Variety and microheterogeneity in the carbohydrate chains of glycoproteins

0020-71IX/88 $3.00 + 0.00 Copyright 0 1988 Pcrgamon Pressplc

ht. .I. Eioehem.Vat. 20, No. 5, pp. 479-491, 1988 Printed in Great Britain.All rightsreserved

MINIREVIEW VARIETY AND MICROHETEROGENEITY IN THE CARBOHYDRATE CHAINS OF GLYCOPROTEINS HITOO IWASE Department of Biochemistry, School of Medicine, Kitasato University, i-15-1, Kitasato, Sagamihara, Kanagawa 228, Japan

(Reepioed24 June 1987) INTRODUCTION There are many kinds of glycoproteins including those that are secreted (plasma, egg white, and mucus materials) and biologically active (enzymes, hormone, antibody and toxin) as well as membranous glycoproteins present in virus envelopes and cell surface membranes. The carbohydrate constituents of glycoproteins are known to stabilize the conformation of proteins, protect glycoproteins from degradation, and by a carbohydrate chain-specific recognition mechanism, determine their localization in cells, their life-time in the circulation system and the type of cell-cell interactions that take place. Great progress has been made in the establishment of techniques for the analysis of carbohydrate chains (Beeley, 1985), such as the development of 500 MHz ‘H-NMR spectroscopy, high performance liquid chromatography, GC-MS (Berger et al., 1982), hydrazinolysis (Takasaki et al., 1982), fluorescence probes (Hase et al., 1984; van Halbeek et al., 1985), lectins (Lis and Sharon, 1986), glycosidases including endo-type enzymes (Kobata, 1979; Plummer et al., 1984) and antibodies against carbohydrate chains (Feizi, 1985). By more accurate methods of analysis, diverse st~ctures of carbohydrate chains have gradually been clarified. Important new info~ation is now available on minute structural changes in carbohydrates accompanying the progress of malignancy, developmental processes and differentiation. In particular, carbohydrate chains in cell surface glycoproteins are important to growth, differentiation and cell-cell interactions (Grabel et al., 1979; Hart, 1982; Takata et al., 1984). The relation of changes in carbohydrate chain structure to malignancy has become clarified through numerous studies conducted on membrane glycoproteins, a-fetoprotein and human chorionic gonadotropin (Warren et al., 1978; Warren and Buck, 1980; Smets and van Beek, 1984; Yamashita ef al., 1983a,b; Mi~~hi et al., 1983, 1985; Bolscher et al., 1986). The mechanism for the production of such variety and microheterogeneity in glycoproteins must be thoroughly clarified for an adequate understanding of the numerous phenomena mentioned above. The biosynthetic processes of carbohydrate chains have been studied extensively and are consequently known to occur by a complicated series of steps including lipid intermediates and the processing of N-glycoside type carbohydrate chains. However, the synthesis of O-

glycoside type carbohydrate chains can be accomplished simply by the stepwise addition of monosaccharide (see reviews by Berger et al. (1982), Komfeld and Komfeld (1985) and Schachter (1986)). Even though they usually consist of 3-5 types of mono saccharide components such as N-acetylN-acetylgalactosamine, glucosamine, mannose, galactose, fucose, sialic acid, xylose or glucose, the reason why the structures of the carbohydrate chains are so complex is that saccharides are multifunctional compounds each with three or four hydroxyl groups exhibiting appro~mately equal chemical reactivity (Sharon, 1975). ~ons~uently, as shown in the figures, the chains generally have branched structures. The production mechanism of such complex structures differs greatly from that of a polypeptide or polynucleotide synthesized according to its own template. A phenomenon so far never observed in protein and nucleic acid is microheterogeneity in the carbohydrate chains of glycoproteins. Microheterogeneity is a quality of a pure glycoprotein such that it is heterogeneous with respect to the structure of its carbohydrate chains (Huang et al., 1970). The physiological activity of a glycoprotein is generally examined on the basis of its protein constituent without the need for further purgation. Analysis of ovalbumin indicated microheterogenity to be reproducible in some manner not yet clarified, but very much in the manner of the preservation of the uniqueness of fingerprints. Such heterogeneity obviously does not result from a random process (Iwase et al., 1981, 1983, 1984, 1987). The synthesis of carbohydrate chains is indispensable for cell growth, as indicated by experiments using defected cells and an inhibitor (tunicamycin) of the enzyme catalyzing the first step of a carbohydrate chain assembly (Struck and Lennartz, 1980; Kukuruzinska and Robbins, 1987). That a cell consumes an enormous amount of energy to produce heterogenous ~bohy~~ chains also reflects that their synthesis is important. This review discusses factors apparently related to the production of heterogenous gtycoproteins, proceeding on the assumption that the variety and even the microheterogenity of a carbohydrate chain do not occur by a random process, but rather according to a pre-programmed mechanism to provide additional information to the protein from the gene. As background for this research, careful reference was made to as many available related papers as possible. 479

480

HITOO IWASE

CLASSIFICAT’ION OF GLYCOPPOTEINS BASED ON CARBOHYDRATE CHAIN TYPE

Glycoproteins are classified as either the Nglycosidic type or 0-glycosidic type based on linkage structures between the protein and carbohydrate chain. In the case of the former, the linkage is between /I-N-acetylglucosamine and the amide group of asparagine in the tripeptide sequence, Asn-X-Ser(Thr) in which X indicates any amino acid except proline or asparatic acid (Struck and Lennartz, 1980; Komfeld and Komfeid, 1985). This linkage is also called a serum type because of its abundance in serum (plasma) glycoproteins. As a special example that is an exception to this, the carbohydrate chain in a nephritogenoside from the glomerular basement membrane, capable of inducing experimental nephritis (Takeda et al., 1982) and the carbohydrate chain in a Aagellin synthesized via the unusual lipid intermediate (Wieland et al., 1985), were both bound to asparagine by their reducing terminal glucose residues. The carbohydrate chain in the latter was also found to be present on the Asn-Leu-Thr sequence. The O-glycosidic linkage was frequently found in mucus glycoproteins and the so-called mucin type showed a linkage between u-N-acetylgalactosamine and the hydroxy group in serine (or threonine). Another type of 0-glycosidic linkage is the collagen type possessing a /I-galactose residue linked with the li-hydroxyl group in hydroxylysine (Spiro, 1967; Ishii et al., 1987). As minor com~nents, O-linked mannose in yeast mannan and O-linked ~-a~tylglu~osamine in a wide variety of cellular components including nucleic membranes have been reported (Holt and Hart, 1986; Schindler et al., 1987). Among these representative glycoproteins, serum and mu& types can be easily distinguished from each other since the 0-glycosidic linkage is cleaved under a weakly alkaline condition by the well known fi-elimination reaction (Rovis, 1973) but N-glycosidic linkage cannot be cleaved in this manner. Distinction is also possible by analysis of the carbohydrate composition since generally the serum type does not contain N-acetylgalactosamine nor does the mucin type have mannose. However, there are some exceptions such as the N-glycosidic type glycoprotein, the epidermal growth factor receptor exhibiting blood group A activity (Cummings et al., 1985) and the pituitary hormone lutropin having a sulfated Nacetylgalactosamine residue on the peripheral part of its carbohydrate chain (Parsons and Pierce, 1980; Green ef ul., 1986). Since there are some glycoproteins containing both N-glycosidic and O-glycosidic carbohydrate chains, as in the case of fetuin, glycophorin, IgA, IgD, hCG (Kessler et al., 1979), low density lipoprotein receptor (Cummings et al., 1983), plasma galactoglycoprotein (Akiyama et al., 1984), thyroid cell surface glycoprotein (Edge and Spiro, 1985), leukosialin (Fukuda et al., 1986) and proteoglycans with N- and/or 0-glycosidic types of carbohydrate chains on the same molecule, care must he taken in their analyses (Nilsson et al., 1982; Takahashi et al., 1985; Gowda et al., 1986). The structural characteristics of serum and mucin type carbohydrate chains are given as follows

(refer to figures for structures): the N-glycosidic type possesses (1) the high-mannose (oligo-mannose) type carbohydrate chain, (2) the high-mannose type carbohydrate chain with one to three glucose residues on the peripheral part, (3) a complex-type carbohydrate chain with a bi-antennary structure, (4) a complex type carbohydrate chain with a structure having more than three antennas, (5) the hybrid type carbohydrate chain, (6) a carbohydrate chain with bisect N-acetylglucosamine bound to the p-linked mannose residue, (7) a carbohydrate chain containing sialic acid, (8) a carbohydrate chain with fucose linked to the proximal N-acetylglucosamine residue, (9) a carbohydrate chain with fucose on the outer part, (10) a carbohydrate chain with xylose bound to the B-linked mannose residue, (11) a carbohydrate chain with a sulfate ester, (12) a carbohydrate chain with a phosphate ester, and (13) a carbohydrate chain with lactosamine repeating units. The 0-glycosidic type possesses: (1) carbohydrate chains with Gal-j? l3GalNAc core, (2) carbohydrate chains with the Gal-8 I-3(GlcNAc$I I-6)GalNAc core, (3) Gal-/l l3(Gal-fi ld)GalNAc core, (4) GlcNAc-/I 1-3GalNAc core, (5) GlcNAc-#I I-3(GlcNAc-/I 1d)GalNAc core and the above (7), (9), (1 l), and (13). Among these characteristics, (12) was closely related to physiological function as a marker (mannose-6-phosphate residue in the carbohydrate structure) for the lysosomal enzyme (Pohlmann et al., 1982) although an exception to this has been reported in dictyostelium discoideum by Mierendorf et ai. (1985). The sulfate group in (11) was rich in the mucin type carbohydrate chain and seldomly found in the N-glycosidic type carbohydrate chain (Yamashita et al., 1983~; Edge and Spiro, 1985; Green et al., 1986). Functions of the sulfate group may possibly be the concentration of glycoprotein in the Golgi apparatus (Reggio and Palade, 1978) and protection of glycoproteins from protease and glycosidase action (Mikuni-Taka~ki and Hotta, 1979; Parsons and Pierce, 1980). Sialic acid in (7) was found related to anti-clearance activity which protects the serum protein from galactose binding protein on the hepatic cell surface (Neufelt and Ashwell, 1980; Harford and Ashwell, 1985). Xylose in (10) was usually found in plant glycoprotein but with one exception, u-hem~yamin (van Kuik et al., 1985). In addition to the above characteristics, micro differences such as positional isomers, anomeric structural differences, and the presence of many kinds of sialic acid as reviewed by Schauer (1982) are reasons for the immense variety of carbohydrate chains. Thus, if each characteristic carbohydrate structure has specific physiological significance, carbohydrate chains, all usually possessing several characteristics, may be considered important as multifunctional groups that provide considerable information to protein constituents. SP~~DEPENDENT DIFFERENT IN THE STRUCTURES OF GLYCOPROTEIN CARBOHYDRATE CHAINS

The heterogeneity of glycoproteins can be explained in part on the basis of species-dependent differences. Generaliy speaking, carbohydrate chain

Carbohydrate

chains of glycoproteins

structures change in a manner peculiar to the species from which they come, as in the case of ovalbumin (van Halbeek et al., 1985; Mutsaers et al., 1985; Iwase et al., 1983, 1984), ~-glut~yltrans~ptidase (Yamashita et al., 1985, 1986), transferrin (Iwase and Hotta, (Yoshima et al., 1981), 1977), tr,-glycoprotein fibronectin (Takasaki et al., 1979), gastric mucus glycoprotein (Slomiany et al., 1984; Tsurui et al., 1986) and glycoproteins from 11 different animal cells (Ceccarini and Atkinson, 1983). The best illustration of this is the production of virus envelop glycoprotein. The carbohydrate chain in the influenza virus envelop glycoprotein has been reported to change in a manner determined by the host infected by the virus (Deom and Schulze, 1985). Depending on the infected host, carbohydrate chain structures at two sites have been noted to change site-specifically in the case of the sindbis virus glycoproteins, E, and Es (Hsieh et at., 1983a) although they are also affected by the heterogeneity of the protein constituent in the case of the vesicular stomatitis virus G protein (Gabel and Bergmann, 1985) and even in the isozyme of cathepsin B (Takahashi et al., 1984). It is thus evident that a carbohydrate chain structure is determined not only by its protein constituent but by enzymes present in the cell as well. In the case of ovalbumin, mouse L cells introduced by the ovalbumin gene produce ovalbumin with a carbohydrate chain fairly different from native ovalbumin produced by hen oviduct (Sheares and Robbins, 1986). It may only be natural for the carbohydrate chains in glycoproteins not to have any direct effect on individual functions performed by the protein constituent. Indeed, the activity of ribonuclease B was found to be the same as that of ribonuclease A without a carbohydrate chain, and interferon produced by genetic engineering was noted to function normally despite loss of the carbohydrate chain constituent (Bose et al., 1976). The assigned role of the carbohydrate chain may possibly be a higher order function, such as a receptor for information from the cell environment, a signal for intracellular movement or localization of glycoprotein in the cell. The carbohydrate chain also may control the amount of glycoprotein by modulating its structure, since the carbohydrate chain changes the life span of the glycoprotein initially determined by the speciesspecific balance of synthesis and degradation. No systematic work has so far appeared on evolutional changes in carbohydrate structures due to insufficient reliable data and their striking heterogeneity. A comparative examination and efforts to classify their structures may possibly provide some clarification of the origin of carbohydrate chains. MICROHETRROGENITY

IN GLYCOPROTEINS

As stated above, as a result of the rapid progress in the analytical techniques of carbohydrate chains, info~ation on glycoprotein ~croheterogeneity has accumulated. Microheterogeneity in glycoproteins means that carbohydrate chains differing in several structural respects are present at the same site at which a carbohydrate is attached to a protein molecule. As shown in Fig. 1, the carbohydrate chain of ovalbumin exhibits typical microheterogenity

481

and appears to have been produced by an incomplete biosynthetic process, in consideration of the precursor-product relationship between the two proper structures. The microheterogeneity of ovalbumin was first analyzed by Huang et al. (1970). The group of Kobata and other workers determined the structures of carbohydrate chains (Fig. 1). Iwase et al. (1981) have quantitatively analyzed individual differences in microheterogenity by direct analysis of glycoproteins with Con A-Sepharose affinity chromatography. They showed that a single hen produces ovalbumin with qualitatively and quantitatively similar carbohydrate chains; moreover, individual differences exist in ovalbumin microheterogeneity which exhibits a constant pattern. This has also been confirmed by the quantitative analysis of each dansylated glycopeptide derived from individual ovalbumin (Iwase et al., 1987). In this case, the microheterogeneity of ovotransferrin and ovomucoid from the same cell differed from that of ovalbumin although they all possessed some common carbohydrate structures (van Halbeek et al., 1985; Yamashita et al., 1983d). The same situation seems applicable to the mucin type carbohydrate chain which contains intermediate structures for some parent molecules in oligosaccharides from mucus glycoprotein of the human colon (see Fig. 2). Two dimensional HPLC analysis also showed similar microheterogeneity (Goso-Kato et al., 1986). However, in the case of mucus glycoprotein, it should be noted that these structures do not represent all the carbohydrate chains of glycoprotein and their dist~bution on a molecule has not been clarified to the same degree as in the case of N-glycosidic glycoprotein. Heterogeneous colon mucin was classified into six distinct glycoprotein species I-VI and selective reduction of species IV has been reported in patients with ulcerative colitis (Podolsky and Isselbacher, 1983; Podolsky, l985a,b). Such microhetero~neity can be examined in greater detail with the establishment of more precise analytical techniques and may come to be considered a general characteristic of mucus glycoproteins. In order to prepare glycoprotein acceptors for various glycosyltransferases, Kato et al. (1984a) separated ovalbumin to make it pure with respect to its carbohydrate chain structures, using various modes of lectin affinity chromatography. Except in the case of separation by charge differences due to sialic acid, sulfate or phosphate content, such a procedure is not necessary even in a strictly controlled experiment, Carbohydrate chains in glycoproteins were formerly not considered when dete~ning the functions of the glycoproteins, since they do not directly affect the functions of protein constituent. Microheterogeneity in the past was apparently considered a mistake in the biosynthesis of glycoproteins. Today, however, there is no justification for this view. Human chorionic gonadotropin is an exceptional glycoprotein which loses hormonal activity due to modification of its carbohydrate chains by chemical or enzymatic treatment (Kalyan et al., 1982; Goverman et al., 1982). A detailed examination of the microheterogeneity of carbohydrate chains in human chorionic gonadotropin showed their components, initially considered to be derived from incomplete synthesis, to indeed be

482

Hrroo IWASE Hiqh-Mmose

TYPO

Mg’2a6 'M a6

vl3 ’ M’

M8-4R

M :3

a2 R a3 M--M a2

M$ III-B

AC-BC

q6

M

M

M (16 M ‘;F3 \ 84 M-R

M ;3

\a684 ,M-R

M’-a3

M a3

M $2

IV

Hybrid

Type

M

a6

’

M e6Gn

Ga I 8,4G! ‘i:‘$$

R

‘M<3Gn $2 III-A

I

II-B

III-C

I I-A

MaeGn Gn i;z \I B4 ,Mg4R M a3

Gn& GnGn (Grill

GnGnGntGn)

AC-CD

Fig. I. Reported carbohydrate structures of ovalbumin and their precursor-product relationships. The name of each carbohvdrate structure was &en in the reports (Tai er al., 1975, 1977a.b; Yamashita er ai., 1978; Longmore andVSchachter, 1982; Nomoto and Inoue, 19i3; Ceccarini er al., 1983, 1984; Yamashita et al., 1984). Abbreviations: M, Gn, SA, Gal, So, and R stand for mannose, N-acetylglucosamine, sialic acid, galactose, sulfate and GlcNAc-j?-1,4-GlcNAc-Am, respectively. A residue appears in parenthesis for one of the two possible structures. Beta-linked mannose residue is indicated by underlined M.

a mixture asymmetrically derived from a- and fl-subunits (Mizuochi and Kobata, 1980). According to Swiedler et al. (1985) major murine histocompatibility antigens show all site-specific oligosacxharide to be highly patterns, i.e. microheterogeneity,

reproducible, independent of the number of in vivo tumor passages. Site-specific microheterogeneity seems well preserved, as also noted for other glycoproteins (Pollack and Atkinson, 1983; Brown and Hickman, 1986; Nsieh et al., 1983b).

Carbohydrate

chains of glycoproteins

483

T-0, 0

r. 0.0’ v. 0

Y

‘I

0.0

*oa

.:-0,

v

.O*O’

v

e-01

0.0

J-0,

cl’

.:-0,

cl’

‘0.0’

oa*

T-0, oa

.OCl

a .04J

.a-ol

oa

:-or .:-0,

OCI

.0-O

O-01

.O-01

.O-Ol

00

J-01

ocl

0

‘043

*o-o,

04J’ o-0

’04s

O-01

0

‘ocl* l *O.D’

o-o.

,043

O-01

.o _0 I

0.0.0

‘0.0

.a-01

l ~0cl l .0.u Fig. 2. Schematic structural representation of oligosaccharides from human colonic mucin and their precursor-product relationships. Some of the structures reported by Podolsky (1985a,b) are arranged according to their structural relationships. Symbols: (e),(e-01),(0),(O) and (v) stand for Nacetylgalactosamine, N-acetylgalactosaminitol, N-acetylglucosamine, galactose and sialic acid, respcctively. 0-o

Recent work on myeloma IgG glycoprotein has indicated microheterogeneity in IgG from myeloma (mono clonal origin) to shift from that in normal human (polyclonal origin) and moreover, the elution profiles of pyridylaminated oligosaccharide prepared from two myeloma patients to differ from each other (Takahashi er al., 1987). Association of rheumatoid arthritis with the shift of microheterogeneity in serum IgG has also been reported (Parekh er ol., 1985). Thus, some observed microheterogeneity initially thought to derive from incomplete biosynthesis may possibly arise from such clonal differences. Similar results have been reported for the Oglycoside type carbohydrate chain. Recently, Fukuda et al. have suggested that each cell type utilizes the same set of core structures for 0-glycoside type carbohydrate, on the basis of results for the analysis of leukosialin from granulocytic cells with three different degrees of maturation (Fukuda er al., 1986). As indicated for the N-glycoside type carbohydrate chain, the microheterogenity of the carbohydrate chain structure in mucin implies that this heterogenity possibly arises from a mixture of different cell types. These reports will indicate that each cell produces a cell-specified set of carbohydrate chains on a glycoprotein molecule. This is consistent with the finding that different species produce speciesdependent carbohydrate chains, and different tissues produce tissuedependent carbohydrate chains for a protein. The microheterogeneity in ovalbumin and digestive tract mucin may possibly reflect to some degree cell populations at various differentiated states or various stages of maturation in oviduct, stomach and colon tissue.

CLOSE RELATION OF THE Bl0SYNTHE.W CARBOHYDRATE CHAINS TO THEIR MICROHETEROGENEI’I?’

OF

Biosynthesis of the N-Giycoside Type Carbohydrate Chain Transfer of oligosaccharide from a lipid intermediate to protein The biosynthesis of glycoproteins has been reviewed by Komfeld and others (Rerger et al., 1982; Komfeld and Komfeld, 1985). Summarized synthetic processes of N-glycoside type carbohydrate chain are shown as a flow sheet in Fig. 3. Glycoprotein is also synthesized on membrane bound polysomes as were other proteins. The first step in the synthesis of N-glycosidic type glycoprotein is the en bloc transfer of the oligosaccharide portion from the lipid-linked oligosaccharide, Glc,Man,GlcNAc,-P-P-Dolichol, to the nascent protein constituent. The biosynthesis of the lipid intermediate occurs by the stepwise addition of a carbohydrate residue from a sugar nucleotide or via a dolichol phosphoryl derivative (Komfeld and Kornfeld, 1985). The reason why two different donors participate is not clear. Induction and modulation by steroid hormone of enzymes participating in the oligosaccharide assembly has been reported in hen oviduct and coupled alteration with hormonal treatment in the production of certain membrane glycoproteins and ovalbumin, has been observed (DeRosa and Lucas, 1982; Hayes and Lucas, 1983). Thus, the amount of a carbohydrate chain synthesized as a lipid intermediate, that is, the amount of glycoprotein to be produced can be controlled at

ose

Type

Ma2M \ a6

I I Glucostdase 1 Glucostdase 4

I II

M9G”2 1 (G

rER mannos tdase

Mg_7Gn2

1 Hannos 1dase

I

Endo mannosidase

t 4

M

\06

M U /as q”

1 1 GlcNAc 4

Gn M5Gn2

84

,M-GneGn U a3

%Gn2 transferase

I

Gal transferase SA transferase ---------------,

SA Gal

Gn USGn2

(SA-Gal)

: I 1 Mannosidase

II U ‘“U EGn ‘IGn

f I 1

G~&$?&,,<2 Alternate

1 +________~~~-------~-~___------------~~~_____--____ I

Gal SA

Gn U3Gn2

G MSGn, thiq 2 nanfiose

transferase transferase

__---------v---L

SA Gal

(SA-Gall transferases

II

to

1

M a6 \ 64 U - Gn ‘IGn

Gn M3Gn2

I i GlcNAc

-

pathway

I

c

u$-

-GneM&-

-

V

I Gal transferase I Fuc transferase I

SA

transferase

J Complex

TYPO

Gn [VI BI-. trl-. tetra-. penta-antennary structure

(SA-Gal

)O_S

/e’; Gntlll GnC IV1 -4 Gn CII

U a,j Gr$III(F~ ‘/JkGn?dn / U a3

<

Fig. 3. Summary of the biosynthetic processes of N-glycoside type carbohydrate chains. Abbreviations: G, M, GlcNAc (Gn), SA, Gal and Fuc (F) stand for glucose, mannose, N-acctylglucosamine, sialic acid, galactosc and fucose, respectively. A residue appears in parenthesis for one of the two possible structures.

Beta-linked mannose residue is indicated by underlined M. The number of the GlcNAc transferase (I-VI) which produces linkages, appears in the parenthesis.

this step. Most of the transferred

oligosaccharide

in

insects, plants and higher animals had the common structure shown above, but MangGlcNAc, in trypanozoma cruzi cells (Parodi and Quesada-Allue, 1982). An alternate pathway for its synthesis will be

later. At this stage, nearly all glycoproteins should have one kind of carbohydrate chain. The number of N-glycosidic type carbohydrate chains bound per molecule can be controlled by the ammo acid sequence. Experiments using many kinds of peptides as acceptors showed the specificity of the discussed

Carbohydrate chains of glycoproteins enzyme for the Asn-X-Ser(Thr) sequence. However, not all the sequence was glycosylated. For example, ovalbumin having two possible asparagine residues on the molecule had a single carbohydrate chain as well as ovalbumin produced by a heterologus cell (Sheares and Robbins, 1986). Pless and Lennartz (1977) has shown that denatured sulfitolysed ovalbumin is capable of accepting an oligosaccharide at both sites in vitro. Kato et al. (1984b, 1986) report that one of two purified ovalbumin intermediates by Con A-Sepharose chromatography has a highmannose type carbohydrate chain at both sites and behaves as an intermediate during its biosynthesis. From this result, it appears that the binding of a carbohydrate to another site may not be stereostructurally impossible since the highly glycosylated intermediate behaves as a native one, as also do other ovalbumin fractions. Yamashita et al. (1983a) reported that an increase in the number of carbohydrate chains was also possible in y-glutamyl transpeptidase produced by hepatoma cells. Nevertheless, the number of carbohydrate chains can be controlled basically by amino acid sequences under gene control and resultant peptide conformations which favour the formation of p-turn or other turn or loop structures (Beeley, 1977). ~~irnrni~g o~glue~~e and matmose residtie The next step in the biosynthesis of glycoprotein is trimming of three glucose and then four tl-I,2 linked mannose residues from the above oligosaccharide transferred to the protein, accompanied by movement of the glycoprotein from rER to the Golgi apparatus. The participation of two specific glucosidases and two specific mannosidases is known. By a detailed examination of the localization of these enzymes on each membrane component, several components could be distinguished from each other, such as cis-, medial-, trans-Golgi (Farquhar and Palade, 1981; Dunphy and Rothman, 1985). Among these, two glucosidases and an ff-l,Z-mannosidase were found to be located on the rER membrane. The trimming of three glucose and one mannose residues occurred co-translationally on the nascent polypeptide (Atkinson and Lee, 1984) in rER producing Man,GlcNAc, and further trimming with Golgi mannosidase I resulted in the production of Man,GlcNAc,. Pollack and Atkinson (1983) examined statistically 50 glycoproteins and reported a significant relationship to exist between the attached carbohydrate structure and the distance from the carbohydrate binding site to the peptide N-terminal position. According to their report, high mannose type carbohydrate is rarely present at the N-terminal side of a previously glycosylated complex site and there is a very definite dist~bution of a complex type carbohydrate chain within the N-terminal region. They also found that the glycoproteins containing both types of carbohydrate chains on the same polypeptide are often present in membrane glycoproteins. Brown et al. (1986) report that the 5 carbohydrate chains of IgM p-chain are trimmed at different speeds. Finally, there were excreated glycoproteins having sites at which carbohydrate chains were trimmed quickly or slowly depending on whether they were destined to take on the high-

485

mannose or complex type. structure. In this case, the final carbohydrate structures were affected by trimming speed, even though the carbohydrate chains were on the same polypeptide. The preferential release of oligosaccharide from a complex type specified site by endo H treatment indicates site specific difference in enzyme availability in sindbis virion glycoprotein (Hsieh et al., 1983b). The heterogeneity produced at this step is that of high-mannose type carbohydrate chain groups composed of Glc,Man,GlcNAc2 to Man,GlcNAc2. For example, the high-mannose type carbohydrate chain of quail ovalbumin was composed of Man,GlcNAc, and Man,GlcNAc, and that of hen ovalbumin was composed mainly of Ma~GlcNAc~ and Man,GlcNAc, (Mutsaers et al., 1985; van Halbeek et al., 1985). Eggs from star fish surprisingly contained a high amount of the high-mannose type carbohydrate chain possessing 3-O glucose residues (Endo et al., 1987). The presence of positional isomers of mannose in IgM has also been reported (Chapman and Kornfeld, 1979a,b). Unusual Man,GlcNAc, and Man,GlcNAc,, each containing a glucose residue, were observed in the glycoprotein intermediates of thyroid cells and rat liver. They were found to be produced by the transfer of glucose to previously trimmed oligosaccharide (Parodi et al., 1983, 1984). l-Deoxynoji~mycin, an inhibitor of the enzyme catalyzing the trimming of the first glucose, was unexpectedly found incapable of completely inhibiting the processing of the oligosaccharide (Romero and Herscovics, 1986). Recently, a novel enzyme in the Golgi fraction, endo mannosidase, was noted to interact with Glc,Man,GlcNAc, to release the disaccharide, glucosyl mannose (Lubas and Spiro, 1987). This enzyme may possibly exhibit the establishment of a new pathway for the trimming of the above unusual high-mannose type carbohydrate chain. This may possibly be a factor affecting the production of heterogeneous carbohydrate structures. The trimming was accompanied by movement of glycoprotein from rER to Golgi. The mechanism for this movement is not completely understood. However, the rates of movement of certain protein and glycoproteins were compared. Tsukada et al. (1985) report that tl -fetoprotein is secreted more slowly than albumin and its transport from rER to Golgi is affected by insulin treatment. I-Deoxynojirimycin treatment resulted in the accumulation of lysosomal enzyme in rER (Lemansky et al., 1984) and treatment with an ionophore such as monensin which prevents movement of glycoprotein from cis-Golgi to transGolgi altered the carbohydrate chain from the complex to high-mannose type (Ledger et al., 1983). Swaisonin treatment accelerates the secretion of a-fetoprotein but not that of albumin (Yea et at., 1985). Tunicamycin treatment gave various results, depending on the particular glycoprotein examined. The variation in these results is considered due to the different contributions of carbohydrates to the stabilization of protein conformation (Sidman, 1981). Still, the intracellular movement of glycoprotein, which is apparently closely related to carbohydrate structure, is an important factor responsible for the production of heterogeneous carbohydrate chains

486

Hnoo IWASE

(Ronin et al., 1984; Gabel and Bergmann, 1985). It is still not clear how the high-mannose type carbohydrate chain is excreted without further trimming. The pmsence of many glycoproteins having highmannose and complex type carbohydrate chains on the same molecule reflects a termination mechanism by which the high-mannose type carbohydrate is protected from trimming. Mannan binding protein found in hepatic cells may possibly function as such a protector (Mori er al., 1984). On the other hand, Williams and Lennarz (1984) report that two factors, conformation of the substrate protein and specificity of the processing enzymes, apparently combine to produce the high-mannose type chain of ribonuclease B. Modtjication of carbohydrate chains

The next process to be discussed is the transfer of the first N-acetylglucosamine residue to the Man,GlcNAc, accompanied by trimming of the remaining two mannose residues. The inhibition of mannosidase II by swaisonine produces a hybrid type carbohydrate chain (Yeo et al., 1985), thus, indicating this enzyme to be a key factor for producing hybrid type carbohydrate chains. By this sequential enzyme action, no Man,GlcNAc, structure should be produced. The structure Man,GlcNAc, found in ovomucoid and other glycoproteins was explained as arising from an alternate pathway involving the participation of another lipid intermediate, Glc,Man,GlcNAc,-P-P-Dolichol (Kornfeld et al., 1979; Yamashita et al., 1983d; Taniguchi et al., 1985). After trimming of the oligosaccharide transferred to protein portion from the lipid intermediate, the first N-acetylglucosamine was transferred to the Man,GlcNAc* to produce GlcNAc,Man,GlcNAc, with a structure the same as that of the product from Glc,MaqGlcNAc2. N-acetylglucosamine, galactose and sialic acid residues from each nucleotide sugar were then transferred to this GlcNAciMan,GlcNAc,. These enzymes related to modification showed different distributions in the Golgi apparatus (Gabel and Bergmann, 1985; Berger and Hesford, 1985; Dunphy and Rothman, 1985). There are at least 6 kinds of GlcNAc transferases (I-VI) for the transfer of N-acetylglucosamine to various positions on the carbohydrate chain to produce the heterogeneous penta-antennary structures in Fig. 3 (Yamashita et al., 198361;Brockhausen et al., 1987). As reviewed by Schachter et al. (1986), owing to the high acceptor specificity of these transferases, even if two reactions are catalyzed by the same set of enzymes, the products from the reactions will not necessarily be the same. They emphasized the importance of the order in which the action of these enzymes occurs and summarized the results of many reports on the substrate specificity of glycosyltransferases and proposed the relationships, GO-NO GO and NO GO-GO among these enzymes. For example, transfer of the bisect N-acetylglucosamine residue to GlcNAc,Man,GlcNAc, inhibits trimming by mannosidase II (GO-NO GO) and that of first GlcNAc to Man,GlcNAc, is essential for subsequent trimming by mannosidase II (NO GO-GO). He applied this relationship to the carbohydrates of ovalbumin and ovomucoid. According to the proposed mechanism,

minor changes in relative activity in the enzyme set may possibly result in small fluctuations in microheterogeneity as demonstrated by quantitative analysis of individual differences in ovalbumin microheterogeneity (Iwase et al., 1987). Biosynthesis of 0-Glycoside Type Carbohydrate Chain

The biosynthesis of 0-glycosidic type carbohydrate chains, except for the stepwise transfer of mono saccharide from a specific nucleotide sugar, has yet to be clarified in detail (Bayer et al., 1981; Ene and Clamp, 1985; Jentjens and Strous, 1985; Schachter, 1986; Spohn and McCall, 1987). This is due to the difficulty in analyzing carbohydrate chain structures originating from prominent microheterogeneity, as shown in Fig. 2. By histochemical and biochemical analysis, the biosynthesis of carbohydrate chains has been found to occur mainly in the trans Golgi lumen. However, Strous (1979) has reported the transfer of N-acetylgalactosamine to the nascent polypeptide and other studies on the low density lipoprotein receptor show the transfer of the N-acetylgalactosamine residue to precede trimming of the N-glycosidic carbohydrate chain on the same molecule (Cummings et al., 1983). The first GalNAc transferase was purified from two sources, a soluble enzyme from bovine colostrum and membrane bound enzyme from mouse lymphoma cells, and characterized (Elhammer and Kornfeld, 1986). The localization of this enzyme on Golgi apparatus has also been confirmed using wellcharacterized rat liver subcellular fractions (Abeijon and Hirschberg, 1987). Beyer et al. (1981) and also Schachter (1986) conducted a study on enzymes related to mucin biosynthesis. As in the case of the N-glycoside type carbohydrate chain, the relationships, G& NO GO and NO GO-GO, among the enzymes were proposed by Schachter (1986). Since the carbohydrate chain of mucin is constructed by the stepwise addition of sugar, the final structure of the carbohydrate chain should depend on the particular enzyme set that is available. He also reported the different species- and tissue-dependent distributions of these enzymes. The report of Ohara et al. (1984, 1986, 1987) that the carbohydrate composition of rat stomach mucin exhibits strain-, tissue-dependent differences and also changes by fasting is in agreement with these findings. Slomiany observed similar structural differences in the carbohydrate chains of mucin (Slomiany et al., 1984). In regard to the intracellular transportation of mucin, recent experiments show that the minimum time required for the initial glycosylation of the cell surface sialomucin of mammary ascite tumor cells is less than 5 min. But the minimum time required from the time of the synthesis of a core polypeptide to its appearance on the cell surface is 70-80min (Spielman et al., 1987). Using cyclohexamide to block protein synthesis, White and Speake (1980) reported N-glycosylation to be inhibited immediately due to lack of a newly synthesized acceptor, whereas 0 -glycosylation continued for 30 min in lactating rabbit mammary gland. Jentjens et al. (1986) applied antibodies to rat stomach mucin to detect biosynthetic intermediates and found a period of 90 min to be required for the synthesis of mature mucin. They also reported only

Carbohydrate

chains of glycoproteins

a small portion of the pulp-la~lied mucin to be secreted after 3 hr chase. Thus, completion of the carbohydrate chain representing 7040% by weight of mucin should require a longer period of time for intracellular transportation. During this period, the initiation of carbohydrate synthesis by the first transfer of the N-a~etylgalactos~ine residue continues. In the case of mucin, the carbohydrate chains are situated quite close together on the peptide (one carbohydrate chain per 5-6 amino acids on the average), Biosynthesis of the carbohydrate chains of mucin may possibly be more complex than that of the N-glycoside type since microheterogeneity is also produced by disturbance of the carbohydrate chain synthesis due to the steric hindrance of neighboring carbohydrate chains even if the sequential transfer of carbohydrate occurs in a concerted manner. Therefore, the manner in which heterogeneous oligosaccharides are distributed on a peptide warrants investigation. CONCLUDING

REMARKS

In this paper, various factors responsible for the variety and microheterogeneity in the carbohydrate chains of glycoproteins are discussed. Also involved are complex factors such as the presence of sugar specific translocators by which nucleotide sugars are transferred from the cytoplasm to the lumen side (Deutscher and Hirschberg, 1986; Abeijon and Hirschberg, 1987) and the possibility of the restoration of carbohydrate chain structures by membrane recycling. This recycling implies reverse movement of cell surface glycoprotein to the Golgi region (Farquhar and Palade, 1981; Snider and Rogers, 1986; Woods et al., 1986). Glycosylation among many post-translational modifications of protein seems to be best marker for the intracellular movement of protein and the striking variety of carbohydrate structures should prove useful for elucidating the mechanism of this movement. It is clear that carbohydrate chain structures undergo a broad range of changes due to species-, tissue-, cellular-, intracellular localization-, maturation (differentiation) state-, and cell environmentdifferences. Since the presence of many lectins with different sugar specificites in plant seeds and animal cells indicates cells to be capable of distinguishing such micro-structural differences in carbohydrate chains (Lis and Sharon, 1986), reevaluation and examination in great detail of the variety and microheterogeneity should facilitate elucidation of the physiological significance of each carbohydrate structure. Acknowledgements-The author gratefully acknowledges the valuable comments of Dr Yukinobu Kato and Dr Susumu Ohara during the course of this study. The author is also indebted to Professor Kyoko Hotta for her helpful advice. This work was supported in part by grants-in-aid for Special Project Research from the Ministry of Education, Science and Culture, Japan. REFERENCES

Abeijon C. and Hirschberg C. B. (1987) Subcellular site of synthesis of the N-acetylgalactosamine (a 1-O) serine B C. zo,s-B

487

(or th~onine~ linkaae in rat liver. J. biol. Chem. 262, 4153-4159. Akiyama K., Simons E. R., Bernasconi P., Schmid K., van Halbeek H.. Vlieaenthart J. F. G.. Hauot H. and Schwick G. H. (1984) The structure of the carbohydrate units of human. pla&a galactoglycoprotein deter&ined by 500megahertz ‘H-NMR soectroscow. .< J. bioi. Chem. 259, 71%7154. Atkinson P. H. and Lee J. T. (1984) Co-translational excision of a-glucose and a-mannose in nascent vesicular stomatitis virus G protein. J. Cell Bioi. 98, 2245-2249. Beeley J. G. (1977) Peptide chain conformation and the glycosylation of glycoproteins. Biochem. biophys. Res. Common. 76, 1051-1055, Beeley J. G. (1985) Glycoprotein and proteoglycan techniques. In Laboratory Techniques in Biochemistry and Molecular Biology (Edited by Burdon R. H. and van Knippenberg P. H.), Vol. 16.-Elsevier, Amsterdam. Beraer E. G. and Hesford F. J. (19853 Localization of galactosyl- and sialyltransferase by im~unofluore~n~: Evidence for different sites. Proc. nafn. Acad. Sci. U.S.A. 82, 47364739. Berger E. G., Buddecke E., Kamerling J. P., Kobata A., Paulson J. C. and Vliegenthart J. F. G. (1982) Structure, biosynthesis and functions of glycoprotein glycans. ExFerie~t~a 38, 1129-1258. Beyer T. A., Sadler J. E., Rearick J. I., Paulson J. C. and Hill R. L. (1981) Glycosyltransferases and their use in assessing oligosaccharides structure and structurefunction relationships. Adv. Enzymol. 52, 23-175. Bolscher J. G. M., Schallier D. C. C., Smets L. A., van Rooy H., Collard J. G., Bruyneel E. A. and Mareel M. M. K. (1986) Effect of cancer-related and drug-induced alterations in surface carbohydrates on the invasive caoacity of mouse and rat cells. cancer Res. 46, 4080-4086. Bose S., Gurari-Rotman D., Ruegg U. T., Corley L. and Antinsen C. B. (1976) Anoarent diswnsabilitv of the ~rbohydrate moiety of h&an integeron for *antiviral activity. J. &of Chem. 251, 1659-1662. Brockhausen I., Grey A., Carver J., Hindsgaul 0. and Schachter H. (1987) The detection and assay of GlcNActransferases I, II, III, IV, V, and VI in hen oviduct extracts using HPLC. In Proceedings of the 9th ~~ter~a~ionai S.yrn~o~i~rnon G~coconj~gafes (Edited by Montreuil J., Verbert A., Spik G. and Fournet B.). Brown P. H. and Hickman S.-(1986) Oligosaccharidd processina at individual dvcosvlation sites on MOPC 104E immu~oglobulin M. i*biol: Chem. 261, 2.575-2582. Ceccarini c. and Atkinson P. H. (1983) Endoglycosidase H-sensitive ~lvco~otides in eleven different animal cells. Biochem. J.-2i2,‘8&885. Ceccarini C., Lorenzoni P. and Atkinson P. H. (1983) Fractionation of ovalbumin glycopeptide AC-C by highpressure liquid chromatography. Determination of structure by ‘H-NMR spectroscopy. Biochim. biophys. Acta 759, 214-221. Ceccarini C., Lorenzoni P. and Atkinson P. H. (1984) Determination of the structure of ovalbumin glycopeptide AC-B by ‘H nuclear magnetic resonance spectroscopy at 5OOMHz. J. molec. Biol. 176, 161-167. Chapman A. and Komfeld R. (l979a) Structure of the high mannose oligosaccharides of a human IgM myeioma protein: I. The major oligosaccharides of the two high mannose glycopeptides. J. biol. Chem. 254, 816-823. Chapman A. and Komfeld R. (1979b) Structure of the high mannose oligosaccharides of a human InM mveloma protein: II. The minor oligosaccharides of high m&nose glycopeptide 1. J. biof. Chem. 254, 824-828. Cummings R. D., Komfeld S., Schneider W. J., Hobgood K. K., Tolleshaung H., Brown M. S. and Goldstein J. L. (1983) Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J. biol. Chem. 258, 15261-15273.

488

Hlnx,

Cummings R. D., Soderquist A. M. and Carpenter G. (1985) The oligosaccharide moieties of the epidennal growth factor receptor in A-431 cells: Presence of complex-type N-linked chains that contain terminal Nacetylgalactosamine residues. J. biol. Chem. 260, 11944-11952. Deom C. M. and Schulze I. T. (1985) Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties. J. bioi C/tent. 260, 14771-14774. DeRosa P. A. and Lucas J. J. (1982) Estrogen-induced changes in chick oviduct membrane glycoproteins. J. biol. Chem. 257,

1017-1024.

Deutscher S. L. and Hirschberg C. B. (1986) Mechanism of galactosylation in the Golgi apparatus. J. biol. Chem. Ml, 96100. Dunphy E. G. and Rothman J. E. (1985) Compartmental organization of the Go@ stack. Cell 42, 12-21. Edge A. S. B. and Spiro R. G. (1985) Thyroid cell surface glycoproteins: Nature and disposition of carbohydrate units and evaluation of their blood group I activity. J. biol. Chem. MO, 15332-15338. Elhammer A. and Komfeld S. (1986) Purification and characterization of UDP-N-acetylgalactosaminepolypeptide N-acetylgalactosaminyltransferase from bovine colostrum and murine lymphoma BW5147 cells. J. biol. Chem. 261, 52495255. Endo T., Hoshi M., Endo S., Arata Y. and Kobata A. (1987) Structures of the sugar chains of a major glycoprotein present in the egg jelly coat of a starfish, Asterias amurensis. Archs Biochem. Biophys. 252, 105-112. Ene M. D. and Clamp 3. R. (1985) Some aspects of the glycoprotein and glycopolypeptide content of human gastric mucus. Chit. chim. Acta 153, 165-171. Farquhar M. G. and Palade G. (1981) The Golgi apparatus (complex)-(19541981)-from artifact to center stage. J. CeN Biol. 91, 77s-103s. Feizi T. (1985) Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onto-developmental antigens. Xafure 314, 5357. Fukuda M., Carlsson S. R., Klock J. C. and Dell A. (1986) Structures of O-linked oligosaccharides isolated from normal granulocytes. chronic myelogenous leukemia cells, and acute myelogenous leukemia cells. J. biol. Chem. MI, 1279612806. Gabel C. A. and Bergmann J. E. (1985) Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vine evidence of Golgi apparatus compartmentalization J. Cell Biol. 101. 460-469. Goso-Kato K.. Iwase H.,.Ishihara K. and Hotta K. (1986) Analysis of reduced oligosaccharides by combined use of gel chromatography and high-performance liquid chromatography. J. Chromatogr. 380, 374-378. Goverman J. M., Parsons T. F. and Pierce J. G. (1982) Enzymatic deglycosylation of the subunits of chorionic gonadotropin. J. biol. Chem. 257, 15059-15064. Gowda D. C., Bhavanandan V. P. and Davidson E. A. (1986) Structures of O-linked oligosaccharides present in the oroteoglycans secreted by human mammary epithelial cells~ J. bike Chem. Ml, 49354939. Grabel L. B.. Rosen S. D. and Martin G. R. (1979) Terato-carcinoma stem cells have a cell surface carbohydrate-binding component implicated in cell-cell adhesion. Cell 17, 477484. Green E. D., Boime 1. and Baenriger J. U. (1986) Biosynthesis of sulfated asparagine-linked oligosaccharides on bovine lutrooin. J. biol. Chem. Ml. 16305-16316. Harford J. and Ashwell G. (1985) Assessment of receptor recycling in mammalian hepatocytes: Prespectives based on current techniques. Meth. Enzymol. 109, 232-246. Hart G. W. (1982) The role of asparagine-linked oligo-

IWWE

saccharides in cellular recognition by thymic lymphocytes. J. biol. Chem. 257, 151-158. Hase S.. Ibuki T. and Ikenaka T. (1984) Reexamination of the pyridylamination used for fluorescence labeling of oligosaccharides and its application to glycoproteins. J. Biochem. 95, 197-203.

Hayes G. R. and Lucas J. J. (1983) Stimulation of lipidlinked oligosaccharide assembly during oviduct differentiation. J. biol. Chem. 258, 15095-15100. Holt G. D. and Hart G. W. (1986) The suboellular distribution of terminal N-acetylglucosamine moieties: Localization of a novel protein-saccharide linkage, O-linked GlcNAc. J. biol. Chem. Ml, 8049-8057. Hsieh P.. Rosner M. R. and Robbins P. W. (1983a) Host-dependent variation of asparagine-linked oligosaccharides at individual glycosylation sites of sindbis virus glycoproteins. J. biol. Chem. 258, 2548-2554. Hsieh P., Rosner M. R. and Robbins P. W. (1983b) Selective cleavage by endo-/?-N-acetylglucosaminidase H at individual glycosylation sites of sindbis virion envelope glycoproteins. J. biol. Chem. 258, 255S2561. Huang C. -C.. Mayer H. E. Jr and Montgomery R. (1970) Microheterogeneity and paucidispersity-of glycoproteins. I. Carbohydrate of chicken ovalbumin. Carbohvdr. Res. 13, 127-337. lshii 1.. Iwase H., Hamazaki H. and Hotta K. (1987) Comparative study of soeciflc a-1.2~alucosidase activitv toward glucosyl g&ctosyl hydroxylyiine in various and ma1 species. Comp. Biochem. Physiol. &3B, 313316. Iwase H. and Hotta K. (1977) Ovotransferrin subfractionation dependent upon carbohydrate chain differences. J. biol. Chem. 252, 5437-5443.

Iwase H., Kato Y. and Hotta K. (1981) Ovalbumin subfractionation and individual difference in ovalbumin microheterogeneity. J. biol. Chem. 256, 5638-5642. Iwase H.. Kato Y. and Hotta K. (1983) Comoarative studv of ovalbumins from various avian’specie’s by Con A; Sepharose chromatography. Comp. Biochem. Physiol. 76B. 345-348.

Iwase H., Kato Y. and Hotta K. (1984) Comparative study of the carbohydrate chain of ovalbumin from various avian species by high pressure liquid chromatography. Comp. Biochem. Physiol. 778, 743-747. lwase H., Kato Y. and Hotta K. (1987) Quantitative HPLC analysis of individual difference in glycoprotein microheterogeneity and estimation of galactosyltransferase activity in ML,o. In Proceedings of the 9th International Symposium on Glycoconjugares (Edited by Montreuil J., Verbert A., Spik G. and Foumet B.). Jentjens T. and Strous G. J. (1985) Quantitative aspects of mucus glycoprotein biosynthesis in rat gastric mucosa. Biochem. J. 223, 227-232.

Jentjens T., van de Kamp A., Spee-Brand R. and Strous G. J. (1986) Biosynthesis, processing and secretion of mucus glycoprotein in the rat stomach. Biochim. biophys. Acra 887,

133-141.

Kalyan N. K.. Lippes H. A. and Bahl 0. P. (1982) Role of carbohydrate in human chorionic gonadotropin. J. biol. Cherk

257,

12624-12631.

-

_

Kato Y.. lwase H. and Hotta K. (1984al Prenaration of eight &albumin subfractions by combined &in affinity chromatography. AMI~I. Biochem. 138, 437441. Kato Y., twase H. and Hotta K. (1984b) Purification and characterization of two biosynthetic intermediates of ovalbumin. J. Biochem. 95, 455-463. Kato Y., Iwase H. and Hotta K. (1986) Characterization of a highly glycosylated biosynthetic intermediate of ovalbumin. Archs Biochem. Biophys. 244, 408-412. Kessler M. J., Mise T., Ghai R. D. and Bahl 0. P. (1979) Structure and location of the 0-glycosidic carbohydrate units of human chorionic gonadotropin. J. biol. Chem. 254, 7909-7914.

Kobata A. (1979) Use of endo- and exoglycosidases

for

Carbohydrate

chains of glycoproteins

structural studies of glycoconjugates. Analyt. Biochem. loo, l-14. Komfeld R. and Komfeld S. (1985) Assembly of asparagine-linked oligosaccharides. A. Reu. Biochem. 54, 631664. Komfeld S., Gregory W. and Chapman A. (1979) Class E Thy- 1 negative mouse lymphoma cells utilize an alternate pathway of oligosaccharide processing to synthesize complex-type oligosaccharides. J. biol. Chem. 254, 11649-l 1654. Kukuruzinska M. A. and Robbins P. W. (1987) Protein glycosylation in yeast: Transcript heterogeneity of the ALG7 gene. Proc. natn. Acad. Sci. U.S.A. 84,2145-2149. Ledger P. W., Nishimoto S. K., Hayashi S. and Tanzer M. L. (1983) Abnormal glycosylation of human fibronectin secreted in the presence of monensin. J. biol. Chem. 258, 547-554.

Lemansky P., Gieselmann V., Hasilik A. and von Figura K. (1984) Cathepsin D and b-hexosaminidase synthesized in the presence of I-deoxynojirimycin accumulate in the endopktsmic reticulum. J.- bi&. &em. U9, 10129-10135. Lis H. and Sharon N. (1986) Application of lectins. In The Lectins: Properties, Functions, and Applications in Biology and Medicine (Edited by Liener I. E., Sharon N. and Goldstein I. J.).,. __ DD. 294-357. Academic Press. New York.

Longmore G. D. and Schachter H. (1982) Productidentification and substrate-specificity studies of the GDP+fucose:2-acetamido-2-deoxy-/I-n-ghtcoside (fucAsn-linked GlcNAc) 6-a+fucosyltransferase in a Golgirich fraction from porcine liver. Carbohydr. Res. 100, 365-392. Lubas W. A. and Spiro R. G. (1987) Golgi endo-aD-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J. biol. Chem. 262, 3775-3781.

Mierendorf R. C. Jr, Cardelli J. A. and Dimond R. L. (1985) Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium disco&ton. J. Cell Biol. 108, 1777-1787. Mikuni-Takagaki Y. and Hotta K. (1979) Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa. Biochim. biophys. Acta 584, 288-297.

Mizuochi T. and Kobata A. (1980) Different asparaginelinked sugar chains on the two polypeptide chains of human chorionic gonadotropin. Biochem. biophys. Res. Commun. 97, 772-778.

Mizuochi T., Nishimura R., Derappe C., Tan&hi T., Hamamoto T., Mochizuki M. and Kobata A. (1983) Structures of the asparagine-linked sugar chains of human chorionic gonadotropin produced in choriocarcinoma. J. biol. Chem. 258, 1412614129. Mizuochi T., Nishimura R., Taniguchi T., Utsunomiya T., Mochizuki M., Derappe C. and Kobata A. (1985) Comparison of carbohydrate structure between human chorionic gonadotropin present in urine of patients with trophoblastic diseases and healthy individuals. Jpn J. Cancer Res. 76, 752-759.

Mori K., Kawasaki T. and Yamashina I. (1984) Subcellular distribution of the mannan-binding protein and its endogenous inhibitors in rat liver. Archs Biochem. Biophys. 232, 223-233.

Mutsaers J. H. G. M., van Halbeek H., Vliegenthart J. F. G.. Iwase H.. Kato Y. and Hotta K. (1985) Structurai analysis of dansyl_ -. &co-asparagines from quail ovalbumin. B&him. biophys. Acta 843, i23-127. _ Neufelt E. F. and Ashwell G. (1980) Carbohydrate recoanition systems for receptor-mediated pinocytosis. In Biochemistry of Glycoproteins and Proteoglycans (Edited by Lennarz W. J.), pp. 241-266. Plenum Press, New York. Nilsson B., De Luca S., Lohmander S. and Hascall V. C. (1982) Structures of N-linked and O-linked oligosaccha-

489

rides on proteoglycan monomer isolated from the swarm rat chondrosarcoma. J. biol. Chem. U7, 10920-10927. Nomoto H. and Inoue Y. (1983) A novel glycoasparagine isolated from an ovalbumin glycopeptide fraction (GPIV). Eur. J. Biochem. 135, 243-250. Ohara S., Kakei M., Ishihara K., Katsuyama T. and Hotta K. (1984) Effects of fasting on mucus glycoprotein in rat stomach. Comp. Biochem. Physiol. 79B, 325-329. Ohara S., Ishihara K. and Hotta K. (1986) Comparative study on mucus glycoproteins in rat stomach and duodenum. Comp. Biochem. Physiol. 83B, 273-275. Ohara S., Ishihara K. and Hotta K. (1987) Regional differences in rat gastric mucus glycoprotein structures. In Proceedings of the 9th International Symposium on Glycoconjugates (Edited by Montreuil J., Verbert A., Spik G. and Foumet B.). Parekh R. B., Dwek R. A., Sutton B. J., Femandes D. L., Leung A., Stanworth D., Rademacher T. W., Mizuochi T., Taniguchi T., Matsuta K., Takeuchi F., Nagano Y., Miyamoto T. and Kobata A. (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316, 452457.

Parodi A. J. and Quesada-Allue L. A. (1982) Protein glycosylation in Trypanosoma cruzi: 1. Characterization of dolichol-bound monosaccharides and oligosaccharides synthesized “in viva”. J. biol. Chem. 257, 7637-7640. Parodi A. J., Mendelzon D. H. and Lederkremer G. Z. (1983) Transient glucosylation of protein-bound MaqGlcNAc,, Man,GlcNAc,, and Man,GlcNAc, in calf thyroid cells. J. biol. Chem. 258, 8260-8265. Parodi A. J., Mendelzon D. H., Lederkremer G. Z. and Martin-Barrientos J. (1984) Evidence that transient glucosylation of protein-linked MaqGlcNAc,, Man,GlcNAc,, and Man,GlcNAc, occurs in rat liver and pha&olus vulgaris cells. 2. biol. dhem. 259, 63516357. Parsons T. F. and Pierce J. G. (1980) Oliaosaccharide moieties of glycoprotein hormones: ’ BovLe lutropin resists enzymatic deglycosylation because of terminal O-sulfated N-acetyl-hexosamines. Proc. natn. Acad. Sci. U.S.A. 77, 70897093.

Pless D. D. and Lennarz W. J. (1977) Enzymatic conversion of proteins to glycoproteins. Proc. natn. Acad. Sci. U.S.A. 74, 134-138.

Plummer T. H. Jr, Elder J. H., Alexander S., Phelan A. W. and Tarentino A. L. (1984) Demonstration of peptide: N-glycosidase F activity in endo+-N-acetylglucosaminidase F preparations. J. biol. Chem. 259, 10700-10704. Podolsky D. K. (1985a) Oligosaccharide structure of isolated human colonic mucin species. J. biof. Chem. 260, 1551&15515. Podolsky D. K. (1985b) Oligosaccharide structures of human colonic mucin. J. biol. Chem. 268, 82628271. Podolsky D. K. and Isselbacher K. J. (1983) Composition of human colonic mucin. J. chit. Invest. 72, 142-153. Pohlmann R., Waheed A., Hasilik A. and von Figura K. (1982) Synthesis of phosphorylated recognition marker in lysosomal enzymes is located in the cis part of Golgi apparatus. J. biol. Chem. 257, 5323-5325. Pollack L. and Atkinson P. H. (1983) Correlation of glycosylation forms with position in amino acid sequence. J. Cell Biol. 97, 293-300.

Reggio H. A. and Palade G. E. (1978) Sulfated compounds in the zymogen granules of the guinea pig pancreas. J. Cell Biol. 77, 288-314.

Romero P. A. and Herscovics A. (1986) Transfer of non glucosylated oligosaccharide from lipid to protein in a mammalian cell. J. biol. Chem 261. 1593615940. Ronin C., Stannard B. !I., Rosenbloom I. L., Magner J. A. and Weintraub B. D. (1984) Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulation hormone subunits: Comparison to nonsecretory cell glycoproteins. Biochemistry 23, 45034510.

490

HIT00

Rovis L., Anderson B., Kabat E. A., Gruezo F. and Liao J. (1973) Structures of oligosaccharides produced by base-borohydride degradation of human ovarian cyst blood group H, I.eb and Lea active glycoproteins. Biochemistry 12, 5340-5354.

Schachter H. (1986) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Biochem. Cell. Biol. 64, 163-18 1. Schauer R. (1982) Chemistry, Metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem. 40, 131-232.

Schindler M., Hogan M., Miller R. and DeGaetano D. (1987) A nuclear specific glycoprotein representative of a unique pattern of glycosylation. J. biol. Chem. 262, 1254-1260. Sharon N. (1975) Introduction. In Complex Carbohydrate (Edited by Sharon N.), pp. l-32. Addison-Wesley, Mass. Sheares B. T. and Robbins P. W. (1986) Glycosylation of ovalbumin in a heterologous cell: Analysis of oligosaccharide chains of the cloned glycoprotein in mouse L cells. Proc. natn. Acad. Sci. U.S.A. 83, 1993-1997. Sidman C. (1981) Differing requirements for glycosylation in the secretion of related glycoproteins is determined neither by the producing cell nor by the relative number of oligosaccharide units. J. biol. Chem. 256, 9374-9376. Slomianv A.. Zdebska E. and Slomianv B. L. (1984) Structures bf the neutral oligosaccharides isolated from Aactive human gastric mucin. J. biol. Chem. 2S9, 14743-14749. Smets L. A. and van Beek W. P. (1984) Carbohydrates of the tumor cell surface. Biochim. biophys. Acta 738, 237-249.

Snider M. D. and Rogers 0. C. (1986) Membrane tragic in animal cells: Cellular glycoproteins return to the site of Golgi mannosidase I. J. Cell Biol. 103, 265-275. Spielman J., Rockley N. L. and Carraway K. L. (1987) Temporal aspects of 0-glycosylation and cell surface expression of ascites sialoglycoprotein-I, the major cell surface sialomucin of 13762 mammary ascites tumor cells. J. biol. Chem. 262, 269-275.

Spiro R. G. (1967) The structure of the disaccharide unit of the renal glomerular basement membrane. J. biol. Chem. 242,48134823. Spohn M. and McCall I. (1987) Biosynthesis of proteins by human gastric mucosa in vitro. Biochem. J. 242,339-346. Strous G. J. A. M. (1979) Initial glycosylation of proteins with acetylgalactosaminylserine linkages. Proc. natn. Acad. Sci. U.S.A. 76, 2694-2698.

Struck D. K. and Lennartz W. J. (1980) The function of saccharide-lipids in synthesis of glycoproteins. In The Biochemistry

of Glycoproteins

and Proteoglycans

(Edited by Lennartz W. J.), pp. 35-83. Plenum Press, New York. Swiedler S. J., Freed J. H., Tarentino A. L., Plummer T. H. Jr and Hart G. W. (1985) Oligosaccharide microheterogeneity of the murine major histocompatibility antigens: Reproducible site-specific patterns of sialylation and branching in asparagine linked oligosaccharides. J. biol. Chem 260, 40464054.

Tai T., Yamashita K., Ogata-Arakawa M., Koide N., Muramatsu T., Iwashita S., Inoue Y. and Kobata A. (1975) Structural studies of two ovalbumin glycopeptides in relation to the endo-b-N-acetylglucosaminidase specificity. J. biol. Chem. 250, 8569-8575. Tai T., Yamashita K., Ito S. and Kobata A. (1977a) Structures of the carbohydrate moiety of ovalbumin glycopeptide III and the difference in specificity of endob-N-acetylglucosaminidases C,, and H. J. biol. Chem. 252, 6687-6694.

Tai T., Yamashita K. and Kobata A. (1977b) The substrate specificities of endo-/l-N-acetylglucosaminidase C,, and H. Biochem. biophys. Res. Commun. 78, 434-441. Takahashi N., Ishihara H., Tejima S., Oike Y., Kimata K.,

IWASE

Shinomura T. and Suzuki S. (1985) The core molecule from type H proteoglycan. B&he&. J. 229, 561-571. Takahashi N.. I&ii I.. Ishihara H.. Mori M.. Teiima S.. Jefferis R., .Endo S.’ and Arata Y. (1987) Com&&ve structural study of the iv-linked oligosaccharides of human normal and pathological immunoglobulin G. Biochemistry 26, 1137-l 144. Takahashi T., Schmidt P. G. and Tang J. (1984) Novel carbohydrate structures of cathepsin B from porcine spleen. J. biol. Chem. 259, 6059-6062. Takasaki S., Yamashita K., Suzuki K., Iwanaga S. and Kobata A. (1979) The sugar chains of cold-insoluble globulin. J. biol. Chem. 254, 8548-8553. Takasaki S., Mizuochi T. and Kobata A. (1982) Hydrazinolysis of asparagine linked sugar chains to produce free oligosaccharides. Meth. Ensymol. 83, 263-268. Takata K., Yamaxaki-Yamamoto K., Ishii I. and Takahashi N. (1984) Glycoproteins responsive to the neuralinducing effect of concanavalin A in cynops presumptive ectoderm. Cell. D@rentiation 14, 25-31. Takeda T., Sugiura Y., Ogihara Y. and Shibata S. (1982) Synthesis of N-(L-glutam-5-oyl)-u-o-glucopyranosylamine and 0 -a-o-glucopyranosyl-( I-+6)-0-/I-o-glucopyranosyl-(l-+6)-N-(L-glutam-5-oyl)-a-o-glucopyranosylamine. Carbohydr. Res. 105, 271-275. Taniguchi T., Mizuochi T., Banno Y., Nozawa Y. and Kobata A. (1985) Carbohydrates of lysosomal enxyrnes secreted by tetrahymena pyriformis. J. biol. Chem. 260, 13941-13946.

Tsukada Y., Hibi N. and Ohkawa K. (1985) Hormonal regulation during secretion of a-fetoprotein in hepatoma cells grown in synthetic medium. J. biol. Chem. 260, 16316-16320. Tsurui M., Harada Y., Ohara S. and Hotta K. (1986) Gastric mucus glycoprotein in different rat strains. Comp. Biochem. Physiol. MC, 359-361.

van Halbeek H., Vliegenthart J. F. G., Iwase H., Li S.-C. and Li Y.-T. (1985) ‘H-NMR Spectroscopic characterization of dansyl glyco-asparagines derived from hen egg white glycoproteins. Glycoconjugate J. 2, 235-253. van Kuik J. A., van Halbeek H., Kamerling J. P. and Vliegenthart J. F. G. (1985) Primary structure of the low-molecular-weight carbohydrate chains of helix pomatia a-hemocyanin. J. biol. Chem. 260, 1398413988. Warren L. and Buck C. A. (1980) The membrane glycoproteins of the malignant cell. Clin. Biochem. 13,191-197. Warren L., Buck C. A. and Tuszynski G. P. (1978) Glycopeptide changes and malignant transformation: a possible role for carbohydrate in malignant behavior. Biochim. biophys. Acta 516, 97-127. White D. A. and Speake B. K. (1980) The effect of cycloheximide on the glycosylation of lactating-rabbit mammary glycoproteins. Biochem. J. 192, 297-301. Wieland F., Paul G. and Sumper M. (1985) Halobacterial flagellins are sulfated glycoproteins. J. biol. Chem. 260, 15180-15185. Williams D. B. and Lennan W. J. (1984) Control of asparagine-linked oligosaccharide chain processing: Studies on bovine pancreatic ribonuclease B. J. biol. Chem. 259, 5105-5114. Woods J. W., Doriaux M. and Farquhar M. G. (1986) Transferrin receptors recycle to cis and middle as well as trans Golgi cistemae in Ig-secreting myeloma cells. J. Cell Biol. 103, 277-286.

Yamashita K., Tachibana Y. and Kobata A. (1978) The structures of the galactose-containing sugar chains of ovalbumin. J. biol.-Chem. 253, 3862-5869: Yamashita K.. Hitoi A.. Taniauchi N.. Yokosawa N.. Tsukada Y. and Kobata A. (19i83a)Comparative study of the sugar chains of y-glutamyltranspeptidase purified from rat liver and rat AH-66 hepatoma cells. Cancer Res. 43, 5059-5063. Yamashita K., Hitoi A., Tsuchida Y., Nishi S. and Kobata

Carbohydrate

chains of glycoproteins

A. (1983b) Sugar chain of a-fetoprotein produced in human yolk sac tumor. Cancer Res. 43, 46914695. Yamashita K., Ueda I. and Kobata A. (1983~) Sulfated asparagine-linked sugar chains of hen egg albumin. J. biol. Chem. 258, 1414414147. Yamashita K., Kamerling J. P. and Kobata A. (1983d) Structural studies of the sugar chains of hen ovomucoid. J. biol. Chem. 258, 3099-3106. Yamashita K., Tachibana Y., Hitoi A. and Kobata A. (1984) Sialic acid-containing sugar chains of hen ovalbumin and ovomucoid. Curbohydr. Res. 130, 271-288. Yamashita K., Hitoi A., Tateishi N., Higashi T., Sakamoto Y. and Kobata A. (1985) The structures of the carbohydrate moieties of mouse kidney y-glutamyltranspeptidase: Occurrence of X-antigenic determinants and

491

bisecting N-acetylglucosamine residues. Archs Biochem. Biophys. 240, 573-582. Yamashita K., Hitoi A., Matsuda Y., Miura T., Katsunuma N. and Kobata A. (1986) Structures of sugar chains of human kidney y-glutamyltranspeptidase. J. Biochem. 99, 55-62. Yeo T.-K., Yeo K.-T., Parent J. B. and Olden K. (1985) Swaisonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells. J. biol. Chem. 260, 2565-2569. Yoshima H., Matsumoto A., Mizuochi T., Kawasaki T. and Kobata A. (1981) Comparative study of the carbohydrate moieties of rat and human plasma a,-acid glycoproteins. J. biol. Chem. 256, 8476-8484.