysine acceptors. The parallel requirements between platelet collagen adhesion (Wilner et al., 1968) and the collagen: glucosyltransferase reaction (R. G. Spiro and Spiro, 1971), especially in their requirement for an unsubstituted r-amino group on the glycosidically substituted hydroxylysine, has strengthened this argument. This would represent a special case of a general mechanism for intercellular adhesion proposed by Roseman (1970) in which glycosyltransferases on one cell surface bind to appropriate acceptors on the surface of another cell by the formation of enzyme-substrate complexes. Treatment of isolated adipose tissue cells with neuraminidase has been found to affect the transport of glucose into the cell, as well as the response to insulin stimulation, and has suggested that surface membrane glycoproteins may be involved in these processes (Cuatrecasas and Illiano, 1971). Digestion of the cells with very low concentrations of the enzyme caused a stimulation of glucose transport, while treatment with larger amounts of neuraminidase resulted in a decrease in this

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enhanced uptake and abolished the stimulating effect which insulin has on it. Since the sialic acid removal did not affect insulin binding by the adipocytes, the glycoproteins may be presumed to be involved in the transmission of the insulin effect rather than being part of the receptor structure itself. The biphasic response of the neuraminidase treatment, moreover, suggested that the carbohydrate units of membrane glycoproteins may have a complex modulating effect on the response to this hormone. Because of the widespread distribution of glycoproteins in animals, plants, and microorganisms (Table I) , the possibility that covalently linked carbohydrate in proteins serves a uniform biological role must be considered. A search for such a unifying purpose is made difficult by the great diversity in the structure of carbohydrate units, as well as in their number, distribution, and manner of attachment to the peptide chains. Moreover, the synthesis of glycoproteins with different types of carbohydrate units by the same cell and the occurrence even in the same protein of units of different structure and with different glycopeptide linkages (Table X) further complicates the picture. If a common biological basis does indeed exist, it must represent a very fundamental phenomenon; it is also possible that the sugar components of proteins, just like the amino acids, may function in a number of unrelated ways. It has been proposed that the carbohydrate may serve as a label which by interaction with a membrane carrier permits transport of proteins after synthesis into the extracellular environment (Eylar, 1965). Such a hypothesis would require demonstration that all extracellular proteins contain covalently bound carbohydrate units, while those remaining inside the cell are devoid of a saccharide moiety and moreover that carbohydrate units in glycoproteins from the same cell would be identical or very similar. As already indicated, what we know of the diversity of carbohydrate units would make unlikely the latter requirement, which suggests a simple lock and key arrangement. Furthermore, evaluation of the carbohydrate content of both extra- and intracellular proteins, as has been done by Winterburn and Phelps (1972) indicates that the primary requirement for this transport theory also cannot be met. Such a survey reveals that a significant number of exported proteins are not glycosylated. These include albumin of plasma ; procarboxypeptidase, chymotrypsinogen, trypsinogen, and ribonuclease A of bovine pancreas; the major alactalbumin of bovine milk; lysozyme of hen egg white; and the silk collagen of the gooseberry sawfly. I n addition, the polypeptide hormones insulin, glucagon, growth hormone, and prolactin must be listed among proteins containing no carbohydrate. While assignment of proteins to a

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truly intracellular location is difficult, proteins with carbohydrate which do occur inside the boundary of the cell include the glycoproteins of mitochondrial, nuclear, and microsomal membranes, kidney y-glutamyl transpeptidase, and liver p-glucuronidase and p-N-acetylglucosaminidase. Since glycoproteins appear to share a common mechanism of carbohydrate unit assembly involving the postribosomal action of membrane bound glycosyltransferases (Section XV) , i t has been suggested that this process might serve to regulate the rate of their synthesis (Spiro, 1969a, 1970a). The control could be exerted by various environmental factors, such as the availability of sugar nucleotides, which influence the glycosylation steps and thereby determine the rate of passage of the glycoprotein precursor through the, channels of the endoplasmic reticulum and the Golgi apparatus. The apparent lack of carbohydrate groups on the membrane-bound intracellular albumin of liver would, however, preclude the assignment of this regulatory process to all proteins (Peters et al., 1971). If a single biological role for the carbohydrate units of proteins is to be invoked, the possibility of the externally located, bulky hydrophilic groups serving as probes for the noncovalent attachment of the proteins to membranes seems most appealing. A model for this concept might be found in the mechanism already discussed (Section XVI) by which liver removes protein from the plasma (Morel1 et al., 1971; Pricer and Ashwell, 1971), in which galactose residues are the determinants for attachment of the protein onto the liver membranes. Similarly, attachment of enzymes, immunoglobulins, and hormones to cellular membranes in the vicinity of which they function could be brought about by the binding of specific sugar components, perhaps by hydrogen bonds, to one or more recognition sites. Differences in the spacing and nature of the carbohydrate units could be geared to interaction with different acceptors. Furthermore, such phenomena as intercellular adhesion, platelet-collagen interaction, and cellular recognition, as in the “homing” of lymphocytes, could be mediated by noncovalent bonds between opposing saccharide or saccharide and peptide residues. Such binding phenomena may play an important role in the organization of proteins with biological activity inside the cell and on the cell surface. Measurements of biological activity are usually made outside of the natural environment and generally reflect only one of the multiple interactions in which the protein may be involved. Confirmation of the binding hypothesis of glycoprotein action will depend to a large measure on the development of sensitive biological assays which will detect. this property of the carbohydrate units of proteins, as well as on general

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knowledge in regard to the three-dimensional structure of these saccharide moieties. ACKNOWLEDGMENTS Work from the author’s laboratory reported in this article was supported by Grants AM 05363 and AM 1048:: from the National Institutes of Health and by a grant from the American Heart A:isociation.

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