Molecular analysis of the lymphocyte membrane

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 8, pp. 757-772, 1984. 0145-305X/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved

MOLECULAR ANALYSIS OF THE LYMPHOCYTE MEMBRANE

Gregory W. Warr, Gerardo R. Vasta, John J. Marchalonis, Robert C. Allen* and Douglas P. Anderson$ Department of Biochemistry and *Department of Pathology, Medical l~niversity of South Carolina Charleston, South Carolina 29425 * U.S. Fish and Wildlife Service, National Fish Wealth Research Laboratory, Box 700, Kearneysville, WV 25430

ABSTRACT Methods for the molecular analysis of lymphocyte membranes are reviewed briefly, and wherever possible presented in a manner relevant to comparative studies. The specific areas reviewed include the bulk isolation of lymphocyte membranes, the use of radioisotooes to covalently label lymphocyte membrane molecules, the use of lectins to characterize membrane glycoconjugates, and our current understanding of lymphocyte membrane immunoglobulins.

INTRODUCTION In the past 20 years the study of the lymphocyte membrane has made tremendous progress. The techniques for specific membrane labeling with radioisotopes, and the techniques for bulk purification of plasma membrane in high purity and adequate yield have been developed, refined and widely exploited. At the same time the techniques for analysis and purification of membrane constituents (particularly by such techniques as affinity chromatography) have also developed to the extent that a number of important membrane proteins have now been characterized to the level of amino-acid sequence, and many others identified and partially characterized. To review such an enormous field of activity within the confines of this article is clearly impossible, and we will restrict ourselves to certain selected topics. In particular, we will concentrate upon comparative studies, which have received relatively little attention from investigators in the area of lymphocyte membrane structure and function, but which we believe can shed valuable light on the function and evolution of the lymphocyte. BULK ISOLATIONOF THE PLASMA MEMBRANE Since the plasma membrane is in essence a bi-molecular film of lipids with associated proteins, it is not the most abundant cellular organelle, and the value of l0 -12 g of membrane per lymphocyte seems to be within the correct range. Thus, for the 757

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o p t i m a l study of l y m p h o c y t e m e m b r a n e proteins, (of which t h e r e may well be fifty or more) it is essential to m a x i m i z e the final yield of m e m b r a n e , without sacrificing its purity (i.e. one must exclude, as far as possible, c o n t a m i n a t i o n with m e m b r a n e s from o t h e r intraeellular, organelles). The initial s t a g e in m e m b r a n e isolation is to disrupt the l y m p h o e y t e s . This is usually a c c o m p l i s h e d by osmotic lysis, by the application of shear forces, or by sudden release of pressure (e.g. nitrogen c a v i t a t i o n techniques). Osmotic lysis, unless coupled with m e c h a n i c a l shear forces, is a v e r y i n e f f i c i e n t way to disrupt l y m p h o e y t e s for the p r e p a r a t i o n of m e m b r a n e s . Both nitrogen c a v i t a t i o n techniques and controlled m e c h a n i c a l shear forces have been used successfully for l y m p h o c y t e disruption as an initial s t e p to m e m b r a n e isolation by a number of groups (1,2) and we have found controlled shear obtained using a m e c h a n i c a l pump (3) to be s a t i s f a c t o r y for disruption of fish l y m p h o e y t e s . Once the l y m p h o e y t e s have been disrupted, by w h a t e v e r method, the plasma m e m b r a n e will tend to spontaneously r e f o r m as closed vesicles, although under c e r t a i n c i r c u m s t a n c e s large sheets of m e m b r a n e can r e m a i n . The lysed cells are then subjected to low-speed e e n t r i f u g a t i o n to r e m o v e i n t a c t nuclei, lysosomes and mitochondria, and then the remaining p a r t i c u l a t e s (mierosomes) are pelleted at high speed. In the typical second phase of purification, the m i e r o s o m e s are f r a e t i o n a t e d by isooycnic c e n t r i f u g a t i o n on a gradient of sucrose. The banding I~osition of the plasma m e m b r a n e (and other) vesicles is influenced by a v a r i e t y of f a c t o r s , including the size of the vesicles. The distribution of m e m b r a n e s derived from all possible cellular sources is monitored by a v a r i e t y of techniques. These include the q u a n t i t a t i o n of e n z y m e a c t i v i t i e s believed to be a s s o c i a t e d with p a r t i c u l a r sub-cellular f r a c t i o n s (Table 1), the use of the e l e c t r o n m i c r o s c o p e (plasma m e m b r a n e vesicles should be smooth) and the following of m a r k e r s s p e c i f i c a l l y introduced into the plasma m e m b r a n e , such as c o v a l e n t labeling with radioiodide by the l a e t o p e r o x i d a s e - c a t a l y z e d technique (see the following Section). Once this has been done, a discontinuous sucrose g r a d i e n t can usually be designed to give purification of plasma m e m b r a n e with minimal c o n t a m i n a t i o n by m e m b r a n e s of other origins. From the l i t e r a t u r e , it a p p e a r s t h a t a t l e a s t 25 !~ercent of the t o t a l plasma m e m b r a n e can g e n e r a l l y be isolated in a s a t i s f a c t o r y p r o c e d u r e .

TABLE I. Some Markers of Sub-Cellular Fractions Fraction

Marker

Plasma Membrane

l) 2) 3) 4) 5) 6) 7)

Externally introduced covalent radiolabels Leetin binding Highcholesterol/phospholipid ratio 5' Nucleotidase Na+, K+ dependent ATPase Certain proteases (e.g. dipeptidyl peptidase IV) Smoothappearance of vesicles in the electron microscope

Nucleus Mitochondria Lysosomes Endoplasmic Reticulum Cytoplasm

DNA Succinic dehydrogenase Acid phosphatase Glucose-6-phosphatase Lactic dehydro~enase

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The plasma membrane, once purified, can be used for many purposes. These include i) the biochemical analysis of total membrane lipids, proteins and glycoproteins, 2) the investigation of the orientation and membrane penetration of molecules of interest, and 3~ the further purification and characterization of membrane molecules. In this latter context, it can be noted that while some investigators use a rigorous plasma membrane purification step in their isolation of a membrane protein (4), this has been partially or completely dispensed with by others without any problems (5). One of the initial stages in characterizing a purified plasma membrane preparation is often to observe the profile of polypeptides obtained by ~D~-polyacrylamide gel electrophoresis. In Fig I we show a comparison, by this method, of plasma membrane purified from the thymocytes of mice and rainbow trout (Salmo gairdneri). Two points of interest can be noted. First, the thymic membrane of the trout apparently lacks a polypeptide migrating in a position (between m.wt. 20,000 and 30,000) comparable to that of the Thy 1 molecule (bands 2 and 3). ~ince the gels were stained by a sensitive silver technique (6) it seems likely that the trout Thy-i homologue is either an extremely minor membrane component, or if it exists in typical mammalian abundance, it must have a different SDS-gel electrophoretic mobility. The second point of interest is the extremely large proportion of membrane polypeptides of both mouse and trout thymocyte membrane that show identical migration (9 out of 14 major bands).

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Although this clearly needs further study by techniques with the c a p a c i t y for analysis bv multiple parameLers (not just a p p a r e n t mass, 3), the initial implication is t h a t t h e r e has been a t r e m e n d o u s c o n s e r v a t i o n (or c o n v e r g e n c e ) of l y m p h o c y t e m e m b r a n e proteins over a period of at l e a s t 300 million y e a r s of evolution. EXTERNAL COVALENT (NON-PERMEANT) V E V B R A N E LABELS The major classes of vectorial reagent which have been used to study lymphocyte membrane proteins (7) generally consist of enzyme systems which either act directly on surface components (e.g. ~alactose oxidase) or generate products that are highly reactive and which do not penetrate the plasma membrane (e.g. protein kinase, lactooeroxidase). One exception is Hexanoyldiiodo-N(4-azido-2-nitrophenyl) tyramine which operates on a different principle; this is a photo-activated probe which is lipid soluble (8). ~'his probe is allowed to dissolve in the lipid phase of the membrane (in the dark) and followin~ activation by light at 480 nm it will react with peptide (and other) bonds. This probe, thus, can be used to identify those portions of a surface associated molecule which occur within the lipid phase of the plasma membrane (8). Since the majority of biologically relevant, exposed surface proteins are glycosylated, it is useful to have means of labeling the exposed ~lycosyl moieties. Terminal sialic acid residues can be labeled specifically bv oxidizing with periodate followed by reduction of the aldehyde using tritiated NaBH4 (9). Furthermore, it is possible to cleave terminal sialic acid using neuraminidase and then to label subterminal galactose using a galactose oxidase procedure (10). The galactose oxidase oxidation step will create a reactive aldehyde either on terminal ~alactose or ~alactosamine and this aldehyde is then reduced to an alcohol using tritiated NaBTI4. Under usual conditions glycolipids label using this approach, but these can be distinguished and separated readily from glycoproteins, e.g. by virtue of their solubility in non-polar solvents such as ether. The most widely used enzyme system for the labeling of lymphocyte membrane proteins or glycoproteins is that of lactoperoxidase-catalyzed radioiodination (LPO). This system was first developed as a rapid and gentle means for the iodination of soluble proteins (ll), and it was subsequently shown to function as an external label for erythrocytes (12), and lymphocytes (13) as well as a variety of nucleated cells. LPO iodinates predominantly exposed tyrosine residues, but an unstable reaction with histidines can also occur (l I), and it has been reported that minor degrees of modification of other reactive groups (such as sulfhydryl) can occur under certain conditions. It is important to emphasize that the capacity of any particular label to react only with external surface molecules of a particular cell type must always be carefully established before interpretation can be made of the results. It is important to demonstrate that the label used is actually restricted to surface components. This is usually done by means of one or more of the following: electron micrographic autoradiography, plasma membrane isolation or by isolation and characterization of components known to be associated with the cytoolasm or with the plasma membrane and determining the relative label in each. The effectiveness of the LPO system as a selective means of labeling membrane oroteins of" intact cells has been verified by electron microscope autoradio~raphy (13), and by direct isolation of the labeled plasma membrane (14). In principle, it should be possible to tailor the LPO system for the needs of virtually any cell system. This process depends upon a balancing of the proper amounts of lactoperoxidase, the proper amount of iodide (carrierfree plus carrier if used) and the amount of H202 present in the reaction mixture. A variety of lymphocyte surface proteins can be labeled using the ]actoperoxidase-catalyzed approach and these will be discussed below. Table 2 gives a partial listing of lymphocyte surface proteins that have been detected and isolated by use of lactoperoxidase-catalyzed radioiodination followed by solubilization procedures involving detergent lysis or shedding into the medium. Specific molecules were isolated most frequently by use of antibodies. ~onoclonal hvbridoma antibodies are now becoming very useful in the study of isolated surface receptors. Other non-permeant membrane labels have not been used to any great degree on lymphocytes,

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although studies with 3H-trinitrobenzene sulphonate show that this label couples to cell membrane H-2, la and IgM and IgD molecules (15,16). A number of surface glycoproteins, notably high molecular weight molecules of T cells and B cells are labeled using the galactose oxidase-tritiated NaBH4 approach (I0,17). The sialic acid labeling procedure using periodate followed by tritiated borohydride has been used to isolate human m lymphocyte differentiation antigens T4 and T5, following solubilization in NP40/deoxycholate and the use of monoclonal antibodies directed against these two surface markers for specific isolation (18). The T4 antigen was reported to be a single chain 62,000-dalton glycoprotein while the T4 antigen is a complex of two glycoproteins, one of apparent mass 30,000 daltons and the other 32,000 daltons. Similar glyeoproteins of mouse T cells have been isolated with antibodies to murine Lyt l and Lyt 23 antigens. An interesting difference in the capacity of lactoperoxidase and the galactose oxidasetritiated NaBH4 approaches was found in that alloantisera to Lyt l.l precipitated a 67,000 dalton glycoprotein and also an 87,000 dalton glycoprotein labeled with borohydrides but these components were not detected when the same cells were labeled with 1251-iodide using the LPO system (19). This result illustrates the fact that a battery of surface labels, preferably reactive with different functional groupings, should be used if one wishes to obtain an accurate picture of the membrane representation of certain

TABLE 2 Partial Listing of Lymphocyte Surface Proteins Known to be Labeled by Lactoperoxidase-Catalyzed Radioiodination Surface Protein

Properties

H-2 alloantigens HLA aIloantigens

glyeoproteins, nom. M.W. 45,000; associated with B2 microglobulins ( B20)

TL alloantigen T/T alloantigen la aIIoantigen

nom. M.W. 45,000; associated with B21J nom. M.W. 45,000; associated with 8 20 glycoproteins; nora. M.W. 28,000; 32,000

Immunoglobulins IgM IgD "IgT"

glycoprotein; (Lu)2 new "7S" isotype; covalent (S-S) dimer of heavy chains of nom. M.W. 65,000-70,000; light chains noneovalently associated or absent

Receptor for dinitrophenyl hapten

B cell IgMm, IgD

Thy-l alloantigen

glycoprotein; nom. M.W. 27,000

Acceptors for lectins

collection of glycoproteins

Heteroantigens

glycoproteins; nora. M.W. 27,000, 150,000-200,000

gp 54

major surface glycoprotein of human B cell subset; nora. M.W. 54,000

p 69,71

glycoprotein, marker for human T cells and some B cell leukemias; nom. M.W. 69,000; 70,000

Lyt 2/Lyt 3

murine suppressor m cell markers; glycoproteins, nom. M.W. 35,000

gp 100

Human acute lymphoblastic leukemia-associated glycoprotein; nora. M.W. 100,000

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components. In addition to the problem of differential labeling, the possibility that a given solvent system might not extract a certain membrane component must also be considered, and this phenomenon can exacerbate problems of isolation as has been reported in detail for immunoglobulin-like receptors of T cells (e.g. see ref. 20). It is interesting that of all the molecules that have so far been radioiodinated using the lactoperoxidase-catalyzed method, the subunit that is associated with the membrane is glycosylated to some degree. Non~lycosylatedsubunits can be associated with the molecules, however. In the case of major histocompatibility complex alloantigens, ~2 microglobulin is noncovalently associated with the 45,000 dalton chain, and the light chain which is usually covalently bound to the heavy chain is likewise non-glycosylated. LYMPHOCYTE MEMBRANE IMMUNOGLOBULINS Studies on the antigen receptors of the two major subdivisions of vertebrate lymphocytes, the T and B cells, have not progressed at equal rates. Whereas a tremendous amount of information on B cell membrane Igs has accumulated, the Ig nature of the T cell receptor is still an area of active research (21) and there can be little doubt that definitive investigations of the nature of the T cell receptor will probably involve pushing current biochemical techniques up to or beyond their current limits of sensitivity. Thus, because to do credit to the current, unresolved status of the T cell antigen receptor would require a major review all to itself, we shall restrict ourselves here to the question of B cell membrane Igs. IgMis a universal B lymphocyte membrane receptor. The monomeric form of IgM (u2L2) has been found on the membrane of B lymphocytes of every species in which it has been sought (Table 3). Other membrane l~s do occur, with a variable representation. For example, IgD, or a putative homolo~ue, has been identified on the membrane of B cells of many primates, rodents and chickens (Table 3), and IgG is also clearly demonstrable on certain B cells. The function of the various isotypes of Ig on the lymphocyte membrane, and the significance of the absence or presence of particular ones has been the subject of extensive investigation and speculation (e.g, see Ref. 22), a detailed discussion of which is beyond the scope of our review. There is no clear pattern governing the expression of IgM, IgG, and the IgD-like molecule, except that IgM is universally present on the B cells of all species so far examined. Membrane IgM differs from the secreted counterpart. Apart from the fact that membrane IgM is a ~2L2 monomer, whereas the secreted form is a (u2L2)n polymer where N equals 4, 5 or 6 (reviewed by Warr and Marchalonis (25)) it is now clear that the carboxyl terminus of the membrane U chain possesses a hydrophobic region (missing from the secreted molecule) that associates specifically with, and probably allows penetration of, the plasma membrane. The major experimental observations, stemming from the earliest experiments of Melcher and Uhr (26,27) suggesting that this phenomenon occurred, are summarized in Table 4. It is now clear that membrane Igs of isotypes other than Ig~I can associate in a similar fashion (i.e. by a hydrophobic tail), with the plasma membrane (31,32,33). One of the major tasks remaining is to determine the significance of this structural feature of membrane Igs for the mechanisms that give rise to lymphocyte activation following binding of antigens to membrane Ig, and presumed transmembrane transmission of a signal leading to this activation. Table 5 summarizes what is known (or deduced) about the amino acid sequences of the transmembrane portions of some mammalian membrane proteins, comparing Ig~¢, IgG, HLA-Dr, the DC-I alloantigen, and glycophorin. While the hydrophobic nature of all these segments is clear (no charged amino acids), and the internal similarity of the Igs and the HLA-DR and DC-I molecules is clear, there is little similarity between the diverse classes of molecule, unless one wishes to speculate on the significance of the 17-22% identity between glycophorin and the HLA-DI~ and DC-I molecules. Current evidence indicates that the transmembrane portion of the Ig molecule probably forms an ce-helix (31,32).

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TABLE 3. Occurrence of Immunoglobulin Isotypes on Vertebrate I,ymphocytes Species

Immunoglobulins Reported

Man and primates Mouse Rat Chicken Reptiles Amphibia Fish

IgM (monomer), IgG, Igr), IgA Ig~ (monomer), IgG, IgO-like IgM (monomer)IgG, IgD-like IgM (monomer), IgY, IgD-like I~M (monomer),IgY IgM (monomer),IgY IgI~q (monomer)

Modified from Wart (23) in which original references are given. Reference to chicken IgD is Leslie (24). TABLE 4 Differences Between Membrane and Secreted Forms of Ig: A Historical Perspective Observation

Reference

Difference in SDS-gel electrophoretic mobility of la chains

Melcher and Uhr (26)

Difference in buoyant density of secreted and membrane forms of IgM

~elcher and Uhr (27)

Detergent binding of membrane IgM

Vassalli et al. (28) Parkhouse et al. (29)

Membrane association of the heavy chain C-terminus

Owen et al. (30)

Inferred sequence of carboxyl terminus of membrane Ig heavy chains

Rogers et al. (31,32) Tyler et al. (33)

LECTINS IN THE STUDY OF LYMPHOCYTE MEMBRANE GLYCOCONJUGATES Lectins (see review Ref. 36-44) are l) sugar binding proteins or glycoproteins of nonimmune origin which agglutinate cells and/or precipitate glycoconjugates (45) 2) devoid of enzymatic activity towards the sugars to which they bind and 3) do not require free glycosidic hydroxyl groups on these sugars for the binding (46). They have been isolated from plants and animals (invertebrates and vertebrates) and can be considered ubiquitous in nature (47). When interacting with cells, plant and invertebrate lectins exert a variety of effects which include binding and agglutination, blastogenesis, cytotoxicity, immunosuppression, release of hormones and effector proteins, etc. (reviewed by Sharon and Lis, (36); Nicolson (38)). Their specificity for carbohydrates has converted them into fine immunochemical tools of wide application in the study of the cell membrane glycoconjugates, particularly of lymphoid cells. Carbohydrate structures recognized by lectins on the cell surface are commonly identified by quantitating the capabilities of monosaccharides, oligosaccharides and glycoproteins to inhibit agglutination of the particular cell or the precipitation of glycoconjugates. The nominal specificity of the lectin (Table 6) is defined by the carbohydrate that requires the lowest concentration to produce a standard degree of inhibition. However,the affinities (measured as association constants Ka) for carbohydrates linked to glycoconjugates are much higher than for the free monosaccharides (see reviews by Lis and Sharon (41); Rapin and Burger (40)) resulting m a strong binding of the lectin to its receptor on the cell surface; this is due to the fact that structural and conformational aspects of the oligosaceharide are important factors for the lectin binding.

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Lectin source

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TABLE 6 Sources and Nominal Specificities of Selected Lectins Common name Nominal specificity (abbreviation)

Solanum tuberosum ~potato)

Solanum agglutinin ---NY~)

8-D-GIcNAc

Ricinus communis (castor bean)

Castor bean lectin (RCAI, RCAII)

~-Gal, B-C,aI~Ac

Arachis Hypogaea ~peanut)

Peanut lectin (P~TA)

Gal 9-I-3 GalNAc

Canavalia ensiformis (Jack bean)

Concavalin A (Con A)

~-~an, ~Glc, GlcNAc

Lens culinaris

Lentil lectin

¢~-~an, p-C,Ic, ~-D-GlcI~Ac

(LCH) Vicia villosa

Vicia villosa lectin

D-GaINAc d-3 DC,al

~ m a x ~ean-~-

Soybean agglutinin (SBA)

% 6-GalNAc

Phaseolus (Red kidney bean)

phytohemagglutinin GalNAc (PHA-E, PHA-L, PHA-LE) -D-Ga]

Lotus tetragonolobus Te~-etTa~Sonolobus purpureas) eeds)

Asparagus pea lectin

~-L-Fuc

Triticum (Wheat germ)

Wheat germ agglutinin (WGA)

-D-GIcNAc sialic acids

Maclura pomifera O-(-O-sage orange)

Osage orange lectin

D-C,al, r)-GalNAc

Helix pomatia agglutinin

~-D-GalNAc

Helix pomatia ~arden snail albumin gland) Limulus polyphemus (horseshoe crab serum)

---raP-h7 Limulin III Limulus polyphemus agglutinin (LPA)

sialic acids

One of the reasons that stimulated research on the interaction of lectins and cell membranes was the fact that certain lectins could selectively agglutinate tumor or virally transformed cells (or at least agglutinate them at higher titer than their normal counterparts, (48-50)). Although many exceptions to this rule are found (see reviews by Burger, 48,50) the differential binding to tumor cells was thought to be due to an increased number of binding sites, their mobility on the cell surface or to such morphological features of the tumor cell membrane as density of microvilli (see reviews by Lis and Sharon, 37, Burger 48, 50). Howeverresults differ from every cell system tested. In the case of Limulus lectin, which agglutinates human chronic lymophocytic leukemic lymphocytes at higher titers than for normal peripheral blood lym~)hocytes (NPBL) (51), exhibits high agglutination titers with phytohemagglutinin-transformed NPBL (52) suggesting that the higher agglutinability is not a property of the leukemic cell but a consequence of the blastic transformation. However,the potential of some plant lectins, including peanut (PNA), wheat germ (WGA) and lentil (LA), as markers for tumor ceils is currently being examined (53-55).

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The lectin properties of binding and agglutinating untreated, enzyme-treated, normal and tumor cells based on their carbohydrate recognition capabilities, oriented early work towards the characterization of lymphoid cells on the basis of the presence or absence of receptors for lectins of well defined specificity, such as potato, ricin (RCA), peanut (PNA) and Limulus (LPA) lectins, and thus map the topochemical arrangements of these receptors on the lymphocyte cell surface (56-61). The number of receptors on the cell surface has been measured by fluorescein, ferritin or radioactive-labeled lectin binding techniques for a wide variety of lymphoid cells and lectins, surprisingly resulting in the order of ]06-I07 binding site per cell for almost all cells considered; in many cases the lectin-cell surface receptor affinity constants were measured simultaneously resulting in Ka values in the order of 105-107 (see review by Lis and Sharon, 41). Once bound to the lymphocyte cell surface, in most cases the lectin-receptor complexes are subject, at the adequate temperature, to redistribution (patching and capping) and rotation (62-65, 41), which reflect the metabolic changes the cells go through after interaction with the lectin; in particular, capping has been shown to be an energydependent process (see review by Nicolson, 66). Certain lectins (mitogenic lectins) trigger the intense biochemical changes (67) that will lead to mitosis and cell proliferation; these early events have been reviewed by Decker and ~archalonis (68). Fluoresceinated lectins have been successfully used to monitor cell surface changes associated with lymphocyte transformation (69). In addition to the analysis of number and distribution of lectin receptor and the measurement of affinity constants involved, several attempts to isolate the receptors have been made thorugh different appraoches: a) membranes were solubilized and the glycoproteins precipitated by the lectin and by the antiserum to the lectin, b) cells were bound to lectin coupled to Sepharose beads, the cells lysed and washed with detergents; the glycoproteins were eluted by the monosaccharide, c) complexed lectin-membrane fragments were glutaraldehyde coupled to a phage, the density perturbant, and separated from the remaining material by centrifugation; the receptors are eluted from the complex. These methods have been reviewed by Sharon and Lis (41). Isolated glycoroteins (lectin receptors) and their oligopeptides are analyzed by gel filtration, electrophoresis, etc. (70). The visualization of the lectin-receptor complexes on the surface of the lymphocytes has been achieved qualitatively and semi-quantitatively by various techniques, including fluorescence microscopy, scanning and transmission microscopy and freeze fracture methods, reflecting the presence, distribution and quantitation of selected cell surface g]ycoconjugates. In a typical procedure, lectins are linked to an electron dense molecule like hemocyanin, colloidal gold or ferritin or to an enzyme molecule like horseradish peroxidase that interacts with a substrate to give a stainable reaction product (71). Limitations of these methods are due to endocytosis and shedding of the labels, lack of exposure of certain receptors, induced active or passive movement of the receptors, etc., on the unfixed cells (71). The biotin-avidin system as an intermediary for the labelling is advantageous with respect to the ferritin-protein methods in that it has but a minor effect on the binding properties of the conjugated protein (lectin) (72). Receptors on human and mouse lymphocytes and thymocytes have been detected by using" a two-step technique which involves glycosylated peroxidase and ferritin and, as in the previous method, avoids direct conjugation of the label to the lectin (73). The presence of lectin receptors in certain cell populations has been the basis for the development of methods of cell characterization and separation. These methods involve the separation of a) different cell types like monocytes from lymphocytes by agglutination with PNA, or separation of mouse erythrocytes from thymocvtes by conconavalin A (Con A)derivatized nylon fibers (74); b) different classes of lymphocytes, like the separation of T and B cells by soybean lectin gBA (75); c) maturation subsets, as in the separation of mature (medullary) from immature (cortical) mouse thymocytes (76) with PNA, which result in different functional subsets (77). Invertebrate lectins have been also useful in the characterization and separation of lymphocytes. Human neuraminidase-treated T and B lymphocytes have been separated successfully employing a

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Sepharose-bound Helix pomatia (HP) lectin column: HP lectins bind only neuraminidase treated peripheral T cells, but not B cells (78-80). However, in untreated chronic lymphocytic leukemia HP binds 90-I00% of the neuraminidase treated lymphocytes (81). Moreover, in human cord blood the majority of cells are NP positive and the HP receptors would be a maturation marker for B cells (82). Natural killer cell populations can be enriched based on the differential distribution of the HP receptor in the lymphocyte populations (83,84). Other functional subsets of lymphocytes have been separated by the use of invertebrate and plant lectins: helper and suppressor mouse ~ cells are agglutinated selectively by LPA and PNA respectively (85). Bone marrow and spleen mouse cells have been fractionated by SBA and PNA yielding subpopulations that can reconstitute lethally irradiate mice without complications due to graft versus-host reactions (86). Mouse and human bone marrow cells can be enriched for spleen or soft agar culture colony-forming cells by the alternative use of SBA and PNA (87). Human lymphocyte subclasses have been separated by Lens culinaris lectin and WGA. These subclasses, although they did not differ f r o m the starting populations with respect to the percentges of T and B lymphocytes and macrophages, responded differently to stimulation in vitro by a panel of plant mitogens (88). By the use of fluorescein-labeled PNA it has been found that the PNA-positive mouse cell subpopulations from thymus and spleen are different in their sensitivity to corticosteroids and irradiation (89). Further work (90) showed that P~A could serve as a marker for human T lymphocyte subsets as well. Flow microfluorometry analysis of mouse thymocytes treated with fluorescein-labeled lectins revealed that cell subpopulations can bind various amounts of lectin which do not correlate with the cell size (91). Maclura pomifera lectin only binds to cells of the T-lymphocyte lineage in the rat (92) and Lotus tetragonolobus lectin agglutinates chicken cells from bursa and spleen but does not agglutinate thymocytes or peripheral blood lymphocytes (93). Cell subsets from mouse bone marrow, thymus and spleen cells were demonstrated by autoradiography when exposed to 1251_labeled lectins (94). Another example of the use of lectins for the identification of lymphocyte subsets is the separation of cytotoxic Ly I-2 + T-lymphocytes by the Vicia villosa lectin. The lectin has a high specificity for the moeity D-GaINAc ~ l-3 D-GaI--T-(~5)and it binds to a unique glycoprotein (T145) present in the cell surface of the Ly I-2 + cytotoxic lymphocytes (96,97). These cells were separated from natural killer and "K" cells by the immobilized lectin (98). Finally, lectins are currently applied to the study and fractionation (99) of glycoconjugates from different sources (See collections of papers edited by T.C. BogHansen, I00-I02, and H. Peeters, I03)including lymphocyte membrane glycoconjugates: a good example is the analysis of the carbohydrate portion of the mouse thymocyte Thy-l glycoprotein by lectin affinity (I 04).

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