329

Biochimiea et Biophysica Acta, 559 (1979) 329 376 © Elsevier/North-Holland Biomedical Press

BBA 85199 PURIFICATION OF CELL MEMBRANE GLYCOPROTEINS BY LECTIN AFFINITY CHROMATOGRAPHY

REUBEN LOFAN and GARTff L. N1COLSON

Department of Developmental and Cell Biology and Department of Plo,siology, College of Medicine, UniversiO, of CaliJbrnia, Irvine, CA 92717 {U.S.A.) (Received January 22nd, 1979)

Contents 1.

Introduction

............................................

330

I1.

(Tell surface membrane organization and dynamics . . . . . . . . . . . . . . . . . . . . . . .

330

1II.

Cell membrane glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Saccharide c o m p o n e n t s o f g l y c o p r o t e i n s . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glyeophorin as a model for integral membrane glycoproteins . . . . . . . . . . . . . .

332 332 334

IV.

Lectins and their saccharide-binding properties . . . . . . . . . . . . . . . . . . . . . . . . .

336

V.

Approaches useful for the purification and characterization of cell membrane lectinbinding c o m p o n e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cell surface labeling of membrane proteins and glycoproteins . . . . . . . . . . . . . .

338 338 339

Non-affinity methods of cell membrane glycoprotein purification . . . . . . . . . . . . . . A. Isolation and characterization of lectin-binding glycoproteins . . . . . . . . . . . . . . B. Isolation and characterization of lectin-reactive glycoproteins . . . . . . . . . . . . . . 1. Solubilization of membrane integral proteins . . . . . . . . . . . . . . . . . . . . . . 2. Purification of solubilized membrane glycoproteins by conventional procedures

340 340 342 342 343

VI.

.

VII.

h n m o b i l i z e d lectin derivatives useful for affinity chroma t ogra phy . . . . . . . . . . . . . .

344

VIII.

Applications of immobilized lectins for fractionation and purification of membrane glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. E r y t h r o c y t e membrane glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. L y m p h o c y t e plasma membrane glycoproteins . . . . . . . . . . . . . . . . . . . . . . . C. Neural glycopeptides and glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Membrane glycoproteins from untransformed and transformed cells . . . . . . . . . . E. Membrane glycoproteins isolated from miscellaneous biological materials . . . . . . .

347 347 35 l 357 360 368

t:inalc o m m e n t s

371

IX.

..........................................

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

372

References

372

.................................................

330 I. Introduction It is now well accepted that the cell membrane and its constituents play vital roles in many fundamental cellular processes. An overwhelming amount of data have implicated the cell membrane as the primary site for control of intercellular interactions, transmission of extraceUular stimuli, regulation of cell movement, growth, division and differentiation [1]. Although the exact molecular details of membrane structure have yet to be elucidated, numerous studies over the last few years have steadily increased our understanding of membrane gross architecture and dynamics [ 2 - 6 ] . The chemical characteristics of the membrane components involved in the various functions described above are now being revealed, mainly due to the development of techniques for solubilization, isolation and reconstitution of the major membrane constituents [7,8]. Since most, if not all, of the external membrane components are glycosylated, the structure and activities of membrane glycolipids and glycoproteins are of prime interest as candidates for mediating cell membrane information as well as function [ 1,8-16]. In this review we will discuss new approaches for isolating, purifying and characterizing cell membrane glycoproteins. An emphasis will be placed on describing affinity techniques for purifying glycoproteins, particularly affinity chromatography on immobilized lectins, because these procedures constitute one of the most important new tools for the membrane biologist. The isolation, structure and function of glycolipids will not be discussed here, and the reader is urged to consult recent reviews on this important subject [11,17-19]. We have also chosen to briefly outline current views on cell membrane organization and dynamics as well as introduce some general properties of glycoproteins and lectins. II. Cell surface membrane organization and dynamics The results of an increasing number of investigations indicate that the structure and organization of cellular membranes conform to several basic principles (reviewed in Refs. 2, 3, 6): (a) the major membrane lipids, the phospholipids, are arranged in a planar bilayer configuration which under physiological conditions is predominantly in a 'fluid' state; (b) numerous proteins and glycoproteins are inserted or intercalated to varying degrees into the bilayer interrupting its continuity and forming a mosaic~like arrangement; (c) the distribution of specific glycolipids, proteins and glycoproteins in the inner and outer halves of the lipid bilayer is asymmetric in most, if not all, cellular membranes; (d) the structures of cell membrane proteins and glycoproteins are quite heterogeneous, and they can exist in various states of aggregation; (e) integral membrane proteins and glycoproteins are globular, amphipathic molecules which interact with both the hydrophobic and hydrophilic regions of the membrane and are characterized by their strong associations with membrane lipids; (f) the extent to which integral plasma membrane proteins are inserted into the lipid bilayer is determined by the amino acid sequence and the three dimensional folding; (g) some integral membrane proteins span the thickness of the bilayer and have portions of their structures protruding at both the inner and outer membrane surfaces; (h) certain integral proteins are thought to associate with similar or different integral proteins to form oligomeric complexes, and there is evidence that a number of these integral proteins interact with peripheral membrane proteins at the external or cytoplasmic membrane surface; (i) peripheral membrane proteins and glycoproteins are only weakly attached to the surfaces of biological membranes, mainly via non-hydro-

331 phobic forces such as ionic interactions or nonionic Van der Waals and London dispersion forces; and (j) some membrane components are capable o f rapid and reversible lateral movements or rearrangements in response to a variety of stimuli, and it is likely that a graded hierarchy of mobilities exist where some components are freely diffusing in the membrane plane while others are restrained. The striking asymmetry of the plasma membrane appears to be maintained by a tremendous thermodynamic barrier whicl~ tends to prevent the transmembrane passage of hydrophilic molecules or polar regions of glycolipids and glycoproteins through the hydrophobic matrix of the membrane. This arrangement of cell surface components allows receptors for hormones, viruses, antibodies and lectins to be present exclusively on the outer surface where they are exposed to the extracellular environment. Since the lipid hydrocarbon chains in the membrane are generally in a fluid state, allowing a considerable freedom of lipid lateral motion, as well as movement of integral membrane proteins and lipoprotein complexes, certain cells have evolved with systems to restrain lateral mobility. Controlled attachment and detachment of integral transmembrane proteins to peripheral components and cytoskeletal elements in the cell cytoplasm can be used to maintain specific topographic arrangements or patterns on the surface of the cells, patterns which can change in response to intra- and extracellular stimuli [2,3,20]. Basic membrane organization is envisaged as an elaboration of the Fluid Mosaic Model of membrane structure [ 2 - 4 , 2 0 ] . In the hypothetical scheme of Fig. 1 additional extracellular and intracellular components are shown such as glycosylaminoglycans and cytoskeletal elements, respectively. Some of the integral membrane glycoproteins are depicted

t:ig. 1. Hypothetical structure of a plasma membrane including possible interactions between glycoproteins (GP) and glycosaminoglycans (GAG) at the outer surface as well as between GP and membraneassociated microtubules (MT) and microfilaments (MF) systems involved in transmcmbrane control over cell surface receptor mobility and distribution.

332 as oligomeric transmembrane complexes, consistent with recent information, and a variety of 'bridging' molecules are shown linking cytoskeletal elements together and t o membrane components. Although membrane dynamics are not depicted here (Fig. 1), it is assumed that the various classes of cell membrane components possess differing abilities to undergo lateral rearrangements and associations.

III. Cell membrane glycoproteins IliA. Saccharide components of glycoproteins Glycoproteins are proteins with covalently bound carbohydrate side chains consisting of one or as many as 60 residues arranged in linear or branched structures [21]. Between 1 and 80% of the mass of most glycoproteins is carbohydrate. The saccharides usually found in animal cell glycoproteins include N-acetylneuraminic acid (NeuNAc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GIcNAc) and N-acetylgalactosamine (GalNAc). Several types of carbohydrate-potypeptide linkages have been described [22-24]. The most commonly encountered linkages are the amide bond between the reducing end of GlcNAc and the ~-amide group of asparagine (N-glycosidic bond) and the O-glycosidic bond between the reducing end of GalNAc and the hydroxyl group of serine or threonine. The latter linkage is susceptible to mild alkali and decomposes via ~-elimination. Glycoproteins may carry N-linked, O-linked or both types of oligosaccharide side chains. N-Glycosidically-bound saccharides are found in most soluble plasma glycoproteins; therefore N-linked side chains are commonly referred to as plasma type. O-Linked saccharides are abundant in mucins; consequently, oligosaccharide chains linked to Ser or Thr are called the mucin type [14]. Most of the detailed structural information on oligosaccharide sequences has been obtained through studies on soluble glycoproteins. This has occurred mainly because of the availability of large quantities of soluble glycoproteins compared to membrane glycoproteins. Therefore, we will consider the soluble glycoprotein structures first, because they provide an insight into the types of oligosaccharide sequences and linkages that cells can produce by their biosynthetic machinery. The N-linked oligosaccharide side chains usually possess a core sequence containing Man and GIcNAc. The disaccharide (GlcNAc)2 is linked to asparagine, and to it Man residues are coupled usually as chain branching points. There are at least two types of N-glycosidically-linked oligosaccharide: (a) a high mannose type which contains several Man residues (>3) peripheral to the Man ~ (GlcNAc)2 core (Fig. 2D), and 0a) a complex type in which NeuNAc residues usually occupy nonreducing terminal positions on both main and branch chains and are linked via Gal ~ GlcNAc- sequences to internal Man residues (Fig. 2A C). Branched residues such as Fuc ~ Gal- or Fuc -~ GlcNAc- have also been found. It has recently been shown that the synthesis of the complex type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated after the transfer and processing of a glucose-containing, high mannose type oligosaccharide chain from a lipid-linked precursor to the nascent polypeptide [28,29]. N-Glycosidically.linked oligosaccharides conforming to the general structures presented in Fig. 2 are found in many plasma glycoproteins [22-25], immunoglobulin molecules [22], glycopeptides isolated from human erythrocytes [ 15,22] and viral membrane glycoproteins [26]. Closer examination of these glycopeptides indicates that although they are similar in structure, characteristic differences exist between them. Analogous structures to N-glycosidically-linked glycopeptides

333

A

B

NeuNAc

~NeuNAc]

1o(2,6

i ~'2,6

Gal

Gal

GIc NAc

G Ic NAc

[c{1,2

[,O1,4(?)

Man ~L--~'Man ~T,//////~'GIc NA c

NeuNAc

NeuNAc

lcf 2,6

l (:z"2,6

Gel

Gal

G IcNAc

GIcNA c

1.81,2

I/31,2

Mon ~i-i~"Man ~'*-~,6M on

/31,4

1 --Asn--

GIcNAc B 1,4 I GIcNAc L --Asn--

D

C NeuNAc

NeuNAc

NeuNAc

Gal

Gol

Gal

1 2.3

F2.3

L 2.3

1/31,4

1/31,4

1/31,4

GIcNAc

GIcNAc

GIcNAc

[,'3 1.2

~

Man

Man [~,,2

Man

Man

Man

[,~,2

[~,,2

1,2//X~l'4

M a n ~-~,3Ma n "*-~i~-6Man

/31,4 GIcNAc

/3 1,4 GIcNAc ~ . 6 Fuc

--Asn--

Man

Man~-i-~ Man ~ •

1/31,4

Man '

GIcNAc

1/31,4 GIcNAc --Asn--

Fig. 2. Examples fl~r N-glycosidically-bound oligosaccharide side chains found in glycopeptides isolated trom: (A) human erythrocyte membrane major sialoglycoprotein (glycophorin) [22]. The interrupted line in (A) represents that a variant glycopeptide lacking terminal NeuNAc was also found; (B) human serum transferrin [251, (C) Vesicular Stomatitis viral envelope glycoprotein [26]; and (D) thyroglobulin unit A [27], human diploid fibroblasts [31], part of the lipid-linked precursor of the complex oligosaccharide units of the Vesicular Stomatitis virus G protein [28].

exist in mouse spleen H-2 alloantigens [30] and membrane glycoproteins isolated from human [31] and rat fibroblasts [32], rat brain [33] and ascites hepatoma cells [34]. The O-glycosidically-linked oligosaccharides (Fig. 3) are usually smaller than N-glycosidically-linked units with the exception of blood group substances which carry 'megalosaccharide' side chains [22]. The saccharide sequences in Fig. 3 as well as those present in many mucins have the core structure Gal-/3(l,3)-GaINAc. Fucose can be linked c~(1,2) to Gal, and NeuNAc can be linked a(2,3) or a(2,6) to Gal or to GalNAc. This common core structure oligosaccharide has been found in mammary carcinoma [36] and lympho-

334

A NeuNAc I ~2.~ Gel

~ .,'31.3

Go INAc ~--~'6NeuNAc

1

--Ser-(Thr)

B

C

Gel

NeuNAc 1a2.3 Gel

, . . . ~~ Gol aT.3 NeuNAc GIcNAc ,ei..~(4) ' 1.~ ,.2(4.6)? Gol

GalNAc

GoINAc

GoINAc

1~31.3

l

--Ser(Thr)

1/3 ,.3(4)

l

-Ser(Thr)

D

1,8 1,3(4)

l

-Ser(Thr)

Fig. 3. Examples for O-glycosidically-boundoligosaccharide side chains found in glycopeptides isolated from: (A) human erythrocyte membrane major sialoglycoprotein(glycophorin) [22,35]; (B-D) TA-3 mammary carcinoma cell surface epiglycanin [22,36]. cyte cell surface glycoproteins [37] and in brain glycopeptides [38]. Removal of NeuNAc by the enzyme neuraminidase results in antigenic modification of many sialoglycopep. tides. For example, the neuraminic acid-free O-linked oligosaccharide of human erythrocytes is part of the T (Thomsen-Friedenreich) antigen [39] which has recently been detected on the surface of human breast cancer cells using immunological procedures [40]. In the last few years techniques for elucidating the structures and sequences of o!igosaccharides have been improved substantially [22]. The use of gas-liquid chromatography to identify volatile sugar derivates [41] and utilization of highly purified endo- and exoglycosidases to sequentially cleave specific saccharide sequences [42,43] have rendered possible the structural analysis of glycopeptides available only in small quantities, Since the main limitation appears to be the amount of pure glycopeptide material available from cells grown in tissue culture, the development of new methods for the efficient and rapid isolation of membrane glycoproteins will be necessary. Li et al. [28] have recently overcome problems in low amounts of starting material by using oligosaccharides prepared from ceils that were grown on radioactive precursors to specifically label the constituent sugars. The oligosaccharide structure was then determined by the use of specific glycosidases, methylation, acetolysis and Smith periodate degradation [28]. One difficulty that has arisen in the isolation and purification of celt surface oligosaccharides and glycopeptides is that microheterogeneity exists in the same oligosaccharide class. This may be due, in part, to artifacts introduced during isolation. When cells are ruptured as an initial step in membrane isolation and purification, glyco~dases are released from the broken cells. Often this leads to enzymic loss of certain terminal saccharide residues such as NeuNAc or Fuc. However, even when artifactual changes are not introduced, there is an inherent microheterogeneity in membrane glycoproteins due to either incomplete glycosylation or partial degradation of a properly glycosytated oligosaccharide during its biosynthesis. Thus, some side chains may contain NeuNAc, while others may have terminal Gal, GlcNAc or even Man residues [14,23,44]. IIIB. Glycophorin as a model for integral membrane glycoproteins While it is not surprising that the oligosaccharides found on soluble and membrane glycoproteins are quite similar, one would expect that the polypeptide moieties of the membrane glycoproteins have special structures or properties distinct from those o f soluble glycoproteins and that these would reflect the differing functions of soluble compared

5o

¥1

13o

t:ig. 4. The amino acid sequence of the major sialoglycopeptide of human erythrocyte membranes (glycophorin A). The shaded squares and the hexagon represent the O-linked and N-linked oligosaccharide side chains, respectively. The interrupted and solid vertical lines indicate the location of the polar groups of the outer and inner halves of the phospholipid bilayer, respectively [451.

20

336 to cell surface molecules. At present our information on membrane glycoproteins is derived mainly from extensive studies on the major sialoglycoprotein of human erythrocytes which culminated in the elucidation of its complete amino acid sequence and the sites of the oligosaccharide chain attachments [5,45]. The sialoglycoprotein, named glycophorin, is an integral membrane glycoprotein which spans the membrane. The glycophorin monomer polypeptide chain is comprised of 131 amino acids (Fig. 4) and contains about 16 saccharide side chains of which 15 are O-glycosidically-linked (see Fig. 3A) and one is N-glycosidically-bound (see Fig. 2A). The amino acid composition of glycophorin is not strikingly different from that of soluble glycoproteins and does not alone provide insight into the ability of glycophorin to strongly associate with membrane lipids. However, the amino acid sequence of glycophorin is unique and distinct from soluble glycoproteins in that there is an unusually high proportion of charged amino acids at both ends of the molecule with a predominantly hydrophobic segment in between. This arrangement (Fig. 4) allows glycophorin to be divided into three domains: (a) a hydrophilic outer surface N-terminal segment of 70 amino acids that contains 22 residues of serine or threonine, 15 of which serve as O-glycosidic saccharide-linkage sites. In this sequence is also a single N-glycosidically-bound oligosaccharide side chain connected at asparagine-26; (b) an internal sequence between residues 73 and 92 that is composed predominantly of hydrophobic amino acids probably arranged in the form of an a-helix. This segment is thought to be entirely membrane embedded in strong association with lipid hydrocarbon tails. A cluster of acidic amino acids at the N-terminal (external) end of the internal hydrophobic segment and of 4 basic amino acids on the C-terminal cytoplasmic side of this segment may participate in electrostatic interactions with phospholipids which could stabilize the intercalation of glycophorin into the membrane lipid bilayer; and (c) a cytoplasmic C-terminal segment that contains hydrophilic amino acids (plus proline) and probably participates in interactions with cytoplasmic peripheral membrane components [5,46-48]. Purified human glycophorin is dimeric in the presence of nonionic detergents, possibly because of strong associations between the hydrophobic segments of each monomer. In its native state the glycophorin dimer appears to be organized with at least one other human erythrocyte sialoglycoprotein, band 3 component, into an oligomer-glycoprotein complex [3,48]. No other integral membrane glycoprotein has been as extensively studied as glycophorin; however, recent investigations on the major human histocompatibility (HLA) antigens have revealed some similarities. The COOH-terminal region of the 44 000 dalton HLA glycoprotein chain consists of a hydrophobic region followed by a cluster of basic amino acids terminating with a relatively hydrophobic sequence [49]. This distribution of amino acids is similar to the analogous exterior hydrophilic and internal hydrophobic segments of glycophorin (Fig. 4).

IV. Lectins and their saccharide.binding properties Lectins are saccharide-binding proteins and glycoproteins [12,13,50-52]. They bind mono- or oligosaccharides with great specificity through complementary sugar-binding sites. Because they generally contain at least two saccharide-binding sites per molecule, their interactions with carbohydrate-containing molecules and cells resemble those of antibodies. Thus, lectins precipitate multivalent glycoproteins and polysaccharides and agglutinate cells by forming multiple crossbridges between saccharide-bearing and saccharide-binding molecules. Since they are readily available in pure form and their interac-

337 tions can be inhibited and often reversed by simple sugars, lectins have been extensively used as probes of the structure and organization of cell membrane saccharide-containing components. Binding of lectins to cell surfaces often results in cell agglutination, and in certain cells specific biochemical changes occur after lectin binding. Detailed information on lectin purification, properties and their biological effects on cells can be found in severn recent reviews [12,13,50-52]; here we will only focus on the saccharide-binding properties of lectins and their use in glycoconjugate purification. Classical methods for detecting the presence of lectins in plant extracts, animal tissue homogenates or serum have depended on hemagglutination of animal or human erythrocytes. Consequently, almost all of the lectins that have been thus far isolated and characterized exhibit specificities for saccharides that are accessible on the surfaces of erythrocytes. Although this usually limits the range of monosaccharide specificities to NeuNAc, Fuc, Gal, Man, GlcNAc and GalNAc and their arrangements into oligosaccharide sequences, it is not a real problem, because erythrocyte membrane oligosaccharide side chains (particularly the blood group substances) seem to be more complex and versatile than similar moieties on many other cell types [22]. The saccharide-binding specificities of lectins have been established by comparing various mono- and oligosaccharides of known structure for their potency in inhibiting hemagglutinating and glycoprotein- (or polysaccharide)-precipitating activities. More direct methods such as measuring sugar binding by equilibrium dialysis or spectrofluorimetric titrations have also been employed [52-54]. Hapten-inhibition studies with free sugars and alkyl glycosides have been used to demonstrate that some lectins specifically recognize either a- or/3-linkages. For example, concanavalin A, Lens culinaris and pea lectins all exhibit a pronounced preference for the a-anomer of manno- and glucopyranose and the anti-A lectins from Helix pomatia, Phaseolus lunatus or Dotichos biflorus are best inhibited by a-D-GalNAc, the dominant blood group A determinant. Other lectins bind both a- and fl-anomers with similar affinity; for example, wheat germ agglutinin is inhibited almost equally well by methyl-a- or methyl-~-GlcNAc and soybean agglutinin by methyl-a or ethyl-fl-GalNAc. Most lectins interact with the non-reducing terminal sugar residues of oligo- and polysaccharides; however, concanavalin A and the lectins from lentil and pea (Lens culinaris and Pisum sativum agglutinins) interact with internal Man residues, while wheat germ agglutinin interacts with internal GlcNAc residues. The saccharide bindingsites are thought to accommodate only one mono- or disaccharide, but some lectins have extended saccharide-binding sites which are composed of several subsites. In this latter case each subsite can be complementary for similar or different sugar moieties. Thus. inhibition of concanavalin A by a series of mannose mono-, di- and trisaccharides comprised of a(1,2)-mannopyranosyl units increased with chain length up to mannotriose [521. Similarly, wheat germ agglutinin affinity for saccharides of fl(1,4)-linked GlcNAc increased up to the triose of GlcNAc. In the case of peanut agglutinin the saccharide con> bining-site was found to be complementary to the heterodisaccharide Gal-~3(1,3)-GalNAc [55,561, and the binding site ofPhaseolus vulgaris agglutinin may be even more complex, as the best inhibitors of this lectin are branched oligosaccharides containing two outer chains with the sequence Gal-/3(1,3 or 4)-GlaNAc-13(1,2)-Man-a-X [12,52,57]. Branched oligosaccharides are also more potent inhibitors of concanavalin A, and in general higher affinity constants were observed for the binding oflectins to soluble glycoproteins or cell surface carbohydrates than to simple sugars. The most plausible explanation for these observations is that very high affinities result from more complex oligosaccharides which are capable of multivalent interactions.

338 A certain degree of 'permissiveness' appears to exist in the recognition of saccharides that are quite different from the best inhibitors which are often complex otigosaccharides. For example, concanavalin A, Lens culinaris and Pisum sativum agglutinins can be inhibited by GlcNAc (Man or Glc > GlcNAc), and the anti-A specific lectins can be inhibited also by Gal (GalNAc > Gal). Although the affinity for these monosaccharides is lower than for the more complementary inhibitors, it may be amplified when several copies of these sugar residues are carried on multi.branched glycoproteins or polysaccharides. This type of 'crossreactivity' may decrease the selectivity of certain lectins, making them less desirable as affinity adsorbants for the separation of different glycoproteins. In order to limit our discussion of lectin saccharide-binding properties to aspects that are relevant to subsequent sections of this review we have omitted numerous examples of lectin-saccharide interactions. For a more complete characterization and precise saccharide-binding specificities of many lectins the reader should consult an excellent recent review of Goldstein and Hayes [52].

V. Approaches useful for the purification and charaeterization of cell membrane lectinbinding components VA. General considerations

A variety of indirect and direct techniques have demonstrated the presence of cell surface lectin-binding components. The binding of fluorescent-lectin and electron-dense lectin derivatives to the surfaces of numerous cell types has been observed using light and electron microscopy, respectively [58 61]. These studies have allowed investigators to follow the distributions and dynamics of lectin.binding components [12,13,58-61]. Quantitative binding of radioactively-labeled lectins to cells has indicated that there are between 106 and 107 binding sites per cell on the surfaces of various cells with binding affinities ranging from 106 to 5 • 107 M -1 [12,51]. Since very few lectin-binding cell surface components have been isolated in pure form and characterized, most of the information on the cell membrane saccharides which bind lectins is indirect and has been inferred from hapten-inhibition experiments. In such studies the ability of mono- and oligosaccharides of known structure to inhibit lectin activities such as cell agglutination is interpreted as evidence for the presence of similar or identical sugars on the cell surface. It has been stressed previously and we re-emphasize that the best hapten inhibitor in solution is not necessarily identical to saccharides which bind the lectin on the cell surface. In addition, different membrane components may have analogous but distinct oligosaccharide side chains with similar or different lectin affinities. Therefore, cell surface lectin-reactive components probably represent a heterogeneous group of molecules with different numbers of oligosaccharide side chains or different degrees of branching and complexity. Two different approaches have been employed for the purification of lectin-reactive components from cell membranes. The first is based on the localization of most, if not all, cell surface carbohydrates on the outer membrane surface allowing enzymic degradation of accessible molecules on intact cells or isolated membranes with proteases or glycosidases. The second is a non-degradative approach in which membranes are isolated from cells followed by solubilization of lectin-binding components from the membranes using various detergents. The first strategy is suitable for studies on the structure of lectin-reactive cell surface glycopeptides and oligosaccharides, whereas the second

339 approach can be used to investigate the structure of intact membrane glycoproteins. The purification of either enzymically fragmented or intact lectin-reactive components can be achieved by non-affinity fractionation methods such as ion-exchange chromatography [32-35,62-66] (this is not applicable for membrane components solubilized with ionic detergents) or gel filtration (see Section VI). Alternatively, affinity chromatography on immobilized lectin derivatives, solely or in combination with the above techniques, has been successfully employed for the purification of enzymically fragmented or intact (solubilized) lectin-binding components (see Section VIB-2). Most of the early studies on membrane glycoprotein purification and characterization were performed using erythrocytes mainly due to the availability of large amounts of cells from stored or freshly drawn blood. Since attempts to purify glycoproteins from cells grown in tissue culture have resulted in rather small amounts of available membrane material, special procedures have been developed to radioactively label cell membrane proteins and glycoproteins before isolation. The labeled components can then be easily detected in column effluents or by autoradiography following separation by gel electrophoresis.

VB. Cell surface labeling of membrane proteins and glycoproteins A variety of techniques have been developed for radiolabeling cell surface proteins, glycoproteins and lipids. Since a detailed discussion of these procedures (reviewed in Refs. 67-71) is beyond the scope of this article, we will only summarize some of the available methods. Several important techniques have been developed for radiolabeling exposed cell surface proteins and glycoproteins. Surface-exposed tyrosyl and histidyl residues of proteins can be lzsl- or 131I-labeled by the lactoperoxidase-catalyzed iodination procedure [72, 73]. Radiolabeling is restricted to the outer surface due to the size of the enzyme (about 78 000 dalton) which does not allow penetration through the membrane during short reaction periods. Cell surface amino groups of proteins have been labeled with the nonpenetrating reagents [3SS]formylmethionylsulfone methylphosphate [74] or [3SS]sulfonic acid diazonium salt [75]. Introduction of tritium into cell surface proteins can be achieved by reacting protein amino groups with pyridoxal phosphate to form a Schiff's base followed by reduction with sodium [aH]borohydride [76]. These labeling methods do not distinguish between proteins and lipids; thus reagents that react with amino groups also label aminophospholipids. Even lactoperoxidase-catalyzed iodination has been reported to introduce iodine into polar and neutral lipids [77]. Another useful approach has been to radiolabel cell surface carbohydrates. This has been achieved by oxidation with IO4 at low concentrations to generate aldehyde groups mainly on sialic acid residues, followed by reduction with [3H]borohydride [78]. Alternatively, radiolabeling can be achieved by oxidation of galactosyl or N-acetylgalactosaminyl residues with cell-impervious galactose oxidase followed by reduction with [3H]borohydride [79] or [3SS]methioninesulfone hydrazide [80]. These reagents also label galactose-containing lipids. Possible artifactual labeling of intracellular components have been discussed [68], and to avoid cross-contamination by nuclear components it was suggested that membranes be isolated from labeled cells or labeled directly after isolation. Since all the methods described above depend on the accessibility of particular amino acid side groups or carbohydrate moieties to the labeling reagents or enzymes, the labeled components may not be representative of all the molecular species on the surface mere-

340 brane. In order to avoid the problem of reagent accessibility, radiolabeling can be achieved metabolically with [3H]fucose or [3H]glucosamine [66,69,81]. This method is especially useful with [3H]leucine-radiolabeled ceil surface glycopeptides which are subsequently released by trypsinization [66].

VI. Non-aff'mRy methods of cell membrane glycoprotein purification VIA. Isolation and characterization of leetin-binding gtycoproteins Most membrane glycoproteins are almost insoluble in neutral aqueous solutions; thus, proteolytic enzymes have been used to cleave surface components on intact cells and release soluble glycopeptides [32-35,62,65,66]. The released glycopeptides can then be fractioned and assayed by monitoring the ability of various fractions to inhibit lectininduced celt agglutination, lymphocyte stimulation or binding of radiolabeled lectin derivatives to intact, untreated cells. Since several recent reviews have described in detail various studies on cell surface lectin-reactive glycopeptides [ 12,13,50-52], the following section will use as examples only the results of two investigations on membrane glycopeptides. The first of these is a study of the glycopeptides of human erythrocytes by Kornfeld et al. (reviewed in Ref. 82), and the other is on the glycopeptides ofhepatoma cells by Walborg et al. (reviewed in Ref. 83). Trypsinization of intact human erythrocytes results in the release of lectin.binding glycopeptides and a 35-50% decrease in the number of remaining cell surface-binding sites for the lectins from Phaseolus vulgaris, Lens culinaris, Robinia pseudoaccacia and Agaricus biflorus *. One glycopeptide released from the trypsinized cells appears to be derived from the N-terminal portion of the major erythrocyte sialoglycoprotein (glycophorin). This glycopeptide (molecular weight 10 000-15 000) is a potent inhibitor of lectin binding to untreated erythrocytes and of lectin-induced hemagglutination. Structural analysis of the glycophorin glycopeptide indicates that it consists of approx. 80% carbohydrate in the form of several oligosaccharide chains of two types designated GP-I and GP-II. GP-I has a branched complex side chain oligosaccharide composed of NeuNAc, Gal, Man and GlcNAc linked by an alkali.stable GlcNAc-asparagine linkage, while GP-II has an alkali-labile side chain which contains NeuNAc, Gal and GalNAc O-glycosidieallylinked to Ser or Thr. Combinations of alkaline borohydride treatment, pronase digestion and fractionation by ion-exchange chromatography have allowed the separation of the two types of glycopeptides. GP-I is a potent hapten inhibitor of P. vulgaris, L. eulinaris and R. pseudoaccacia agglutinins, while GP-II inhibits only the binding of A. biflorus agglutinin to human erythrocytes. To identify the 'determinants' recognized by each of the tectins on the isolated glycopeptides, the oligosaccharides have been sequentially degraded with glycosidases and the resulting products tested for lectin inhibitory activity after each step. Experiments with GP-I reveal that binding o f P vulgaris, R. pseudoaccacia and L. culinaris agglutinins is not diminished after removal of terminal NeuNAc; however, * Experiments with model glycopeptides isolated from partially degraded plasma glycoproteins and human myeloma immunoglobulins indicate that these three leetins are best inhibited by a branched oligosaccharide having Man residues in the core and Gal --*GlcNAc in the extending chains. The Man appears to be important for binding all three lectins, Gal for binding to P. vulgaris and R. pseudoaccacia lectins and GlcNAc for binding to L. cultnaris aggtutinin. The results obtained with the human myeloma immunoglobulin glycopeptides also indicate that the saccharide-bindingsite on the three lectin moleculesis extended [82,84].

341 subsequent removal of penultimate Gal decreases GP-I inhibitory activity to 10% of the original activity against P. vulgaris and R. pseudoaccacia lectins, but inhibition against L. culinaris agglutinin is not diminished. Further studies have demonstrated that the Man and GlcNAc residues on GP-I are also involved in the binding of the three lectins. The inhibitory activities of intact GP-I against the three lectins are several hundred to several thousand times higher than the potency of the monosaccharides Man (inhibitor for L. culinaris and R. pseudoaccacia agglutinins) or GalNAc (inhibitor for P. vulgaris agglutinin). Even though removal of NeuNAc indicates that it does not play a role in the binding of P. vulgaris, R. psuedoaccacia or L. culinaris lectins to GP-I, it has an inhibitory effect on the binding of A. biflorus agglutinin to GP-II. Removal of terminal NeuNAc residues increases the inhibitory activity of GP-II 8-fold toward A. biflorus agglutinin, while subsequent removal of Gal completely abolishes binding. Interestingly the inhibitory activity of the oligosaccharide Gal -~ GalNAc is only 15% of Gal ~ GalNAc -+ Ser suggesting the participation of the amino acid in defining the binding affinity of GP-II to A. biflorus agglutinin [82]. The studies of Kornfeld et al. [82] have demonstrated that various lectins may bind to different sugar side chains located on the same polypeptide chain and that internal (Man, GlcNAc) as well as terminal (NeuNAc) saccharides determine lectin binding. Despite the similarity in the inhibitory potency of GP-I and its partially degraded derivatives toward the activities of P. vulgaris and R. pseudoaccacia agglutinins, several differences in the binding of these lectins to human erythrocytes have been observed. There are twice as many binding sites for P. vulgaris compared to R. pseudoaccacia lectin, and in competition binding studies P. vulgaris lectin effectively binds to all R. pseudoaccacia lectinbinding sites, whereas R. pseudoaccacia agglutinin can block only 50% of P. vulgaris agglutinin-binding sites on the erythrocyte surface. Since neuraminidase treatment of the cells increases R. pseudoaccacia lectin binding by 50-60% without affecting the binding of P. vulgaris agglutinin, it seems reasonable to assume that NeuNAc interferes with R. pseudoaccacia agglutinin binding. It is interesting that removal of NeuNAc from isolated GP-I did not decrease its potency as a hapten inhibitor ofR. pseudoaccacia agglutinin suggesting that the effect of NeuNAc on binding of this lectin to the cell surface may be due to a nonspecific steric effect rather than lower affinity for the complementary saccharide sequence on the sialoglycoprotein [82]. Another example of extensively studied surface glycopeptides is the family of glycopeptides isolated from two rat hepatoma cell lines and normal rat hepatocytes which may mediate cell agglutination by wheat germ agglutin.in and concanavalin A [83]. Novikoff hepatoma cells are strongly agglutinated by both of these lectins, whereas AS-30D hepatoma cells are more agglutinable by wheat germ agghitinin compared to concanavalin A, and normal hepatocytes are not agglutinated by either lectin. Papain digestion of the rat hepatoma cells releases a crude cell surface mixture of glycopeptides and sialoglycopeptides. Upon gel filtration the sialoglycopeptides can be resolved into three fractions, the first of which contains most of the lectin inhibitory activity (tested by inhibition of guinea-pig erythrocyte agglutination). In contrast to the glycopeptides released from hepatoma cells, the papain-released material from normal rat hepatocytes separates upon gel filtration into two fractions both of which do not possess lectin-inhibiting activity. When the lectin-inhibiting fraction from the hepatoma cells is subjected to pronase digestion followed by gel filtration, all of the concanavalin A- and wheat germ agglutinininhibiting activity elutes in the first sialic acid-containing peak. Further purification of this sialic acid-containing material from Novikoff hepatoma cells by DEAE-celhilose

342 chromatography yields eight subfractions which elute from the ion-exchange column in order of increasing sialic acid content. Most of the concanavalin A-inhibiting activity is eluted in the first three subfractions, whereas the fourth and fifth subfractions contain most of the wheat germ agglutinin-inhibiting activity. DEAE chromatography of sialic acid-containing material from AS-30D hepatoma cells results in separation of three subfractions of which the second contains all of the wheat germ agglutinin-inhibitory activity and only minimal concanavalin A-inhibitory activity. These conventional purification techniques allow separation and isolation of cell surface glycopeptides possessing distinct inhibitory activities against two different lectins; however, the material isolated by ionexchange chromatography is not homogeneous and probably includes various polypeptides highly substituted with heterosaccharide chains which may protect against proteolytic attack. The structures of the lectin reactive components have not been determined; however, the concanavalin A.inhibiting sialoglycopeptide from AS-30D hepatoma contains 27% peptide by weight with a relatively high proportion of amino acids involved in carbohydrate-peptide linkages (Asp, Thr and Ser). The major saccharides of the sialoglycopeptide are GlcN, Gal (18.8 and 19.7%, respectively) with lower contents of Man, GAIN, NeuNAc, Fuc and Glc suggesting that both N-glycosidic- and O-glycosidic-linked side chains may branch off the peptide backbone [83]. These studies demonstrate the complexity and heterogeneity of cell membrane saccharide-containing macromolecules found on tissue culture cells.

VIB. Isolation and characterization of leetin-reactive glycoproteins The elucidation of cell membrane glycoprotein structure cannot be based exclusively on studies with proteolytic cleavage products. The main disadvantage is that one obtains a mixture of glycopeptides which could have been derived from different portions of a single macromolecule or from entirely different membrane glycoproteins. In addition, only glycopeptides sensitive to proteotytic attack are released, and these may not be representative. Therefore, alternative approaches aimed at isolating unfragmented membrane glycoproteins have been developed.

VIB{1). Solubilization of membrane integral proteins Membrane glycoproteins in their native form have an extremely low propensity to be extracted into aqueous buffered solutions. The isolation of integral membrane glycoproteins (and proteins) requires disruption of protein-lipid interactions as well as disruption of protein-protein interactions. Since the associations between integral membrane proteins and lipids, as well as between the proteins themselves, involve both hydrophobic and ionic forces, efficient extraction requires agents that are able to disrupt both types of forces [7,8]. Strong chaotropic agents like urea and guanidine-hydrochloride or inorganic ions (e.g., SCN-, C107~,I-) rupture hydrogen bonds and denature solubilized globular proteins into randomly coiled polypeptide chains [7,8]. Chaotropic agents have been used with varying success to extract integral membrane proteins and glycoproteins. Partial release of membrane proteins can be achieved with organic solvents such as ethanol, chloroethanol, butanol, n-pentanol, acidified phenol or aqueous pyridine. These solvents extract lipids into the organic phase leaving most of the membrane glycoproteins and proteins either insoluble or in the aqueous phase [8] ; however, organic solvents alsohave certain disadvantages. They have deleterious effects on the structure of membrane proteins, and in many instances membrane lipids may partition into the aqueous phase in the form

343 of protein-lipid complexes. Another drawback of organic solvents is that they do not efficiently disaggregate integral membrane protein complexes. By far the most successful class of membrane solubilizing agents are the surfactants (detergents) [85]. These amphiphilic compounds exhibit high affinities for both membrane lipids and integral membrane proteins and are effective at relatively low concentrations. Membrane disrupting surfactants are classified according to their charge into nonionic (Triton X-100, Nonidet P40), zwitterionic (dimethyldodecyl glycine), anionic (sodium deoxycholate and other bile salts, lithium diiodosalicylate, sodium dodecyl sulfate) and cationic (dodecyltrimethylammonium bromide) detergents (for review see Ref. 85). The nonionic detergents have been used in numerous studies where it was desirable to retain enzymic, receptor or antigenic activities of membrane proteins. An additional advantage is the lack of charge on the detergent which allows the application of ionexchange chromatography for the purification of the solubilized proteins. However, the dissociative capacity of nonionic detergents is low, and they often fail to solubilize integral membrane proteins [86,87]. Use of anionic deoxycholate or zwitterionic dimethyldodecyl glycine allows better dissociation - more efficient solubilization of membrane components without loss of biological activity [88]. An even more efficient solubilizing agent is sodium dodecyl sulfate (SDS); it forms soluble complexes with both lipids and proteins, primarily by hydrophobic interactions. The dissociative capacity of SDS is so strong that most integral membrane proteins are dissociated to their monomeric polypeptide chains and lose their native structure in 1% solutions. Due to a constant ratio of binding (on a weight/weight basis) of SDS by different proteins, proteins can be separated according to their molecular weight by gel filtration or gel electrophoresis in detergent solutions [7,89,90].

VIB(2). Purification of solubilized membrane glycoproteins by conventional procedures Once solubilized and stabilized in buffered detergent solutions, membrane glycoproteins can be purified by gel filtration mad ion-exchange chromatography or by affinity chromatography on immobilized lectin derivatives (Section VIII). Here we will discuss a few examples of the application of conventional separation methods to the purification of integral membrane glycoproteins. Marchesi and Andrews [91] have isolated glycophorin, the major glycoprotein of human erythrocytes, after membrane solubilization with lithium diiodosalicylate. The solubilized material was extracted with phenol, and the aquous phase containing most of the soluble glycoprotein was dialyzed, washed with ethanol and dialyzed again to remove the detergent. Finally, contaminating proteins were removed by passing the soluble fraction through a phosphocellulose column at acidic pH. Glycophorin does not bind to phosphocellulose at low pH and emerges as a pure material with an apparent monomer molecular weight of 55 000 by SDS-polyacrylamide gel electrophoresis. Its homogeneity was confirmed by analysis of end groups and cyanogen bromide cleavage products. Glycophorin constitutes 70-80% of the total sialoglycoproteins of the erythrocyte membrane [5]. Marchesi and Andrews [91 ] reported that blood group A, B and MN activities as well as binding sites for influenza virus, P. vulgaris and wheat germ agglutinins were associated with the purified glycoprotein, although more recent studies have questioned the presence of AB0 determinants [92,93]. To test whether some of these activities were due to contaminating glycolipid, the glycoprotein preparation was extracted with chloroform/ methanol. After extraction glycophorin was water-soluble and retained all of the above activities. In contrast to lithium diiodosalicylate, the chaotropic agents urea (8 M) and

344 guanidine-hydrochloride (6 M) were ineffective in extracting glycophorin from erythrocyte membranes. Lithium diiodosalicylate probably acts like the anionic detergent: SDS, but unlike the latter, it can be quantitatively removed by dialysis [91]. Glycoproteins from the plasma membranes of lymphoid cells, platelets, liver ceils and various tumor cells have also been solubilized with lithium diiodosalicylate. For example, human platelet membranes contain a 100 000 dalton glycoprotein that can be extracted with lithium diiodosalicylate and purified on phosphonoceltutose [94]. Glycophorin has also been isolated and purified by Fukuda and Osawa [64] using different procedures. Erythrocyte membranes were solubilized with 1% Triton X-100 and 0.5% SDS, extracted with phenol, and the aqueous phase was removed and dialyzed. Further purification of this crude mixture was achieved by successive chromatography on DEAE-Sephadex (in the presence of 0.4% Triton X-t 00 and 5 mM EDTA)which retarded the sialic acid-containing glycoprotein, then on CM-Sephadex which removed contaminating proteins. One final gel filtration on Sephadex G-100 yielded an almost pure glycoprotein which was extracted with chloroform/methanol to remove trace quantities of contaminating gtycolipids. After these procedures the 53 000 dalton glycophorin monomer appeared homogeneous by polyacrylamide gel etectrophoresis, gel filtration and ultracentrifugation in the presence of SDS. Carbohydrate analysis indicated that 55% of the weight of the glycoprotein was sugar of which NeuNAC (23.6%), GalNAc (10.3%) and Gal (9.9%) were predominant; and GlcNAc, Man and Fuc constituded a small proportion of the total. Glycophorin purified from type 0 erythrocytes contained H(0) and MN blood group activities as well as binding sites for the lectins from P. vulgaris, L. culinaris, wheat germ, P. sativurn, Vicia faba, Sophora japonica, Ulex europaeus, Bauhinia purpuraea and Is'. graminea. After alkaline borohydride removal of part of the O-glycosidically-linked saccharide side chains, inhibitory activity against the latter three lectins decreased concomitant with increased inhibitory activity against concanavalin A and Ricinus communis agglutinin [64]. These findings indicated that different lectins which bind to a particular glycoprotein can bind to complementary saccharides on the same or on different side chains. The non-affinity procedures described above for the purification of glycoproteins from erythrocytes have been only partially effective in purifying glycoproteins from membranes of other cell types. Therefore, new approaches for the purification of glycoproteins by affinity chromatography on immobilized lectins have been utilized to effectively obtain all membrane glycoproteins in purified form. VII. Immobilized leetin derivatives useful for affinity chromatography Because of the analogy of lectin-saccharide interactions to those of antibody-antigen reactions, the purification of glycoproteins by immobilized lectins was a natural extension of the use of immobilized antibodies for antigen isolation [95]. Although the utilization of lectins is limited to certain saccharide-containing molecules, it has several advantages. First, lectins can be easily purified in large quantities; second, the specifically adsorbed glycoproteins can be eluted with readily available monosaccharides; and third, elution of bound material with saccharides can be performed at constant ionic strength in neutral or near neutral pH buffers with minimal deleterious effects on the glycoprotein. In contrast, elution of antigens from insolubilized antibody often requires use of denaturing conditions and extreme pH. Various examples of soluble glycoprotein purification on immobilized lectins have been reviewed elsewhere [96,97].

345 TABLE I SOME PROCEDURES FOR LECTIN IMMOBILIZATION References to each method are given in Tables II-VII. A. Direct lectin insolubilization 1. Crosslinking with glutaraldehyde 2. Crosslinking with L-leucine-N-carboxy anhydride B. Direct lectin conjugation to CNBr-activated agarose C. Lectin coupling to agarose via a spacer 1. Activated N-hydroxysuccinimide esters of succinylated aminoalkyl agarose 2. Carboxyhexyl agarose (Carbodiimide) 3. Aminohexyl agarose (Carbodiimide) 4. Glutaraldehyde derivative of Polyacrylichydrazido-agarose

Several different methods have been described for the preparation of insoluble or immobilized lectins (Table l). Lectins directly coupled to CNBr-activated agarose have been used extensively, while some studies have utilized other insolubilized lectin derivatives. We will not dwell here on the techniques of coupling proteins to insoluble matrices; these can be found in recent reviews [100,101] and in most of the specific work referred to below. It should be kept in mind that in some cases nonspecific (hydrophobic or ionic) adsorption to immobilized lectins has been encountered, and care must be taken to control such artifacts. For example, human fibroblast interferon was found to adsorb nonspecifically to concanavalin A (immobilized on CNBr-activated agarose) in the presence of the saccharide methyl~-mannoside. That this binding was due to hydrophobic interactions with concanavalin A was shown by elution of the interferon with ethylene glycol [102]. After comparing various coupling conditions, it was found that coupling at pH 9.0 apparently modified concanavalin A conformation and exposed hydrophobic groups on the lectin which were responsible for the subsequent nonspecific adsorption of interferon. When coupling was performed at lower pH (pH

Purification of cell membrane glycoproteins by lectin affinity chromatography.

329 Biochimiea et Biophysica Acta, 559 (1979) 329 376 © Elsevier/North-Holland Biomedical Press BBA 85199 PURIFICATION OF CELL MEMBRANE GLYCOPROTEIN...
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