Erythrocyte Glycoproteins

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME

11

Erythrocyte Glycoproteins MZCHAEL J . A. T A N N E R Department of Biochemistry University of Bristol Bristol, United Kingdom

I. Introduction . . . . . . . . . . . . . . . . . . . 11. The Origin and Turnover of the Erythrocyte . . . . . . . . . 111. The Glycoproteins o f t h e Erythrocyte Membrane . . . . . . . . A. Periodate-Stainable Glycoproteins . . . . . . . . . . . B. Non-Periodate-Stainable Glycoproteins . . . . . . . . . IV. Organization of the Glycoproteins in the Erythrocyte Membrane . . . V. Structure of the Glycoproteins . . . . . . . . . . . . . A. The Major Human Sialoglycoprotein (Glycophorin A) . . . . . B. The Minor Periodate-Stainable Proteins of the Human Erythrocyte . C. Polypeptide3 . . . . . . . . . . . . . . . . . VI. Functions of Glycoproteins . . . . . . . . . . . . . . A. Polypeptide3 . . . . . . . . . . . . . . . . . B. Periodate-Stainable Glycoproteins . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

1.

279 280 281 282 284 285 288 288 295 297 304 304 306 316

INTRODUCTION

Erythrocyte membrane proteins have been the subject of several recent reviews (Zwaal et al., 1973; Juliano, 1973; Steck, 1974; Marchesi et al., 1976) which reflect the considerable progress made in recent years in characterizing these proteins and understanding their organization within the membrane. Only membrane glycoproteins are discussed in this chapter, and an attempt is made to explore the functional significance of these components with an emphasis on functional attributes which might depend on the carbohydrates they contain. It is sometimes felt that the erythrocyte and its membrane are too atypical and specialized to be useful as a representative model for study of the involvement of plasma membranes in many of the complex phenomena which occur in the mammalian organism. It is true that the erythrocyte is highly specialized for the transport of oxygen Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153311-5

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MICHAEL J. A. TANNER

and bicarbonate ions and lacks intracellular organelles and some features, such as hormone sensitivity, found in many other cells. This degree of specialization is reflected in the properties of the erythrocyte membrane. However, it should be recognized that, in adult mammals, most tissue cells are equally highly differentiated and specialized and that their plasma membranes often have equally unique morphological and biochemical characteristics which give rise to the particular features of different tissues. The technical advantages which result from the ability to obtain large quantities of homogeneous plasma membranes from the erythrocyte are well known. In addition, the mode of generation of erythrocytes provides several useful features in studying the involvement of plasma membrane components in cellular differentiation and cellular interactions. The erythropoietic system is the largest “organ” in the adult animal and is dedicated to the production of a single cell type. The high rates of turnover of this regenerating system (particularly in the anemic condition) make it possihle to isolate erythrocytes in the intermediate stages of maturation in quantities which allow biochemical studies of the plasma membranes. These intermediate stages are well defined with regard to both morphology and histochemistry. Finally, erythroid cells undergo an unusual, defined transition from a tissue-bound phase in the bone marrow to a circulating phase in the bloodstream. The cells in the latter phase undergo few interactions with other cells, and this stage provides a particularly useful experimental system for study of the function of cell surface glycoproteins. II. THE ORIGIN AND TURNOVER OF THE ERYTHROCYTE

Before considering the functional role of the glycoproteins present in the erythrocyte membrane, it is useful to discuss-the life history of the erythrocyte, since in addition to having certain functions during the circulating phase of the life of the erythrocyte, these membrane components may also be involved during its generative and degradative stages. A few pertinent features of the events involved in the maturation and turnover of the erythrocyte are summarized in the following discussion. An excellent and most detailed review of this subject is available (Wickramasinghe, 1975). The early nucleated stages of erythroid cells occur in an organized system in the sinusoids of the bone marrow. Erythroid cells are derived from erythropoietin-sensitive stem cells which are not well characterized. The earliest recognizable erythroid cell is a large, dividing, nucleated cell designated the pronormoblast. The successively more mature stages of this cell,

ERYTHROCYTE GLYCOPROTEINS

28 1

the basophilic normoblast, polychromatic normoblast, and orthochromatic normoblast, have distinctive histochemical characteristics. At each of these nucleated stages except the last, the cell undergoes mitosis, but the final nucleated stage, the orthochromatic normoblast, is not capable of mitosis and DNA synthesis. Thus a single pronormoblast yields 16 orthochromatic normoblasts. During this progression the cells decrease in diameter from approximately 18 p m to 10p m and increase in hemoglobin content. A large proportion of the hemoglobin of the mature erythrocyte has been synthesized by the time the orthochromatic normoblast stage of the cell is reached. The final stage of erythroid cell maturation in the bone marrow is characterized by loss of the nucleus from the orthochromatic normoblast to yield the reticulocyte. The nucleus is extruded from this cell in a form which is surrounded by a layer of plasma membrane containing about 5% of the cytoplasm of the cell. Some investigators suggest that this process of nuclear extrusion is concurrent with, and indeed allows, release of the reticulocyte from the sinusoids of the bone marrow into the circulation (Tavassoli and Crosby, 1973),while others believe that the most immature form of the reticulocyte resides in the bone marrow for a short time before its release into the circulation. It takes about 7 days for the human pronormoblast to become a reticulocyte, and about onehalf this time is spent in cell division. The final maturation phase, reticulocyte to erythrocyte, results in the loss of protein-synthesizing ability and intracellular membranous organelles from the cell and is accompanied by a further decrease in the size of the cell from about 9 pm to 7 pm. The mature cell has an average circulating life span of 115 days in the human and is probably destroyed by the reticuloendothelial organs (liver, spleen, and bone marrow). There is some evidence which suggests that carbohydrates on the erythrocyte surface are involved in the removal of erythrocytes from the circulation. Neuraminidase-treated erythrocytes have a much reduced survival time in the rabbit and rat, and it has been suggested that this decreased survival time is due to the exposure of galactose residues subterminal to sialic acid residues (Gattegno et al., 1974; Durocher et al., 1975). This system appears to be analagous to that found for clearance from the circulation of many plasma glycoproteins (Morel1 et al., 1968, 1971). 111.

THE GLYCOPROTEINS OF THE ERYTHROCYTE MEMBRANE

The membrane of the human erythrocyte, the most intensively studied red cell membrane, contains 8-10% carbohydrate, and a large pro-

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MICHAEL J. A. TANNER

portion of this carbohydrate is bound to membrane proteins (Winzler, 1969, 1971). A. Periodate-Stainable Glycoproteins

1. HUMANERYTHROCYTE MEMBRANE When the proteins of human erythrocyte membranes are separated

by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Fairbanks et al., 1971), only one protein (polypeptide 3)which can be visualized with coomassie blue is a glycoprotein (see Section V,C). If the gel is treated with the periodic acid-Schiff (PAS) stain, one major

and several minor bands are observed. The distribution and resolution of the minor PAS staining depends markedly on the conditions of gel electrophoresis. The clearest resolution of the minor bands is obtained with the discontinuous SDS gel electrophoresis system of Laemmeli (1970), see Dahr et al., (1975b). Figure 1 shows the nomenclature used here to designate these periodate staining bands. These bands are not all unique glycoproteins. The relative proportions of PAS-1 and PAS-2 bands obtained depend on the conditions used to dissolve the glycoprotein preparations in the detdrgent for SDS gel electrophoresis (Marton and Garvin, 1973; Tuech and Morrison, 1974). If dissolution is carried out in the presence of phosphate buffers, the PAS-1 form predominates, while heating and the use of tris-containing buffers results in the formation of more of the PAS-2 form. These two bands are interconvertible forms of the major sialoglycoprotein (glycophorin A, Tomita and Marchesi, 1975). Studies with an erythrocyte variant lacking the major sialoglycoprotein confirm this conclusion (Tanner and Anstee, 1976b). It appears that the PAS-1 and PAS-2 bands have a dimer-monomer relationship, and that the site of association of the dimer is the hydrophobic domain in the polypeptide chain of the major sialoglycoprotein (Furthmayr and Marchesi, 1976). The PAS4 band also appears to be a complex, in this case a heterocomplex, consisting of the major sialoglycoprotein and PAS-3. Membranes from En (a-) cells, which lack the major sialoglycoprotein, do not yield the PAS-4 band (Tanner and Anstee, 1976b), and S - s human erythrocytes, which have an altered PAS-3 glycoprotein, also show alterations in the PAS4 band (Dahr et al., 1975c; Tanner et al., 1977). Presently available evidence suggests that polypeptide 3, the major sialoglycoprotein, PAS-S', and PAS-3 are all distinct entities. It

283

ERYTHROCYTE GLYCOPROTEINS

PAS-].

0.l!

0.1c

O.D.

,i” POLYPEPTIDE

0.05

0

1

1

3

2

CM.

AIDNC

4

5

6

GEL

FIG. 1. Periodate-stainable glycoproteins of the human erythrocyte membrane. Human erythrocyte ghosts were separated by SDS gel electrophoresis on a gel containing 10%acrylamide with an overlay containing 4% acrylamide, using the buffer system described by Fairbanks et al. (1971). The gels were stained with the PAS stain and scanned at 560 nm. 0. D., Optical density.

is not known whether the remaining minor components are unique glycoprotein species or complexes of other glycoproteins. The apparent MWs obtained for these glycoproteins by the SDS gel electrophoresis method are unreliable. The major bands all yield apparent MWs which change with the acrylamide concentration used in the gel electrophoresis system (Bretscher, 1971b). Thus the monomer of the major sialoglycoprotein, the PAS-2 band, yields apparent MWs of 53,000 and 40,000 from 5% and 8% acrylamide gels, respectively (Tanner and Boxer, 1972), but the amino acid sequence and carbohydrate content of the protein suggest that it has a true MW of 31,000 (Tomita and Marchesi, 1975). These anomalies have been attributed to different extents of binding of detergent to the oligosaccharide-rich and the hydrophobic regions of these substantially gl ycosylated membrane proteins, compared with the binding of detergent to the polypeptide chain of the nonmembrane proteins used to calibrate MWs in the gel electrophoresis system (Grefrath and Reynolds, 1974; Tanford and Reynolds, 1976).

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MICHAEL J. A. TANNER

2. ERYTHROCYTE MEMBRANESOF OTHER SPECIES

The periodate-stainable glycoproteins of erythrocytes from several other species have also been studied. The major periodate-stainable components of different species (presumably analogous to the human major sialoglycoprotein) show considerable variation in their apparent MWs on SDS gel electrophoresis (Lenard, 1970; Kobylka et al., 1972; Capaldi, 1973; Carraway et al., 1975; Ralston, 1975). It is not clear how much this variability in electrophoretic mobility reflects changes in carbohydrate content rather than MW. The rabbit erythrocyte is unusual in that it lacks any major periodate-stainable glycoprotein equivalent to the human sialoglycoprotein (Lodish and Small, 1975; Light and Tanner, 1977). Polypeptide 3 shows relatively little variation among species, although it has a somewhat higher MW in the camel erythrocyte (Ralston, 1975). 0. Non-Periodate-btoinobleGlycoproteins

Staining of glycoproteins with the PAS stain depends almost entirely on the presence of sialic acids in these glycoproteins (Dahr et aZ., 1974, 1976).The staining method used by Liao et aZ. (1973), utilizing mild periodate oxidation followed by reduction with radioactive borohydride, is also dependent on sialic acid and gives patterns which closely resemble those obtained with the PAS stain. This restricted specificity of the PAS staining technique has become increasingly apparent on examining the erythrocyte membrane with probes having a specificity for sugars other than sialic acid. When erythrocyte membranes are examined for galactose and N-acetylgalactosamine-containing oligosaccharides by oxidation with galactose oxidase followed by reduction with radioactive borohydride, a heterogeneous mixture of components is labeled in the region between the PAS-1 and PAS-2 bands (Steck, 1972a; Gahmberg and Hakomori, 1973; Steck and Dawson, 1974; Gahmberg, 1976). Many of these bands do not correspond to any of the bands detected with the protein or the PAS stain. Similar results are obtained when galactose and N-acetylgalactosaminespecific lectins (such as those from Ricinis communis and Phaseolus vulgaris) are used as probes (Tanner and Anstee, 1976a). It is generally assumed that these are glycoproteins, but some of them may be complex glycolipids containing large oligosaccharides of the type mentioned by Gardas and Koscielak (1974a,b). No estimates of the abundance of these components in the membrane are available, but they are generally considered minor components.

ERYTHROCYTE GLYCOPROTEINS

IV.

285

ORGANIZATION OF THE GLYCOPROTEINS IN THE ERYTHROCYTE MEMBRANE

Several reviews are available which cover the general subject of the organization of proteins in the erythrocyte membrane (Steck, 1974; Zwaal et al., 1973; Marchesi et al., 1976). As a group, the glycoproteins of the erythrocyte membrane all appear to b e integral membrane proteins and are hydrophobically bound to the lipid bilayer of the membrane. Thus these proteins remain associated with the lipid of the membrane when simple extraction procedures are used on erythrocyte ghosts (see for example, Hoogeveen et al., 1970; Fairbanks et al., 1971; Tanner and Boxer, 1972; Steck and Yu, 1973) but can be solubilized from the membrane by detergents (Yu et al., 1973), chaotropic agents (Winzler, 1969; Marchesi and Andrews, 1971), and certain organic solvents (Blumenfeld, 1968; Hamaguchi and Cleve, 1972; Anstee and Tanner, 1974a). A wide variety of impermeable protein-modifying probes has been used to define the location of these glycoproteins with respect to the lipid bilayer of the membrane (see Hubbard and Cohn, 1976, for a detailed review). The experimental approach generally follows that used by Bretscher (19714, Bender et al. (1971),and Phillips and Morrison (1971). Intact erythrocytes and various membrane preparations which are permeable or impermeable to the probe are exposed to the reagent. The proteins accessible to the probe in each of these preparations are then identified. Given a knowledge of the permeability of the membrane to the probe, and of the orientation or sidedness of the membrane preparations, it is possible to infer the location of the membrane proteins relative to the permeability barrier of the membrane. An interesting variation on this procedure is to compare the labeling patterns obtained b y using chemically analogous permeant and impermeant labeling agents on the intact erythrocyte (Whitely and Berg,

1974).

Several major assumptions are implicitly made when using these types of chemical approaches to locate proteins. It is assumed that (1) the modification procedure does not itself alter the structure of the membrane, (2) the proteins have the same orientation in each of the membrane preparations being compared, and (3) the membrane is impermeable to the probe. In practice, it has been difficult to show rigorously that all these criteria have been met in many of the experimental systems used. Nevertheless, the use of a wide variety of protein-specific probes and of a carbohydrate-specific probe leads to the conclusion that all the erythrocyte glycoproteins are accessible at the

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MICHAEL J. A. TANNER

external surface of the cell. The reagents used in this way include several low-MW compounds (Bender et al., 1971; Bretscher, 1971a,b,c; Whiteley and Berg, 1974; Staros and Richards, 1974; Cabantchik et al., 1975), proteases (Bender et al., 1971; Steck, 1972a), and lactoperoxidase (Phillips and Morrison, 1971). While all these probes are protein-specific, galactose oxidase has been used as a carbohydratespecific probe (Steck, 1972a; Gahmberg and Hakomori, 1973; Steck and Dawson, 1974). Ambiguities of interpretation which arise when these methods are used, in the case of proteins that span the membrane and are exposed at both membrane surfaces, have been overcome by showing that unique regions of polypeptide chain are accessible at each membrane surface (Bretscher, 1971b,c; Boxer et al., 1974), and by using dual isotope labeling of different membrane preparations (Reichstein and Blostein, 1973, 1975; Mueller and Morrison, 1974; Shin and Carraway, 1974). Polypeptide 3 and the major sialoglycoprotein have both been shown to penetrate right through the membrane and span it in this way. There is evidence that the minor glycoproteins (PAS-2' and PAS-3) also span the membrane (Mueller and Morrison, 1974), but Marchesi et al. (1976) suggest that these minor glycoproteins are bound to the lipid bilayer but do not extend beyond the cytoplasmic surface of the membrane. The use of the carbohydrate-specific probe galactose oxidase in a similar experimental approach (Steck, 1972a; Gahmberg and Hakomori, 1973; Steck and Dawson, 1974) has led to the important conclusion that all the carbohydrate of the erythrocyte is present at the external surface of the cell, confirming the conclusions of morphological studies (Winzler, 1971). It has proved difficult to define unambiguously the organization and associations of the membrane glycoproteins in the plane of the membrane. The associations of the proteins in the membrane have been examined using a variety of chemical cross-linking procedures. These experiments suggest that polypeptide 3 may exist in a dimeric form in the membrane (Steck, 1972b; Wang and Richards, 1974), a possibility strengthened by the observation that the isolated protein assumes a dimeric form in the presence of a nonionic detergent (Yu and Steck, 197513). The remaining glycoproteins are markedly unreactive to cross-linking agents, but in this case the large amounts of carbohydrate associated with these proteins may impose steric restrictions on their ability to become cross-linked. The persistence of a dimeric form of the major sialoglycoprotein in the presence of SDS has led to the suggestion that this protein may also be in a dimeric state in the mem-

ERYTHROCYTE GLYCOPROTEINS

287

brane (Marchesi et al., 1976). The presence of an aggregate of the major sialoglycoprotein and PAS-3 (the PAS-4 band) under similar conditions could lead one to suppose that a complex of the major sialoglycoprotein and PAS-3 also exists in the membrane. However, there is no other evidence either supporting or refuting these two suggestions at the present time. Our knowledge of the longer-range organization of these glycoproteins comes mainly from morphological studies utilizing the electron microscope. These have shown that a variety of erythrocyte antigens and receptors is present in a random array at the surface of the erythrocyte (Pinto da Silva et al., 1971; Tillack et al., 1972; Nicolson, 1973; Pinto da Silva and Nicolson, 1974). Freeze-cleavage of erythrocyte membranes exposes particles (intramembranal particles) which are probably proteinaceous and are embedded in the lipid bilayer (Pinto da Silva and Branton, 1970; Pinto da Silva and Nicolson, 1974). A large number of particles is exposed on the cleavage faces retaining the cytoplasmic half of the bimolecular lipid leaflet (the A or P F face), and these appear to be relatively homogeneous in size. The cleavage faces retaining the extracellular half of the bimolecular lipid leaflet (the B or E F face) are less densely populated with particles, and these vary a great deal in size. Most of the studies reviewed here relate to the particles on the A fracture face. The abundance, properties, and location of the major sialoglycoprotein and polypeptide 3 suggest that these glycoproteins might be components of the intramembranal particles. Studies of the distribution on the membrane of various receptors known to be associated with the major sialoglycoprotein and polypeptide 3 have shown them to be correlated with the intramembranal particles and have led to the suggestion that both these glycoproteins are components of the intramembranal particles (Pinto da Silva and Nicolson, 1974; Nicolson, 1976). However, there are some ambiguities in this interpretation, particularly with regard to the major sialoglycoprotein, since non of these receptors or markers have been shown to be exclusively associated with the major sialoglycoprotein. The markers used for the major sialoglycoprotein include blood-group-A antigenic activity (Pinto da Silva et al., 1971), phytohemagglutinin receptors (Tillack et al., 1972), and anionic sites (Pinto da Silva et al., 1973). However, recent evidence suggests that ABO(H) blood group antigens are not present on the major sialoglycoprotein (Hamaguchi and Cleve, 1972; Brennessel and Goldstein, 1974; Anstee and Tanner, 1974a,b, 1975), while phytohemagglutinin binds to polypeptide 3 and other membrane components in addition to the major sialoglycoprotein (Tanner and Anstee, 1976a).

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MICHAEL J. A. TANNER

Sialoglycolipids and the minor periodate-stainable glycoproteins also provide anionic sites which are available at the cell surface (Swee'ley and Dawson, 1969; Marchesi et al., 1976). Bachi et al. (1977) showed that there are no significant differences in the distribution and density of intramembranal particles in membranes from normal erythrocytes and those from erythrocytes which lack the major sialoglycoprotein. A proportion of polypeptide-3 molecules appears to carry concanavalin-A (Con-A) receptors (Findlay, 1974; Tanner and Anstee, 1976a; Jenkins and Tanner, 1977b), and the results of experiments in which the binding of concavavalin A-ferritin conjugates was studied suggest that polypeptide 3 is a component of the intramembranal particles (Pinto da Silva and Nicolson, 1974). However, there have recently been suggestions that polypeptide 3 may not be entirely located in the intramembranal particles (Howe & Bachi, 1973; Bachi & Schnebli, 1975). Many problems remain in transferring the information from freezecleavage replicas to a detailed model of the architecture of the erythrocyte membrane. It is not known whether the intramembranal particles represent intact proteins or only the hydrophobic regions of proteins (Bretscher and Raff, 1975). Nor is it possible to assess the extent of heterogeneity of the particles in the A fracture face and their physical and chemical relationship to the particles in the B fracture face. V.

STRUCTURE

OF THE GLYCOPROTEINS

A. The Major Human Sialoglycoprotein (Glycophorin A)

1. ISOLATIONAND STRUCTUREOF THE HUMAN SIALOGLYCOPROTEIN

This glycoprotein carries the human erythrocyte blood group-M and -N antigens and, because of its immunochemical interest, has been the subject of study for many years. The earliest preparations were obtained by methods based on phenol extraction (Klenk and Uhlenbruck, 1960; Kathan et al., 1961; Springer et al., 1966; Winzler, 1969). More recently developed isolation methods include the extraction of erythrocyte ghosts with an organic solvent such as pyridine (Blumenfeld, 1968; Zvilichovsky et al., 1971),chloroform-methanol (Hamaguchi and Cleve, 1972), or butanol (Anstee and Tanner, 1974a). In each case, the major sialoglycoprotein is extracted in a water-soluble form,

ERYTHROCYTE G LYCOPROTEI NS

289

while the remaining erythrocyte ghost protein is precipitated, and the bulk of the lipid is extracted into the organic phase or remains associated with the insoluble protein. The sialoglycoprotein preparations used by Marchesi and co-workers for structural studies were prepared by a procedure based on the use of lithium diiodosalicylate (Marchesi and Andrews, 1971). The minor PAS-stainable components behave in much the same way as the sialoglycoprotein during all these different isolation procedures and this, together with the tendency of the sialoglycoprotein to form aggregates during preparation, has complicated its purification. Nevertheless, by applying suitable further purification steps after the extraction, homogeneous preparations of the sialoglycoprotein can be obtained (Zvilichovsky et al., 1971; Tanner and Boxer, 1972; Hamaguchi and Cleve, 1972; Anstee and Tanner, 1974a; Furthmayr et al., 1975). The purified human major sialoglycoprotein contains about 60% carbohydrate by weight (Winzler, 1969) and is particularly rich in sialic acid (approximately 25% by weight). This protein is the major carrier of cell surface carbohydrate in the erythrocyte. The subunit MW of the protein has proved difficult to ascertain by physical methods, because of the anomalous detergent-binding effects and the presence of different aggregation states. [Marchesi et al. (1976) discuss this aspect more fully.] But the MW derived from amino acid sequence data and the carbohydrate content is 31,000 (Tomita and Marchesi, 1975). The amphiphilic nature of the major sialoglycoprotein was first recognized by Morawiecki (1964), and this property was confirmed and its significance elaborated on by Winzler (1969). Treatment of watersoluble preparations of the sialoglycoprotein with trypsin yields an insoluble peptide which contains little or no carbohydrate and a soluble sialic acid-rich glycopeptide. A similar sialic acid-rich glycopeptide was obtained by treatment of intact erythrocytes with trypsin. Winzler (1969) suggested that oligosaccharide chains were present only in the extracellular region of the protein (the N-terminal segment), while the insoluble C-terminal region was hydrophobic and interacted with the lipid bilayer of the membrane. This orientation of the major sialoglycoprotein (Fig. 2 ) has been confirmed using various labeling techniques (Bretscher, 1971b; Segrest et al., 1973), and it has also been shown that the C-terminus of the protein is exposed at the cytoplasmic face of the membrane (Bretscher, 1975; Mueller and Morrison, 1974). The complete amino acid sequence of the major sialoglycoprotein has been recently determined by Marchesi and co-workers (Tomita and Marchesi, 1975).Figure 3 shows this sequence and also shows the

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MICHAEL J. A. TANNER

MEMBRANE

I

FIG.2. Orientation of the major sialoglycoprotein in the human erythrocyte membrane. The continuous line represents the polypeptide backbone and the solid circles attached to this line indicate the oligosaccharide chains.

distribution of oligosaccharides on the amino acid chain. The amphiphilic nature of the protein is evident, the N- and C-terminal portions of the molecule being separated by a segment of about 20 nonpolar amino acids which probably penetrates the lipid bilayer of the membrane (Segrest et al., 1972). The adjacent regions on the N- and C-terminal sides of this segment contain clusters of charged residues which may interact with hydrophilic components at the surfaces of the membrane (Marchesi et al., 1976). The asymmetry in the distribution of oligosaccharides on this molecule is striking. The protein contains 16 oligosaccharide units. Fifteen are relatively small sialic acid-rich units which are O-glycosidically linked to serine or threonine residues (sialotetrasaccharides). The remaining carbohydrate is present in a larger and more complex mannose- and N-acetylglucosamine-rich oligosaccharide which is N-glycosidically linked to the protein via an asparagine residue. The polypeptide is particularly densely substituted with sialotetrasaccharides at the extreme N-terminus of the sialoglycoprotein where each of the residues from positions 10 to 15 carries one of these oligosaccharide units. The majority of the O-glycosidically linked oligosaccharides appear to be tetrasaccharides of the type shown in Fig. 4A (Thomas and Winder, 1969), but incomplete forms of this oligosaccharide are probably also present. It is possible that some of the oligosaccharides may have the related structure shown in Fig. 4B (Springer and Desai, 1975). The structure of the large N-glycosidically linked oligosaccharide remains uncertain. Two structures have been proposed for

291

ERYTHROCYTE GLYCOPROTEINS

I LEU

Ism 1

10

20

30

40

50

60

90

100

110

I20

I30

FIG.3. Amino acid sequence and carbohydrate distribution on the major human sialoglycoprotein. The solid circles indicate the residues in the hydrophobic segment which probably passes through the lipid bilayer. The attached diamond shapes indicate the positions of the O-glycosidically linked oligosaccharides, while the position of the N-glycosidically linked oligosaccharide is shown by the hexagonal shape. (From Marchesi e t al., 1976, reproduced with permission.)

A -

B -

NANA %GAL NANA

&GAL

3@ @

GALNA~

% SENTHR)

FIG.4. Proposed structures of O-glycosidically linked oligossaccharides. (A) Structure suggested by Thomas and Winzler (1969).(B)Structure proposed by Springer and Desai (1975).

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MICHAEL J. A. TANNER NAM

&a

GAL

2-6

GAL

&B

J, B 1-3(4)

CLCNAc

1-3(4)

GLCNAc

ASN

FIG.5. Structure proposed by Kornfeld and Kornfeld (1970)for the N-glycosidically

linked oligosaccharide of the major sialoglycoprotein.

this unit, one (Fig. 5) by Kornfeld and Kornfeld (1970) and a second (Fig. 6) b y Thomas and Winzler (1971).Marchesi et al. (1976) suggest that this oligosaccharide is larger and contains more N-acetylglucosamine than either of these proposed structures. Interpretation of the results of the earlier workers is complicated by the possibility that their sialoglycoprotein preparations were contaminated by other glycoprotein components. Marchesi et al. (1976)also suggest that the microheterogeneity of their sialoglycoprotein preparations could give rise to misleading results with regard to its composition.

2. ANTIGENS AND RECEPTORS PRESENTON THE

HUMANERYTHROCYTE SIALOGLYCOPROTEIN

The blood group-M and -N antigens of the human erythrocyte are located on the major sialoglycoprotein. The isolated protein has potent M and N antigenic activity, and antigenically active glycopeptides can be obtained after proteolytic digestion (Lisowska and Jeanloz, 1973). However, when the 0-glycosidically linked oligosaccharides are released from the polypeptide by alkali treatment, neither the released oligosaccharides nor the remaining polypeptide have antigen activity (Lisowska, 1969).Treatment of the glycoprotein with amino group-blocking reagents (Lisowska and Morawiecki, 1967; Lisowska and Duk, 1975)results in the loss of antiFUC JCY

GAL

.le

1-2(6)

GLCNAc

-&

GAL

NANA

GLCNAc

GAL

&B

J B

(MAN)3&

4

4

3.8

GLCNAc

GLCNAC

.l+B

ASN

r l G . 6. Structure proposed by Thomas and Winzler (1971) for the N-glycosidically linked oligosaccharide of the major sialoglycoprotein.

ERYTHROCYTE GLYCOPROTEINS

293

genic activity, suggesting that residues of the polypeptide chain as well as carbohydrate moieties are part of the antigenic determinant. Studies using periodate oxidation and a variety of M- and N-specific lectins suggest that one of the sialotetrasaccharides of the type shown in Fig. 4A is part of the M antigenic determinant (Dahr et al., 1975b). However, Springer and co-workers (Springer et aZ., 1966; Springer and Desai, 1974) suggest that the oligosaccharide shown in Fig. 4B is part of the antigenic determinant. The structural basis for the difference between the allelic M and N antigens has been the subject of some controversy. No difference has been found in the composition of the 0- and N-glycosidically linked oligosaccharides from blood-group-M and -N cells (Thomas and Winzler, 1969, 1971; Adamany and Kathan, 1969; Kornfeld and Kornfeld, 1970),but it seems unlikely that the presence or absence of a single residue in a large number of oligosaccharide chains would have been detected by these workers. It has been suggested (Springer and Desai, 1974)that the N antigen contains an oligosaccharide of the type shown in Fig. 4B, but deficient in one terminal sialic acid, and that this structure is the substrate for a sialyltransferase dependent on the blood group-M gene. However, other studies do not support this view and suggest that the difference between the M and N antigens does not depend on sialic acid but reflects changes in the amino acid sequence of the polypeptide chain of the major sialoglycoprotein (Dahr et al., 1975b; Anstee et aZ., 1977).Recent studies have shown that the two amino acids found by Tomita and Marchesi (1975) at residues 1 and 5 in the amino acid sequence of the major sialoglycoprotein reflect differences in the amino acid sequence of the blood group M and N-active sialoglycoproteins in their pooled preparations (see Fig. 3 ) . The M glycoprotein contains serine and glycine at residues 1 and 5 while the N glycoprotein contains leucine and glutamic acid at these positions (Wasniouska et ul., 1977; Dahr et al., 1977).It seems likely that these amino acids are involved, at least in part, in distinguishing the M and N antigenic determinant. It has also been shown that in certain situations blood-group-M and -N antigenic activity can be found on membrane components other than the major sialoglycoprotein. Thus the traces of N activity found on homozygous M erythrocytes appears to be present on the PAS-3 glycoprotein (Hamaguchi and Cleve, 1972; Dahr et ul., 1975b), and some individuals whose erythrocytes lack the major sialoglycoprotein carry M antigen on membrane components other than the major sialoglycoprotein (Anstee et al., 1977). Several reports have associated blood-group-ABH and -I activity

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MICHAEL J. A. TANNER

with the major sialoglycoprotein (Springer et al., 1966; Marchesi and Andrews, 1971; Fukuda and Osawa, 1973; Liao et al., 1973).These antigenic activities appear to be due to the presence of tightly bound contaminating glycolipids in these sialoglycoprotein preparations (Hamaguchi and Cleve, 1972; Brennessel and Goldstein, 1974; Anstee and Tanner, 1974a,b, 1975; Gardas and Koscielak, 1974a,b). The major sialoglycoprotein contains receptors for influenza virus (Winzler, 1971; Jacksonet al., 1973). In addition, since the protein carries both 0- and N-glycosidically linked oligosaccharides, receptors for a wide variety of lectins are present on the molecule. The oligosaccharide and carbohydrate residues which are binding sites for some of these lectins are summarized in Table I.

3. SIALOGLYCOPROTEINS OF OTHERSPECIES In the past, interest has been almost entirely directed toward the human sialoglycoprotein. However, the number of studies being done on analogous glycoproteins from other species is increasing (see Section III,A,2). Bovine glycoprotein (Capaldi, 1973; Emerson and Kornfeld, TABLE I

LECTINRECEPTORS ON THE MAJOR SIALOGLYCOPROTEIN~

SOME OLIGOSACCHARIDE

0-Glycosidically linked

N-glycosidically linked

Maclura aurantiaca (GalNAc)* Bauhinia purpurea (GalNAc)* Arachis hypogea (Cal)c

Ricinis communis (Gal)" Robinia pseudoaccncio (Gal)" Triticurn uulgaris (WGA, GlcNAcY Phaseolus uulgaris (Gal + Man, complexp Lens culinaris (GlcNAc + Man, complex)h

The sugar residues in parentheses are probably the major determinants for the binding of each lectin to these particular oligosaccharides. These assignments are mainly based on inhibition studies with monosaccharides. GalNAc, N-acetylgalactosamine; Gal, galactose; WGA, wheat germ agglutinin; GlcNAc,N-acetylglucosamine, Man, mannose. * Dahr et al. (1975a). Reacts after removal of terminal sialic acid (Lotan et al., 1975; Terao et al., 1975). Fukuda and Osawa (1973). Leseney et al. (1972). Jackson et al. (1973). Komfeld and Kornfeld (1970). Kornfeld et al. (1971). f

ERYTHROCYTE GLYCOPROTEINS

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1976) contains 80% carbohydrate, and this is mainly present in the form of O-glycosidically linked oligosaccharides. However, the majority of these oligosaccharides differ from human sialotetrasaccharides in that they contain N-acetylglucosamine and have a lower content of sialic acid. Glockner et al. (1976) examined the carbohydrate content of the glycoproteins from a variety of species and found sialotetrasaccharides similar to human ones in many species. However, bovine and porcine glycoproteins contained much lower amounts of these components than the glycoproteins of the other animals examined. 6. The Minor Periodate-Stainable Proteins of the Human Erythrocyte

1. PAS-2’ This glycoprotein component was not detected until recently because it has a mobility on SDS gel electrophoresis similar to that of the PAS-2 form of the major sialoglycoprotein. Mueller and Morrison (1974) recognized this component because, on lactoperoxidase radioiodination, it labeled more strongly from the cytoplasmic side of the erythrocyte membrane than from the extracellular side. The converse is true for the PAS-1 and PAS-2 forms of the major sialoglycoprotein. The uniqueness of this component has been confirmed by studies on the glycoproteins of cells which lack the major sialoglycoprotein (Tanner and Anstee, 1976b) and cells which are defective in the PAS-3 glycoprotein (Dahr et al., 1975c; Tanner et al., 1977). PAS-2’ takes up about 10% as much of the periodate stain as the major sialoglycoprotein. The fact that it (PAS-2’) contains receptors for lectins from Maclura aurantiaca and Arachis hypogea (Tanner and Anstee, 1976a; Anstee et al., 1977),as well as sialic acid as demonstrated by PAS staining, suggests that O-glycosidically linked sialic acid-rich oligosaccharides, similar to the sialotetrasaccharides found in the major sialoglycoprotein, are also present in this molecule. In addition, it binds P . vulgaris phytohemagglutinin and probably also contains Nglycosidically linked carbohydrates. The studies of Dahr et al. (1975d) on erythrocytes from individuals with the Tn syndrome are consistent with this conclusion. No data are available on the abundance or composition of PASS’, but it is usually regarded as a minor component of the erythrocyte membrane. Although Fujita and Cleve (1975)isolated a sialic acid-rich glycoprotein with an electrophoretic mobility similar to that of PAS-2’, this preparation did not contain the N-acetylga-

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MICHAEL J. A. TANNER

lactosamine which might be expected from the reactivity of PAS-2' with lectins. 2. PAS3 There are conflicting reports about the disposition of this protein in the erythrocyte membrane. It is certainly accessible at the extracellular surface of the membrane, and Mueller and Morrison (1974) also reported that it could be labeled by the lactoperoxidase iodination technique from the cytoplasmic side of the membrane and thus spans the membrane. However, Marchesi et al. (1976) suggest that the protein does not project through the cytoplasmic surface of the membrane. The protein stains with about 10% of the intensity of the major sialoglycoprotein on PAS staining and has receptors for M . aurantiaca and A. hypogea lectins as well as wheat germ agglutinin, but no receptors for P. vulgaris and €3. communis lectins (Robinson et al., 1975; Tanner and Anstee, 1976a; Anstee et al., 1977). Thus 0-glycosidically linked sialic acid-rich sialotetrasaccharides of the type found in the major sialoglycoprotein are probably present, while N-glycosidically linked oligosaccharides, if present, may be different from those found on the major sialoglycoprotein. Furthmayr et al. (1975) have published analytical data on a crude preparation of PAS-3. The carbohydrate content of their preparation is consistent with the above interpretation, and their results suggest that PAS-3 represents 5-10% by weight of the total periodate-stainable glycoproteins. Fujita and Cleve (1975) also isolated a glycoprotein similar in electrophoretic mobility to PAS-3. However, their glycoprotein lacks N-acetylgalactosamine, while the data of Furthmayr et al. (1975) and the lectin-binding properties of PAS-3 suggest that this sugar is present. A similar inconsistency has been noted in the carbohydrate content found by Fujita and Cleve (1975) for a glycoprotein similar to PAS-2' (see Section V,B,l). It has been suggested that PAS-3 has a carbohydrate composition similar to that of the major sialoglycoprotein, but that the polypeptide portions differ. Marchesi et al. (1976) also report that PAS-3 is similar to the sialoglycoprotein in having a glycosylated N-terminal region and a hydrophobic polypeptide segment, but it lacks the hydrophilic intracellular C-terminal segment found in the major sialoglycoprotein. In addition to lectin receptors, PAS-3 probably carries the erythrocyte Ss antigens (Hamaguchi and Cleve, 1972; Fujita and Cleve, 1975; Anstee and Tanner, 1975). Cells lacking these antigens have an altered PAS-3 (Dahr et al., 1975c; Tanner et al., 1977). There is some evidence that the small amounts of N antigen found in homozygous M

ERYTHROCYTE GLYCOPROTEINS

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erythrocytes are also present on this component (Dahr et al., 1975b; Anstee and Tanner, 1975). However, it seems likely that the ABH and I antigenic activities found associated with this glycoprotein (Hamaguchi and Cleve, 1972; Fujita and Cleve, 1975)are the result of tightly bound contaminating glycolipids (Gardas and Koscielak, 1974a,b; Anstee and Tanner, 1975). In the intact erythrocyte PAS-3 resists trypsin treatment but can be degraded with chymotrypsin (Dahr et al., 197513). The erythrocyte Duffy antigens (Fy” and Fyb) have similar properties (Miller et al., 1975), and the F y a antigen has chromatographic properties similar to those of PAS-3, although so far there is no evidence to show that it is the same as PAS-3 (Anstee and Tanner, 1975).This is of interest, since it has been shown that erythrocytes lacking Duffy antigens are resistant to infection by Plasmodium uivax, a human malaria (Miller et al., 1975). C. Polypeptide 3

Polypeptide 3 is the most abundant of the erythrocyte membrane proteins, comprising about lo6copies per erythrocyte (25% of the coomassie blue-stainable proteins, Steck, 1974). This glycoprotein migrates as a characteristically diffuse band on SDS gel electrophoresis, the front edge of the band having an apparent MW of 86,000-90,000, and it stains very weakly with the PAS stain (Fig. 1).Much evidence suggests that this protein spans the erythrocyte membrane (see Section IV) and that it is an integral membrane protein. Unlike the major sialoglycoprotein, polypeptide 3 has not yet been obtained in a water-soluble form in the absence of detergents, and this has hindered purification of the protein. However, several methods for purifying polypeptide 3 have recently been reported. Most of these methods utilize various types of preliminary extraction procedures performed on erythrocyte ghosts to remove the bulk of the extrinsic membrane proteins, followed by solubilization in a detergent such as Triton X-100 (Yu et al., 1973; Yu and Steck, 1975a; Furthmayr et d.,1976) or SDS (Tanner and Boxer, 1972; Ho and Guidotti, 1975; Tanner et al., 1976). Although apparently native protein is isolated by solubilization in nonionic detergents (Yu and Steck, 1975a; Drickamer, 1976), solubilization in SDS has proven convenient for obtaining large amounts of protein for structural studies (Tanner et al., 1976; Jenkins and Tanner, 1977b). Affinity chromatography methods have also been used to purify polypeptide 3 (Findlay, 1974; Adair and Kornfeld, 1974).

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The isolated protein is rich in leucine and glutamic acid and/or glutamine and, although the amino acid composition is more hydrophobic than that of typical water-soluble proteins, it is less hydrophobic than that of some other integral membrane proteins (Yu and Steck, 1975a). Unlike the major sialoglycoprotein, which has no sulfhydryl groups, polypeptide 3 contains about six half-cystine residues (Jenkins and Tanner, 1977b), at least one of which is in the reduced form (Steck, 197213).There is no evidence for the presence of any form of covalent interchain cross-links in the protein (Bailey et al., 1976; Jenkins and Tanner, 1977b). The protein is a glycoprotein (Ho and Guidotti, 1975; Yu and Steck, 1975a; Tanner et al., 1976; Gahmberg et al., 1976) with a distribution of sugars quite different from that found in the major sialoglycoprotein (Table 11). The small amounts ofN-acetylgalactosamine and sialic acid suggest that few, if any, sialic acid-rich 0-glycosidically linked oligosaccharides are present and that most of the carbohydrate is linked N-glycosidically to the protein via asparagine residues. The protein contains 15% carbohydrate, and it has been estimated that it carries about 18% of the surface carbohydrate of the erythrocyte (Tanner et al., 1976; Jenkins and Tanner, 1977b). Thus it contributes about one-third as much carbohydrate as the major sialoglycoprotein to the total cell surface carbohydrate. The protein contains receptors for Con A and lectins from P . uulgaris and R. communis (Findlay, 1974; Adair and Kornfeld, 1974; Tanner and Anstee, 1976a) but does not have receptors for any of the lecTABLE I1

CARBOHYDRATE CONTENTSOF POLYPEPTIDE 3 MAJOR SIALOGLYCOPROTEIN

AND THE

Mole carbohydrate per mole protein Component

Major sialoglycoprotein"

Polypeptide 3b

Fucose Mannose Galactose N-Acetylglucosamine N-Acetylgalactosamine Sialic acid

2 5 18 9 15 18

4 7

a

Based on data from Furthmayr et al. (1975).

* Tanner et al. (1976).

2A 25 4 5

ERYTHROCYTE GLYCOPROTEINS

299

tins which bind to-O-glycosidically linked oligosaccharides such as those from M . uurantiaca and A. hypogea (Tanner and Anstee, 1976a; Anstee et al., 1977).This confirms the absence of the O-glycosidically linked type of oligosaccharide from the molecule. It can also be labeled using the galactose oxidase technique (Gahmberg and Hakomori, 1973; Steck and Dawson, 1974; Yu and Steck, 1975a). The apparent MW of polypeptide 3 obtained from SDS gel electrophoresis may be unreliable, not only because it is glycosylated but also because it is an intrinsic membrane protein. It has been shown that both the heavily glycosylated segments and the hydrophobic segments of membrane proteins behave anomalously in binding SDS (Grefrath and Reynolds, 1974; Tanford and Reynolds, 1976)compared with the water-soluble proteins used as MW standards in this system. The broadness of the polypeptide3 band further complicates MW estimation and has led to suggestions that polypeptide 3 may be heterogeneous, with regard to either its polypeptide chains or its carbohydrates. It is true that several other erythrocyte membrane proteins migrate in the same region as polypeptide 3. These include acetylcholinesterase (Bellhorn et a1., 1970),the phosphorylated intermediate of Na+,K+-activatedATPase (Avruch and Fairbanks, 1972), and a membrane-penetrating protein of MW 90,000 (Reichstein and Blostein, 1975; Jenkins and Tanner, 1977a). However, the amounts of these components are sufficiently small that it is unlikely that they represent significant contamination of polypeptide3 preparations in a protein chemical sense. The behavior of the protein on proteolysis (Bender et ul., 1971), oxidative dimerization (Steck, 1972b), and cleavage with a variety of reagents (Steck et al., 1976; Drickamer, 1976; Jenkins and Tanner, 1977a) suggests that the polypeptide is homogeneous, or else that the polypeptide3 band contains a family of very closely related proteins. If any such heterogeneity exists, studies on fragments of the protein suggest that it is likely to be present in the C-terminal region of the molecule (Jenkins and Tanner, 197713). An increasing amount of evidence suggests that there is heterogeneity in the oligosaccharides present on polypeptide 3 and that the diffuseness of the band obtained on SDS gel electrophoresis is associated with this heterogeneity. The leading and trailing edges of the band differ in reactivity toward galactose oxidase (Yu and Steck, 1975a) and ability to bind Con A (Tanner and Anstee, 1976a). Proteolytic removal of the region of the protein most heavily glycosylated yields a large protein fragment which migrates as a much sharper band on SDS gel electrophoresis. A C-terminal fragment of polypeptide 3 which retains this glycosylated region yields a more diffuse

300

MICHAEL J. A. TANNER

band than intact polypeptide 3, and the components in this fragment preparation differ in their ability to bind Con A and lectins from P . uulgaris and R. communis (Jenkins and Tanner, 1977b).This carbohydrate heterogeneity appears to be restricted to the C-terminal region of the protein. Polypeptide 3 in the intact erythrocyte is much more resistant to proteolysis than the major sialoglycoprotein, Thus it is not cleaved by trypsin (Triplett and Carraway, 1972; Boxer et al., 1974), while treatment of erythrocytes with a variety of other proteases (Bender et al., 1971; Bretscher, 1971a; Boxer et al., 1974; Jenkins and Tanner, 1975) yields a fragment which remains reactive toward extracellularly applied reagents. The resistance of both the extracellular and intracellular regions of the protein to proteolysis depends on the ionic strength of the medium. Both regions are much more resistant to trypsin at isoosmotic ionic strengths than at low ionic strengths. This suggests that the structure of the protein depends on the ionic strength of the medium (Jenkins and Tanner, 1977a). Changes in the accessibility of tyrosine residues of the protein to lactoperoxidase radioiodination support this conclusion. The protein probably has a tighter structure at ionic strengths similar to those found in vivo than under the lowionic-strength conditions usually used for preparing and manipulating erythrocyte ghosts. It is possible that some of the functional properties associated with polypeptide 3 depend on the ionic strength of the medium in which they are studied. Since polypeptide 3 is involved in erythrocyte anion transport, the way in which the polypeptide is folded in the membrane has been of interest. Some intrinsic membrane proteins such as the major erythrocyte sialoglycoprotein and microsomal cytochrome b, appear to contain a singIe hydrophobic polypeptide segment which interacts with the lipid bilayer of the membrane (Segrest et al., 1972; Tomita and Marchesi, 1975; Spatz and Strittmatter, 1971; Visser et al., 1975). However, there is no reason to suppose that the purpose of the hydrophobic segment in this class of protein is other than to lock the protein into the membrane. Membrane proteins involved in transport are likely to have a much greater proportion of their polypeptide within the lipid bilayer, since the structures which allow penetration of the substrates through the membrane must be constructed from these intramembranous regions. Thus a protein of this class, the light-dependent proton pump bacteriorhodopsin, contains seven helical rods which traverse the membrane in a porelike structure (Henderson and Unwin, 1975).This protein has a MW of 25,000 (Bridgen and Walker, 1976) and is almost entirely embedded in the membrane. An a-helical

ERMHROCME GLYCOPROTEINS

301

rod needs to be only 20 to 30 amino acids long to traverse a membrane (Tanford and Reynolds, 1976). Polypeptide 3 contains about 750 amino acid residues, and a number of traverses of the polypeptide chain similar to that found in bacteriorhodopsin would not be unexpected and would require only about one-fourth of the polypeptide to be embedded in the membrane. We studied the folding of polypeptide 3 in the erythrocyte membrane by utilizing the tyrosine sites on the protein which can be radioiodinated with lactoperoxidase as markers for the location of particular portions of the polypeptide with respect to the membrane. These tyrosine-containing sites were distinguished by their mobility on peptide maps after thermolysin digestion of the radioiodinated protein. Unique sets of these sites are present in the extracellular and intracellular regions of the protein (Boxer et al., 1974). Two distinct labeled peptides were obtained from polypeptide 3 on proteolysis of erythrocyte membranes, and each of these peptides contained a set of extracellular labeled sites linked to a different and nonoverlapping set of intracellular labeled sites (Jenkins and Tanner, 1975, 1977a).Thus the polypeptide traverses the membrane at least twice. It should be noted that this type of technique can only yield a minimum figure for the number of traverses of the polypeptide chain, for two main reasons. The method used for determining the sidedness of individual peptide segments may not detect all the different polypeptide sections exposed at the membrane surface, either because of limited accessibility of potential labeled sites or because appropriate residues of the reactive amino acid are not randomly distributed throughout the length of the polypeptide. Similarly, it is not possible to establish, a priori, that peptides containing only a single traverse are obtainable from all the traverses under any given set of cleavage conditions. Thus in practice it is very difficult to show that a transmembrane segment of a protein does not cross the membrane more than once without the aid of detailed amino acid sequence data. Our studies also suggest that the tyrosine sites in the extracellular region of polypeptides are duplicated. Each of the regions containing a set of these duplicated extracellular sites is separated by an intracelMar region of the polypeptide chain (Jenkins and Tanner, 1975, 1977a).It is difficult to be certain that this interpretation has not been made in error as a result of the presence of overlapping partial digestion products. However, the two sets of sites behave differently on radioiodination at isoosmotic and low ionic strength. The presence of two sets of sites is consistent with the earlier results of Bretscher (19714, who used a labeling reagent with a different amino acid spec-

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MICHAEL J. A. TANNER

ificity. The extent and significance of the amino acid sequence duplication these sites represent are unknown, but it may reflect some symmetry in the molecule, possibly at the anion-binding site of the protein. The relatively low inhibitory potency of sulfanilic acid derivatives toward erythrocyte anion transport compared with that of their " dimeric" forms-diaminostilbene disulfonic acid derivatives-lends some weight to this suggestion (Ho and Guidotti, 1975; Cabantchik and Rothstein, 1972, 1974a). The composition and terminal group analysis of the isolated protein, and fragments of it, suggest that the protein has a blocked Nterminus and that the termini are oriented in the membrane as shown in Fig. 7. Oligosaccharides are located in three regions of the protein. The most highly glycosylated portion, which contains lectin receptors, is present in the C-terminal region of the protein (Jenkins and Tanner, 1977b). Drickamer (1976) suggests that the protein may have valine at the C-terminus, while Jenkins and Tanner (197713) believe that C-terminal leucine is present. However, rather low yields of Cterminal amino acids were obtained in both cases. Several other laboratories have recently reported the results of other structural studies on polypeptide 3 (Steck et al., 1976; Drickamer, 1976). These reports suggest that the N-terminus of the protein is on the cytoplasmic side of the membrane rather than extracellular, as shown in Fig. 7, and that the polypeptide traverses the membrane only once. We feel that the content and known sidedness of lactoperoxidase-labeled sites in fragments obtained from this part of the protein unambiguously show that it is a transmembrane segment of the

FIG.7. Structure of polypeptide 3 in the human erythrocyte membrane. The continuous line represents the polypeptide backbone, while the solid circular shapes represent the attached carbohydrates. These solid shapes are intended to indicate the relative amounts of carbohydrate in each region and not exact numbers of oligosaccharide chains. The general location of several binding sites is also shown.

ERYTHROCYTE GLYCOPROTEINS

303

protein which is distinct from the other membrane-penetrating segment. The model shown in Fig. 7 is the simplest one consistent with our results. The N-terminal part of the chain could of course penetrate the membrane again and protrude from the cytoplasmic side of the membrane, leaving lactoperoxidase-labeled sites in an extracellular loop. Drickamer (1976) was unable to detect any labeling by lactoperoxidase in this region of the protein. This may be a result of the lower concentrations of lactoperoxidase used during labeling in his study, as even under our labeling conditions this N-terminal region was only weakly labeled. A situation similar to this exists with respect to lactoperoxidase labeling of the tyrosine residue of the major sialoglycoprotein which is situated adjacent to the hydrophobic segment in the cytoplasmic region of the protein. This tyrosine residue is relatively unreactive toward labeling, and it is possible that the labeling obtained is due to the generation of excess iodine radicals (Marchesi et al., 1976). Nevertheless this tyrosine residue appears to be located on the cytoplasmic side of the membrane permeability barrier. Even if labeling by excess iodine radicals occurring in polypeptide 3 under our labeling conditions, we showed that under these conditions the sites iodinated by extracellularly located lactoperoxidase are distinct from and do not overlap with the sites labeled by intracellularly located lactoperoxidase (Boxer et al., 1974). Thus the sidedness of labeling is retained under these conditions. The most reasonable interpretation of the distinct domains of labeling observed is that this reflects labeling at each surface of the membrane. In this connection it is interesting that the same tyrosine sites are labeled in polypeptide 3 when intact erythrocytes are labeled either with '251Cl or lactoperoxidase, although some additional sites are labeled by '251Clwhich are not labeled by lactoperoxidase in any membrane preparation. When IC1 is used to label erythrocyte ghosts, new sites of labeling occur in polypeptide 3 , and among these new sites is a group of peptides labeled b y lactoperoxidase only from the cytoplasmic side of the membrane (Tanner, unpublished observations). The apparent sidedness of IC1 labeling of the intact erythrocyte may result in part from the destruction of any intracellular labeling reagent b y the reducing environment inside the erythrocyte, and perhaps also from the presence of a "sink" of intracellular protein which can compete with the polypeptide cytoplasmic region for the reagent. In this case, reduced glutathione, a major source of cellular reducing power, may define the apparent permeability barrier by its ready accessibility to the cytoplasmic surface of the membrane. It has not been possible to identify positively any intramembranously located tyrosine resi-

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MICHAEL J. A. TANNER

dues using lZ5ICllabeling. Hubbard and Cohn (1976) have discussed

the general problem of labeling studies with permeant reagents in

more detail. (See also Juliano, this volume.) The mechanism of biosynthesis of a protein such as polypeptide 3 is obscure. This problem also applies to other intrinsic membrane proteins of this type, such as bacteriorhodopsin, in which the peptide backbone crosses the membrane more than once. Many of the current concepts of the biogenesis of membrane proteins are based on studies of the transfer of secreted proteins across membranes (see Sturgess et al., this volume). No experimental data are available on the biosynthesis of intrinsic membrane proteins which contain multiple traverses of the polypeptide across the membrane. Yu and Steck (1975b)have reported the results of some interesting studies which suggest that isolated, nondenatured polypeptide 3 behaves as a dimer in nonionic detergent solutions. The dimeric polypeptide-3 complexes were able to bind two tetrameric erythrocyte glyceraldehyde-3-phosphate dehydrogenase molecules. A tryptic peptide of MW 22,000, derived from the cytoplasmic region of polypeptide 3, was also able to bind tetrameric glyceraldehyde-3-phosphate. They also provided evidence which suggests that polypeptide 3 remains associated with band 4.2 in nonionic detergent solutions. The latter component (4.2) is probably itself tetrameric (Steck, 1972b).

VI.

FUNCTIONS OF GLYCOPROTEINS

Apart from polypeptide 3, there is no evidence to suggest that other periodate-stainable glycoproteins have a role in any of the enzymic or pseudoenzymic (e.g., transport) processes which occur in the erythrocyte. A. Polypeptide 3

Erythrocyte anion transport is an exchange transport system proba-

bly utilized in vioo for the exchange of C1- and HC0,- across the

erythrocyte membrane: The system has a broad specificity and carries both mono- and divalent anions, but equilibrates small monovalent anions such as C1- particularly quickly (Tosteson, 1959; Passow and Wood, 1974). Even large organic anions appear to penetrate the membrane through this system, albeit slowly (Cabantchik et al., 1975, 1976; Staros et al., 1975).

ERYTHROCYTE GLYCOPROTEINS

305

Evidence suggesting an involvement of polypeptide 3 in the erythrocyte anion transport system is based mainly on the use of inhibitors of transport which covalently react with erythrocyte membrane proteins. One of the most potent inhibitors of erythrocyte anion disulfonate (DIDS) (Catransport is 4,4’-diisothiocyano-2,2’-stilbene bantchik and Rothstein, 1972). The binding of a radioactively labeled reduced form of DIDS to polypeptide 3 correlates well with the inhibition of anion transport, polypeptide 3 being essentially the only component bound by the compound (Cabantchik and Rothstein, 1974a,b; Lepke et al., 1976). This compound does not penetrate the membrane, and it binds to an extracellular region of polypeptide 3 between the cleavage site for extracellularly applied pronase and intracellularly applied trypsin (see Fig. 7; Cabantchik and Rothstein, 1974b; Lepke and Passow, 1976). About 1 x lo6 sites per cell are labeled with DIDS, that is, approximately one molecule of inhibitor per polypeptide-3 monomer (Lepke et al., 1976; Ship et al., 1977). The conclusion that polypeptide 3 is involved in erythrocyte anion transport has been confirmed in studies using a related, less potent inhibitor, 1-isothiocyano4benzenesulfonate (Ho and Guidotti, 1975). Vesicles containing polypeptide 3 as a major component are also able to transport anions (Rothstein et al., 1975; Cabantchik et al., 1977)and recently Ross and McConnell(l977) reconstituted the anion transport system by incorporating purified polypeptide 3 into liposomes. The possibility that the major sialoglycoprotein is involved in this transport process has been ruled out. Specific inhibitors of anion transport show little binding to this protein under conditions which completely abolish transport (Cabantchik and Rothstein, 1974a; Ho and Guidotti, 1975; Lepke et al., 1976; Ship et al., 1977). In addition, erythrocytes which lack the major sialoglycoprotein have normal anion permeability (Tanner et al., 1976). Initial attempts to identify the protein involved in erythrocyte Dglucose transport led to the suggestion that polypeptide 3 might also be involved in this process. This was based on studies of the binding sites of cytochalasin B, a noncovalently bound inhibitor of &glucose transport (Taverna and Langdon, 1973a; Lin and Spudich, 1974), affinity labeling studies employing D-glucosylisothiocyanate (Taverna and Langdon, 1973b), selective extraction (Kahlenberg, 1976),and experiments using reconstituted vesicles (Kasahara and Hinkle, 1976). Recent results suggest that this interpretation is incorrect. Studies using impermeant maleimides under selective conditions (Batt et al., 1976) and further reconstitution studies (Kasahara and Hinkle, 1977)suggest that the component involved in D-glucose transport is a minor protein

306

MICHAEL J. A. TANNER

(band 4.5) which is relatively difficult to detect and copurifies with polypeptide 3 during selective extraction procedures. A large fraction of the total erythrocyte glyceraldehyde-3-phosphate dehydrogenase remains associated with the membrane after the hypotonic lysis of erythrocytes (Mitchell et al., 1965; Tanner and Gray, 1971). The enzyme is specifically and reversibly bound to the membrane and can be dissociated from it at high ionic strengths (Kant and Steck, 1973; McDaniel et al., 1974). Detergent-solubilized polypeptide 3 binds the enzyme, and the resulting complex can also be dissociated at high ionic strengths (Yu and Steck, 1975b). The association of the enzyme with both erythrocyte ghosts and with isolated polypeptide 3 is also influenced by both oxidized and reduced pyridine nucleotide coenzymes (Kant and Steck, 1973; McDaniel and Kirtley, 1974; Yu and Steck, 1975b). It is not known whether there is a relationship between the ionic strength-dependent structural changes which occur in polypeptide 3 (Jenkins and Tanner, 1977a) and the ability of polypeptide 3 to bind with glyceraldehyde-3-phosphate dehydrogenase. The significance of the association of these two proteins in vivo is not understood, but it seems likely that, if the binding of glyceraldehyde-3-phosphate dehydrogenase to the erythrocyte membrane occurs in the intact erythrocyte, its extent will be dependent on the metabolic state of the cell. Fossel and Solomon (1977) report that the glycolytic enzymes phosphoglycerate kinase and monophosphogl ycerate mutase also interact with glyceraldehyde-3-phosphate dehydrogenase and suggest that these enzymes provide a link between metabolism and cation transport in the erythrocyte. Erythrocyte ghosts have also been reported to bind another glycolytic enzyme, aldolase (Strapazon and Steck, 1976). B. Periodate-Stainable Glycoproteins

The functional role of periodate-stainable glycoproteins is poorly understood. The structure of the major sialoglycoprotein with its heavily glycosylated N-terminal region, high local concentration of charged sialic acid residues and carbohydrates, and single-membranepenetrating polypeptide chain segment suggest an extended shape rather than the globular shapes usually found for enzymes. No enzymic activity is known to be associated with this protein. Insofar as information is available on the structure of PAS-2' and PAS-3 they appear to be similar to the major sialoglycoprotein (Marchesi et al., 1976). These glycoproteins appear to share the common characteristic of being heavily glycosylated, and it seems reasonable to suppose that

ERYTHROCYTE GLYCOPROTEINS

307

the carbohydrates associated with them play a major part in their functional role, while the peptide portions may act as a framework for the distribution and orientation of these oligosaccharides.

1. ERYTHROCYTE GLYCOPROTEIN VARIANTS One approach to studying the function of erythrocyte membrane glycoproteins has been to investigate erythrocyte variants which are defective in these components with a view toward correlating these defects with the hematology of the individuals carrying these cells. This approach is immensely simplified in the case of the human erythrocyte, because of the importance of erythrocyte genetics in blood transfusion. Thus much information has been accumulated by blood group serologists covering a variety of human populations, SO that genetically typed cells, even those which are quite rare in human populations, are available. Although only a small number of studies on human erythrocyte variants so far has been done, a few erythrocytes with interesting glycoprotein defects have already been found. Erythrocytes homozygous for the type En(a-) have been shown to lack the major sialoglycoprotein and also to have alterations in the carbohydrate content of their polypeptide 3 (Tanner and Anstee, 1976b; Tanner et ul., 1976; Gahmberg et al., 1976; Anstee et al., 1977).These cells lack a normal erythrocyte antigen, Ena, and have a low sialic acid content, weak M and N antigens, and lowered mobility on cell electrophoresis (Darnborough et al., 1969; Furuhjelm et al., 1969, 1973).The location of the Ena antigen is unknown, but it is not present on the major sialoglycoprotein (Tanner and Anstee, 197613). The nomenclature for these cells is somewhat confusing, and we have suggested that it might be more appropriate to describe them phenotypically as sialoglycoprotein negative (SGP -) erythrocytes (Anstee et al., 1977). Loss of the major sialoglycoprotein in En(a-) cells must result in drastic changes at the surface of these cells, since this protein carries nearly half the total carbohydrate and an even larger proportion of the sialic acid present at the surface of the normal erythrocyte. The increased carbohydrate content of the polypeptide 3 probably compensates to some extent for this loss, though not in a gross way (Tanner et al., 1976; Gahmberg et al., 1976). It is particularly interesting to note that, although the major source of O-glycosidically linked sialic acidrich oligosaccharides found in the normal cell is lost in these cells, these oligosaccharides units do not appear in the polypeptide 3 of En(a -) cells. However, these oligosaccharides are probably present on the PAS-2’ and PAS-3 which remain in En(a-) cells.

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The loss of the sialoglycoprotein does not appear to have a detrimental effect on individuals with homozygous En(a -) erythrocytes. However, while no clinically significant abnormalities appear to be associated with the En(a-) condition, there have been no studies to determine whether or not there are less dramatic alterations in the physiological properties of these cells. The influence of the sialoglycoprotein deficiency on the in vivo lifetime of human erythrocytes, their fragility, and their flow properties, will be of particular interest. The rarity of the sialoglycoprotein-deficient condition in human populations suggests that the sialoglycoprotein confers some advantages. Several other erythrocytes have characteristics suggesting the presence of defects in the major sialoglycoprotein. These include the Mk, Mg,and Miltenberger antigens (Race and Sanger, 1975).However, in most of these cases the homozygous condition is unknown or very rare. We have found that it is difficult to pin down the nature of these defects unambigously unless a homozygous defective cell is available (Anstee and Tanner, unpublished observations). Erythrocytes lacking Ss antigens (S - s -) have also been studied (Dahr et al., 1975c; Tanner et al., 1977).This type is unknown in Caucasians but is relatively common among African tribal groups (Race could not detect any PAS-3 in and Sanger, 1975). Dahr et al. (1975~) these cells, but our results suggest that the polypeptide chain of PAS-3 remains but is defectively glycosylated, probably having lost the 0glycosidically linked sialic acid-rich units characteristic of normal PAS3 (Tanner et al., 1977). Again, this defect is not associated with any abnormality of clinical significance. These are two examples of viable erythrocytes which are defective in one of the periodate-positive glycoproteins. A cursory glance at the authoritative summary of information on human erythrocyte antigens (Race and Sanger, 1975) suggests that many other examples must exist. The examples discussed above are of inherited defects. T and Tn erythrocytes provide an interesting group of acquired changes in the erythrocyte surface (Race and Sanger, 1975). In both cases, erythrocytes become polyagglutinable, that is, become agglutinable by most normal human sera. T polyagglutinability is a transient phenomenon, often associated with infection by microorganisms. These erythrocytes carry a new antigen (the T antigen) which reacts with A. hypogea lectin (Bird, 1964).In vitro treatment of normal erythrocytes with bacterial neuraminidases also exposes the T antigen (Prokop and Uhlenbruck, 1969). The antigenic determinant appears to consist of galactose residues in the O-glycosidically linked sialotetrasaccharides of

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the membrane which are exposed by the removal of sialic acid (Dahr

et al., 1975a). The Tn syndrome is a related, acquired, but more per-

sistent condition. It is interesting that in this case the disorder is associated with hematological abnormalities (Bird et al., 1971; Race and Sanger, 1975). Blood from individuals with this syndrome contains varying proportions of defective cells. Studies on the defective cells (Dahr et al., 1975c) have shown that the O-glycosidically linked oligosaccharides of the major sialoglycoprotein and PAS3 (and possibly also PAS-2’) are probably incomplete and consist solely ofN-acetylgalactosamine linked to the polypeptide chain.

2. APPEARANCE OF SURFACE GLYCOPROTEINS DURING MATURATIONOF THE ERYTHROCYTE Another experimental approach to obtaining information on the functional role of the erythrocyte glycoproteins is to correlate the appearance of the glycoproteins of the mature erythrocyte with the physiological requirements imposed on the cell during the period when these components are present. The rabbit erythropoietic system is a convenient one to study, particularly because the rabbit erythrocyte lacks the major sialoglycoprotein found in humans and most other species (Lodish and Small, 1975; Light and Tanner, 1977).Polypeptide 3 is the major surface glycoprotein of the rabbit erythrocyte, and three other minor glycoproteins are also present. This lack of a major sialoglycoprotein is not entirely unexpected, since the rabbit erythrocyte has been shown to have an unusually low sialic acid content, surface charge, and mobility on cell electrophoresis (Walter et al., 1967; Durocher et d., 1975). It is interesting to note that no O-glycosidically linked oligosaccharides appear to be present on the surface of rabbit erythrocytes since none of the glycoproteins bind to M . auruntica lectin (Light and Tanner, 1977). We studied the changes in surface glycoproteins which take place during the differentiation of rabbit erythroid cells in the bone marrow and during the release of nonnucleated erythroid cells into the circulation (Light and Tanner, 1977). Nucleated bone marrow-bound erythroid cells and circulating erythroid cells were found to have unique and mutually exclusive groups of surface membrane proteins. The two types of membranes were shown to give distinct protein patterns on gel electrophoresis and after lactoperoxidase radioiodination. They also had different distributions of lectin-binding components when tested with four lectins of differing specificity. Bone marrowbound erythroid cells can be separated according to their age by ve-

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locity sedimentation using methods similar to those of Miller and Phillips (1969) and Denton and Arnstein (1973). The plasma membranes of different age groups of nucleated erythroid cells were found to have the same surface components. We can infer from these results that neither polypeptide 3 nor the other minor glycoproteins found in circulating erythroid cells have a functional role during any of the earlier nucleated erythroid cell stages of the erythrocyte. This makes it possible to narrow down considerably the events in the life history of the erythrocyte in which the carbohydrates bound to polypeptide 3 might play a part. These stages are (1) the dissociation of the reticulocyte from the bone marrow, (2) the circulatory phase of the erythrocyte, and ( 3 )the turnover of senescent erythrocytes. The carbohydrate contents of rabbit reticulocyte and erythrocyte polypeptide 3 are shown in Table I11 (Light, 1976).The rabbit protein has a carbohydrate composition similar to that of the human protein and lacks N-acetylgalactosamine and sialic acid. Since the turnover of erythrocytes in the rabbit appears to involve the removal or loss of sialic acid residues (see Section 11),it is unlikely that the carbohydrates associated with polypeptide 3 play any part in this process. The source of the sialic acid residues involved in the process of erythrocyte turnover is not known, but they may be present on rabbit minor glycoproteins or on sialic acid-containing glycolipids. The latter may be a significant source, since Sweeley and Dawson (1969) estimate TABLE I11

RABBIT RETICULOCYTEAND ERYTHROCYTE POLYPEPTIDE3"

CARBOHYDRATE COMPOSITION OF

Nanomoles per milligram of protein in polypeptide 3 Component

Reticulocytes

Erythrocytes

Fucose Mannose Galactose N-Acetylglucosamine .N-Acetylgalactosamine Sialic acid

5 30 62 40

5 24 49

-

37

a Data from Light (1976). The protein was purified from erythrocytes and reticulocytes as described by Tanner et al. (1976).

ERYTHROCYTE GLYCOPROTEINS

31 1

that a single erythrocyte carries several million molecules of total glycolipid, although the relative proportions of the neutral and the sialic acid-containing-types vary considerably in different species. The functional role of the protein portion of polypeptide 3 in the equilibration of anions during the circulating phase of the erythrocyte has already been discussed (Section V1,A). Since the nucleated erythroid cells present in the bone marrow are unlikely to need to equilibrate anions at as a rate fast as the erythrocyte, the absence of polypeptide 3 from these cells is not altogether surprising. Light (1976) found that the anion permeability of bone marrow erythroid cells was much lower than that of erythrocytes. The correspondence between the dissociation of erythroid cells from the bone marrow and the appearance of polypeptide 3 suggests that the carboh.ydrates bound to this protein may be involved in the alteration in intercellular interactions which must take place during the transition from a tissue-bound erythroid cell to a freely circulating erythroid cell.

3.

SOME CONSIDERATIONS AND SPECULATIONS ON THE FUNCTION OF THE GLYCOPROTEIN-BOUND CARBOHYDRATES AT THE SURFACE OF THE ERYTHROCYTE

Although glycoproteins are found in a wide variety of situations in mammals and in many other organisms, in general we have only a minimal understanding of the attributes the bound carbohydrates bestow on these proteins. Even less is known about the detailed structural and conformational properties of oligosaccharides which cause them to be so widely distributed. Therefore the considerations presented in this section reflect the limited theoretical and experimental knowledge available at this time and contain a considerable element of speculation. The justification for including it at all is to highlight some unresolved questions which to some extent have not been a focus of attention for investigators interested in the biochemistry of erythrocyte membrane glycoproteins. Two types of functional attributes can be broadly distinguished for most biologically important compounds:

1. Functions involving specific chemical interactions. In this case, the requirement to be able to interact specifically with some other structure can impose considerable chemical and conformational limitations on the molecule. The absence of the molecule often results in the cessation of some sequence of biological reactions.

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2. Functions which may be broadly described as structural. Here, the function depends on the general properties of the particular class of compounds and does not impose such restrictive requirements on the chemical structure of the molecules involved. Many classes of compound can have both types of functional role. Thus the catalytic serine residue in serine proteases is an example of the first type, while the large number of amino acid residues which can be substituted for by a residue of a similar type without affecting biological activity in many proteins represents the second type. The requirement of many membrane-bound enzymes for particular phospholipids for enzymic activity, and the general and relatively nonspecific ability of phospholipids to form bimolecular leaflets in biological membranes, illustrate a similar dual role for this class of compounds. There is no reason to suppose that cell surface carbohydrates cannot have a similar duality in functional role. Several mechanisms have been suggested which involve cell surface carbohydrates in specific processes such as cell recognition and cell-cell interactions (see Wallach, 1975, for a recent review). We can ask whether or not the carbohydrates associated with the periodatestainable glycoproteins of the erythrocyte, and in particular the major sialoglycoprotein, have any involvement in this type of specific process. Possible functions of the carbohydrates associated with polypeptide 3 are considered in Section VI,B,2. The apparent absence of clinical abnormalities in En(a -) human erythrocytes, which lack the major sialoglycoprotein, suggest that the normal function and viability of the human erythrocyte are not critically dependent on the presence of this protein and the oligosaccharides present on it, and that loss of this carbohydrate can be compensated for by a relatively small increase in the carbohydrate content of polypeptide 3. The major source of the O-glycosidically linked sialotetrasaccharides of the normal human erythrocyte is absent in the En(a-) cell, and these sialotetrasaccharides do not appear in the new sugar units found on the altered polypeptide 3 (Tanner et al., 1976). Since no hematological disorders occur in this case, it seems likely that these O-glycosidically linked sialic acid-rich oligosaccharides of the sialoglycoprotein are not involved in interactions of the specific type. A similar argument. can be presented for the O-glycosidically linked oligosaccharides of PAS-3 which are absent in S - s - erythrocytes. The very large number of these O-glycosidically linked sialic acid-rich oligosaccharides present at the surface of the normal erythrocyte (approximately 8 x lo6 per erythrocyte) tends to support

ERYTHROCYTE GLYCOPROTEINS

31 3

this view. It is difficult to see why there would need to be so many of these if the oligosaccharide had a specific receptor function. For similar reasons it is even more difficult to envisage the necessity for 15 apparently similar oligosaccharides on a single sialoglycoprotein molecule, many on adjacent amino acid residues, when a single accessible oligosaccharide on each protein molecule might be expected to be adequate. In the rabbit erythrocyte the absence of glycoproteins analogous to the periodate-stainable glycoproteins of the human cell, together with the lack of O-glycosidically linked oligosaccharides, is also consistent with this suggestion. Nevertheless, the almost ubiquitous presence of the sialoglycoprotein in humans, and the presence of analagous glycoproteins in nearly all other animals, suggests that the carbohydrates associated with this protein do have a function. It seems probable that this is of the more general or structural type referred to at the beginning of this section. The compensatory change in carbohydrate content in polypeptide 3 of En(a-) erythrocytes is consistent with this, and it is interesting to note that the only other known sialoglycoprotein-deficient erythrocyte, the rabbit cell, is also unusual in containing a N-acetylglucosaminyl lipid as its major glycolipid (Sweeley and Dawson, 1969), which might take over the role of the sialoglycoprotein-like molecules in this case. The sialic acid on the erythrocyte (which is mainly associated with the sialoglycoprotein) has been thought to serve to keep erythrocytes separate (Klenk, 1956). Although this may be the case, neither rabbits nor individuals with En(a-) erythrocytes appear to suffer any detrimental effects because of increased agglutinability of their erythrocytes. It is unlikely that the sialic acid residues associated with erythrocyte sialoglycoproteins are involved in the turnover of erythrocytes (see Section VI,B,2). The plasma membrane of the erythrocyte is a remarkably stable entity. The cell remains intact and does not fuse with other cells or vesicularize durihg the long circulating phase of the cell (120 days at 37°C). During this period the cell is subjected to continuous and very severe mechanical stresses, particularly while in the microcirculation. Glycoprotein-bound carbohydrates may contribute to the stability of this membrane in some way. In particular, they may provide a hydrophilic and highly hydrated layer at the cell surface which could serve as a thermodynamic barrier to cell fusion. It has been suggested that the aggregation of intramembranal particles (which in the case of the erythrocyte might contain the membrane glycoproteins; see Section IV) is an important prerequisite to cell fusion (Poste and Allison, 1973), and that cell fusion involves the interaction of protein-free, bare, lipid bilayers (Maroudas, 1975; Ahkong et al., 1975; Vos et al.,

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MICHAEL J. A. TANNER

1976).Thus it might be necessary to break the continuity of the carbohydrate layer in order for cell fusion to proceed. The effectiveness and continuity of such a barrier would be improved by extensive interactions between oligosaccharides on the same and different glycoprotein molecules, so as to form an extended network over the surfaceof the cell. This type of structure has been suggested by Bretscher (1973). It seems likely that the periodatestainable glycoproteins have extended structures, because of the high density of negatively charged sialic acid residues they contain (Winzler, 1971).An extended network could be built up between carbohydrate moieties both by interconnecting hydrogen bonds and by divalent cation bridges between sialic acid residues. Approximately 6 x lo6 Ca2+-bindingsites per cell are located on sialic acid residues on the erythrocyte surface (Seaman et al., 1969). Only 6 basic amino acid residues are available in the extracellular region of the human major sialoglycoprotein for intramolecular interactions with the 20 or 30 sialic acid molecules present on the protein, and it seems probable that most of the latter interact with divalent cations. This glycoprotein network need not be entirely continuous but may contain polypeptide islands which do not contain carbohydrates. These could be determined b y proteins like polypeptide 3 and form an access path for anions to the transport system and in general terms allow natural ligands to reach their receptors (Fig. 8).One of the functions of the relatively small proportion of carbohydrate found in polypeptide 3 may be to allow the protein to become integrated into a glycoprotein network. The presence of 15apparently similar O-glycosidically linked oligosaccharides in the major sialoglycoprotein, particularly the 6 adjacent oligosaccharides on residues 10 to 15, suggests that some sort of repetitive structure can be formed by these oligosaccharides. Strong selection pressures, presumably structural in nature, must operate on the polypeptide and the carbohydrate of this protein to preserve this large number of similar chains. It is striking that closely related oligosaccharides all having the core structure found in the sialotetrasaccharides, GalPl- 3GalNAc 4 Ser(Thr), are found in a great variety of vertebrate glycoproteins, very often in dense clusters on adjacent or nearly adjacent amino acid residues (Kornfeld and Kornfeld, 1976), again suggesting that groups of oligosaccharides contribute to some sort of a repeating structure. The similarities in the core structures of the generally more complex N-glycosidically linked oligosaccharides and the often repetitive sequences present in the outer chains of these oligosaccharides are also becoming evident (Kornfeld and Kornfeld,

ERYTHROCYTE GLYCOPROTEINS

315

FIG.8. Schematic representation of the carbohydrate barrier at the erythrocyte surface. The solid areas represent the glycoprotein network, while the hatched areas show regions of polypeptide. The periodate-stainable glycoproteins are indicated by extended polypeptides, and the globular protein represents polypeptide 3, the anion transport protein.

1976). When the relatively restricted variety of oligosaccharide sequences found is compared with the immense variety of theoretically possible sequences, the conclusion that groups of these sequences have special structural properties which has caused their selection and restricted their divergence seems a reasonable one. Since virtually nothing is known about the three-dimensional structure of these heterooligosaccharides, it is not possible to surmise the nature of these structures. However, it is conceivable that in many biological situations it is advantageous for proteins to possess localized or extended hydrogen-bonded networks which could provide hydrophilic, highly hydrated, impenetrable barriers in certain exposed regions of proteins or to cover particular surface areas. If a carbohydrate lattice is present at the surface of erythrocytes, the viability of En(a -) human erythrocytes and rabbit erythrocytes might be explained by the ability of the compensatory change in carbohydrate content of En(a-) erythrocyte polypeptide 3, and the unusual glycolipid and the carbohydrate on polypeptide 3 of rabbit erythrocytes, to provide a sufficiently continuous network of this type of yield a membrane with adequate stability. Whether or not a similar situation applies to the surface membrane of other mammalian cells cannot yet be predicted, since very little information is available on the structure of the oligosaccharides bound to the surface glycoproteins. However, in view of the general compositional similarities among these and other glycoproteins, it would not be surprising to find similar surface barriers in other cells, particularly in tissues where plasma membranes are in close juxtaposition and yet cells must retain their integrity. An interesting consequence of the presence of a restricting barrier of this type is that either surface receptors must be external to this barrier or ligands must penetrate or disrupt this network in order to interact with components in the cell

31 6

MICHAEL J. A. TANNER

membrane. The well-known and diverse effects of lectins on cellular events and on membrane structure (see, e.g., Lis and Sharon, 1973; Wallach, 1975; Nicholson, 1976) may be a consequence of the disruption of this lattice by these polyvalent, carbohydrate-specific ligands. It is evident that, in order to understand the function and importance of membrane glycoproteins, it will be necessary to understand the detailed three-dimensional structures and interactions of the heterooligosaccharides found in these molecules. The resolution of these structures remains a problem of major significance in current biology. ACKNOWLEDGMENTS

I thank my colleagues Drs. D. H. Boxer, R. E. Jenkins, and N. D. Light for their en-

thusiastic contributions to the work that has been done in our laboratory, and especially Dr. D. J. Anstee for his collaboration and the many hours of stimulating discussion we have had. I am indebted to Annual Reviews, Inc., and to Dr. V. T. Marchesi, for perniission to use Fig. 3. The work from our laboratory has been supported in part by grants from the Medical Research Council and the Wellcome Trust. REFERENCES

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