Binding Specificities of the Sialoadhesin Family of I-type Lectins
SIALIC ACID LINKAGE AND SUBSTRUCTURE REQUIREMENTS FOR BINDING OF MYELIN-ASSOCIATED GLYCOPROTEIN, SCHWANN CELL MYELIN PROTEIN, AND SIALOADHESIN*

(Received for publication, February 19, 1997, and in revised form, April 9, 1997)

Brian E. Collins Dagger §, Makoto Kiso , Akira Hasegawa , Michael B. Tropak par , John C. Roder par , Paul R. Crocker ** and Ronald L. Schnaar Dagger Dagger Dagger

From the Dagger  Departments of Pharmacology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the  Department of Applied Bioorganic Chemistry, Gifu University, Gifu 501-11, Japan, the par  Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto M5G 1X5, Canada, and the ** Imperial Cancer Research Fund Laboratories, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The carbohydrate binding specificities of three sialoadhesins, a subgroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an alpha 2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1balpha >>  GD1a = GT1b >>  GM3 = GM4 >>  GM1, GD1b, GD3, GQ1b (nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal alpha 2,3- or alpha 2,8-linked sialic acids with the following rank-order potency of binding: GQ1balpha  > GD1a = GD1b = GT1b = GM3 = GM4 > GD3 = GQ1b >>  GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid acetamido group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.


INTRODUCTION

Sialoadhesins (1) are a structurally and functionally related family consisting of five immunoglobulin superfamily lectins (I-type lectins) (2) including myelin-associated glycoprotein (MAG),1 Schwann cell myelin protein (SMP), CD22, CD33, and sialoadhesin. MAG and SMP are found on oligodendroglia and Schwann cells in the nervous system (3, 4), CD22 is expressed on a subset of B lymphocytes, sialoadhesin on a subset of macrophages, and CD33 on cells of myelomonocytic lineage. Sialoadhesins have been proposed to mediate cell-cell recognition, perhaps via their carbohydrate binding activities (5-7). Each sialoadhesin family member has two or more Ig-like domains: an amino-terminal V-set domain followed by one or more (up to 16) C2-set domains (8). Domain deletion and site-directed mutagenesis of sialoadhesin and CD22 localize their carbohydrate-binding sites to the amino-terminal V-set domain, with contributions (for CD22) from the adjoining C2-set domain. These first two domains share very high amino acid sequence similarity between MAG and SMP (>70%) and significant similarity across all I-type lectins (>30% in pairwise comparisons) (2, 8, 9).

Each I-type lectin binds to carbohydrate structures bearing a nonreducing terminal sialic acid (1, 6, 10). Sialic acids are a common nonreducing terminus of vertebrate glycoconjugates and appear to play uniquely important roles in recognition phenomena. Because sialic acids may be linked to Gal, GalNAc, or other sialic acid residues at various positions and because they may carry different substituents on their 9-carbon base structure, the sialic acids represent a diverse family of carbohydrate determinants (11). In certain sialic acid-dependent recognition systems, determinant stringency is low. For example, selectins bind to oligosaccharides bearing truncated sialic acids (12) or appropriately placed anionic groups (sulfates, carboxylic acids) otherwise unrelated to the sialic acid structure (13-16). In contrast, sialoadhesins appear to have more stringent sialic acid specificities (see "Discussion") (9). In this study, we used cells expressing different sialoadhesins to explore and compare the fine structural preferences of their binding to target sialylated glycoconjugates.


EXPERIMENTAL PROCEDURES

Gangliosides

The ganglioside structures used in this study are shown schematically in Fig. 3. Purified bovine brain GM1, GD1a, GD1b, GD3, and GT1b were from EY Laboratories (San Mateo, CA) or Matreya, Inc. (Pleasant Gap, PA), and GQ1b was from Accurate Chemical & Scientific Corp. (Westbury, NY). GM3 (NeuAc form) was from Sigma. GQ1balpha , GT1beta , GM1alpha , GM4 and its derivatives, and GM3 derivatives were synthesized de novo using previously described methods (17-19). GD1a gangliosides bearing sialic acids with truncated glycerol side chains (7/8-aldehydes) were prepared by mild periodate oxidation followed (as indicated) by sodium borohydride reduction to form the 7/8-alcohols (20). GD1a gangliosides bearing sialic acid ethyl esters, 1-amides, and 1-alcohols were prepared as described (20). Products were analyzed by thin-layer chromatography and fast atom bombardment mass spectrometry at the Middle Atlantic Mass Spectrometry Laboratory (21).


Fig. 3. Structure-function studies of MAG-, SMP-, and sialoadhesin-mediated cell adhesion to gangliosides. Binding data are summarized from Figs. 1 and 2. Potency (concentration supporting approximately half-maximal adhesion) is indicated in the following ranges: +++, <10 pmol/well; ++, 10-100 pmol/well; +, >100 pmol/well; +/-, very low but statistically significant adhesion over background; and -, no adhesion over background at any concentration tested. Statistically significant adhesion above background (two-tailed Student's t test) is indicated as follows: *, p < 0.001; and Dagger , p < 0.01. §, this preparation of GQ1b contains a small amount of contaminating GT1b (20). NeuAc-NeuAc linkages are all alpha 2,8; other NeuAc linkages are as noted in the key.
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I-type Lectin Transfection of COS Cells

Full-length I-type lectin cDNAs were cloned into the eukaryotic expression vector pcDNA1/Amp (sialoadhesin only) or pCDM8. The lectins used in this study included mouse sialoadhesin (8), both the long (L-MAG) (22) and short (S-MAG) splice variants of rat MAG, quail SMP (4), and human CD22 (seven-Ig-like domain variant, CD22beta ) (23, 24).

Plasmids were propagated in Escherichia coli MC1061/p3 and purified by polyethylene glycol precipitation. COS-1 cells, routinely maintained in 10% fetal calf serum in Dulbecco's modified Eagle's medium at 37 °C in a humidified atmosphere of 90% air and 10% CO2, were transiently transfected with lectin-expressing plasmids via a high efficiency procedure (using 40 µg/ml DEAE-dextran) (25). Transfected cells were returned to culture for 40-50 h to allow lectin expression to proceed and then were detached from plates for adhesion experiments (see below). Lectin expression was confirmed by flow cytometry and/or immunocytochemistry using the following monoclonal antibodies: mAb 513 (MAG/SMP cross-reactive) (4, 7), SER-4 (sialoadhesin) (26), and Chemicon 2112 (CD22; Chemicon International, Inc., Temecula, CA).

Microplate Cell Adhesion to Adsorbed Glycolipids

Adhesion was performed as reported previously (20, 22, 27). Aliquots (50 µl) of ethanol/water (1:1) containing phosphatidylcholine (0.5 µM), cholesterol (2.0 µM), and gangliosides (concentrations as indicated) were added to microwells (96-well Serocluster, Costar Corp., Cambridge, MA). Plates were incubated for 90 min uncovered at ambient temperature to allow partial evaporation and lipid adsorption (28, 29), after which the wells were washed with water. Wells were preblocked by addition of 100 µl/well Hepes-buffered Dulbecco's modified Eagle's medium containing 1.5 mg/ml bovine serum albumin. Plates were covered and incubated for 10 min at 37 °C prior to cell addition (see below).

Transfected COS cells were harvested using hypertonic Ca2+/Mg2+-free phosphate-buffered saline containing 1 mM EDTA as described (22), collected by centrifugation, and resuspended at 107 cells/ml in Dulbecco's phosphate-buffered saline containing 2 mg/ml bovine serum albumin. Transfected cells were pretreated with neuraminidase, which enhances cell adhesion without changing carbohydrate binding specificity (20), as follows. Aliquots of cells (500 µl) were placed in 1.5-ml microcentrifuge tubes, and 10 milliunits of Vibrio cholerae neuraminidase (Calbiochem) were added. Suspensions were incubated for 1.5-2 h at 37 °C with end-over-end mixing. Cells were collected by centrifugation, washed twice with Dulbecco's phosphate-buffered saline containing 2 mg/ml bovine serum albumin, and resuspended at 250,000 cells/ml in Hepes-buffered Dulbecco's modified Eagle's medium containing 1.5 mg/ml bovine serum albumin. Cell viability was determined by trypan blue exclusion on representative transfected cells. Prior to pretreatment, cells were 84% viable. After neuraminidase or control pretreatment, viability ranged from 81 to 85%, essentially unchanged from the freshly collected cells. Quantitation of cell adhesion was via an enzyme assay (see below) that measured only viable cells.

Aliquots of the cell suspension (200 µl) were added to preblocked, lipid-adsorbed microwells and incubated at 4 °C for 10 min to allow the cells to settle and then at 37 °C for 45 min. To gently remove nonadherent cells after the incubations, plates were immersed in phosphate-buffered saline, inverted, and placed in an immersed Plexiglas box that was sealed with a gasket to exclude air (27). The inverted plate in its fluid-filled chamber was placed in a centrifuge carrier and centrifuged at 110 × g. The box was again immersed in phosphate-buffered saline; the plate was removed and righted (while immersed); and excess surface buffer was removed by aspiration, leaving 300 µl/well. Adherent cells were lysed by addition of 20 µl of 10% Triton X-100 to each well, and 80 µl were removed to a fresh 96-well plate for quantitation. Cell adhesion was quantitated by measuring lactate dehydrogenase activity in the cell lysate after addition of 120 µl of phosphate-buffered saline containing 0.7 mM NADH and 4.7 mM pyruvate. The decrease in absorbance at 340 nm as a function of time was measured simultaneously in each well using a Molecular Devices UV multiwell kinetic plate reader. This method is amenable to testing large numbers of samples. The data presented are compiled from approx 4000 individual data points and are presented as the mean ± S.E. of the mean for 3-103 replicate determinations. Where indicated, the statistical significance of adhesion to ganglioside-adsorbed surfaces compared with control surfaces (adsorbed with phosphatidylcholine and cholesterol, but no ganglioside) was determined using a two-tailed Student's t test.


RESULTS

Ganglioside Binding Specificities of Sialoadhesins

MAG-, SMP-, and sialoadhesin-transfected COS cells bound specifically to ganglioside-adsorbed surfaces (Figs. 1, 2, 3). Adhesion to the most potent target gangliosides was typically very high (>80% of the cells added), whereas background adhesion to surfaces adsorbed with phosphatidylcholine and cholesterol without ganglioside was low. COS cells transfected with CD22 failed to adhere to any ganglioside tested (GD1a, GD1b, GD3, GT1b, GQ1b, and GQ1balpha ). COS cells transfected with either of the two splice variants of MAG (L-MAG and S-MAG) demonstrated the same extent and specificity of adhesion to a representative set of ganglioside-adsorbed surfaces (GM1, GD1a, GD1b, GT1b, and GQ1balpha ) (data not shown). Therefore, L-MAG-transfected COS cells were used in subsequent experiments, and all data presented on MAG-mediated adhesion refer to the long splice variant.


Fig. 1. Adhesion of COS cells expressing sialoadhesins to adsorbed gangliosides. COS cells transiently transfected to express MAG (A), SMP (B), or sialoadhesin (C) were collected from culture dishes; pretreated with neuraminidase to enhance adhesion; and placed in microwells previously adsorbed with phosphatidylcholine, cholesterol, and the indicated gangliosides. After incubation, nonadherent cells were removed by centrifugation, and adherent cells were quantitated enzymatically (see "Experimental Procedures"). Adhesion is expressed relative to the total number of cells added to each well and represents the mean ± S.E. of 3-103 replicate determinations. Background adhesion, represented by a horizontal line in each panel, was determined on wells adsorbed with phosphatidylcholine and cholesterol without gangliosides (7.5 ± 0.6% for MAG, 11.0 ± 0.8% for SMP, and 5.9 ± 0.6% for sialoadhesin). open circle , GD1a; black-square, GT1b; triangle , GM1; down-triangle, GM3; diamond , GQ1b; square , GD1b; black-triangle, GD3; bullet , GM4.
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Fig. 2. Adhesion of COS cells expressing sialoadhesins to adsorbed alpha  and beta  series gangliosides. Adhesion of COS cells transiently transfected to express MAG (A), SMP (B), or sialoadhesin (C) to microwells adsorbed with phosphatidylcholine, cholesterol, and the indicated gangliosides was determined as described under "Experimental Procedures" and in the legend to Fig. 1. black-square, GT1b; square , GT1beta ; triangle , GQ1b; black-down-triangle , GQ1balpha ; bullet , GM1alpha .
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The two neural sialoadhesins, MAG and SMP, had similar ganglioside binding specificities (Figs. 1, 2, 3). The abundant brain gangliosides GD1a (at >= 12.5 pmol/well) and GT1b (at >= 25 pmol/well) supported highly significant adhesion (p < 0.0002) of both MAG- and SMP-transfected COS cells (Fig. 1, A and B). Other gangliosides including GM3 and GM4 also supported significant adhesion of both lectins, although only at >= 10-fold higher ganglioside concentrations compared with GD1a. In contrast, neither MAG nor SMP bound to GM1, GD1b, or GD3, indicating that both lectins require a terminal alpha 2,3-linked sialic acid. All gangliosides that supported statistically significant adhesion of SMP contained the NeuAcalpha 2,3Gal terminal structure (see Fig. 3), whereas all nonsupportive gangliosides lacked this terminal structure. MAG supported adhesion to the same gangliosides, although typically with higher efficiency (greater number of adherent cells). This may be due to more efficient transfection with the MAG plasmid, higher expression of the transfected MAG, and/or more effective ganglioside binding by MAG. Flow cytometry using a MAG/SMP cross-reactive antibody (mAb 513) indicated that more MAG-transfected cells (48.2%) expressed the highest level of lectin compared with SMP-transfected cells (28.3%). Within these highest expressing populations, the mean fluorescence intensities were similar (496 and 441 relative units for MAG and SMP, respectively).

In addition to gangliosides bearing the NeuAcalpha 2,3Gal terminus, GQ1b (which bears only alpha 2,8-linked sialic acid termini) supported a low amount of adhesion by MAG-transfected cells. This preparation of GQ1b, however, was contaminated with a small amount of GT1b (20). We conclude that MAG and SMP bind with similar rank-order potency to gangliosides terminated with NeuAcalpha 2,3Gal (see Fig. 3).

In contrast to MAG and SMP, sialoadhesin had a distinctly broader binding specificity. Several gangliosides with terminal NeuAcalpha 2,3Gal structures (GD1a, GT1b, GM3, and GM4) as well as GD1b (which bears only a terminal NeuAcalpha 2,8NeuAc structure) supported nearly equivalent sialoadhesin-mediated adhesion (Fig. 1C). GD3 and GQ1b, which also bear only NeuAcalpha 2,8NeuAc termini, supported sialoadhesin binding with moderate potency. Binding was structurally specific in that GM1 did not support sialoadhesin-mediated adhesion.

Prior studies indicated that MAG bound with markedly high affinity to one of the "Chol-1" gangliosides (22). These minor brain gangliosides bear a sialic acid linked alpha 2,6 to the GalNAc(III) of the gangliotetraose core (structures in Fig. 3) (30). Fig. 2 presents a comparison of adhesion of MAG-, SMP-, and sialoadhesin-transfected COS cells to synthetic Chol-1 and related gangliosides. MAG and SMP again had markedly similar binding specificities (Fig. 2, A and B). GT1beta was equipotent to GT1b in supporting MAG and SMP binding, whereas GQ1balpha was 10-fold more potent. GM1alpha , which contains a single alpha 2,6-linked sialic acid, failed to support adhesion of either lectin. Therefore, the terminal NeuAcalpha 2,3Gal structure is required for both SMP- and MAG-mediated cell adhesion, and additional sialic acids on the internal GalNAc(III) and Gal(II) of the gangliotetraose core enhance binding of MAG and SMP to a similar extent. In contrast, GQ1balpha was only modestly (<3-fold) more potent than GT1b in supporting sialoadhesin-mediated adhesion. Binding potencies for all gangliosides tested using MAG-, SMP-, and sialoadhesin-mediated cell adhesion are summarized in Fig. 3.

The MAG/SMP cross-reactive antibody mAb 513 (4, 7), shown previously to block MAG binding to neurons (31) and gangliosides (22), demonstrated the carbohydrate-binding site structural similarity between MAG and SMP and their difference from sialoadhesin. As shown in Fig. 4, mAb 513 eliminated or markedly reduced binding of MAG and SMP to GT1b, whereas binding of sialoadhesin was unaffected. The anti-sialoadhesin blocking mAb 3D6 (32) inhibited binding of sialoadhesin to GT1b (data not shown).


Fig. 4. Inhibition of MAG- and SMP-mediated adhesion to gangliosides by mAb 513. COS cells transfected with MAG, SMP, or sialoadhesin were collected and pretreated as described under "Experimental Procedures." Cells were resuspended (133,000 cells/ml) in medium alone (open and dark stippled bars), in medium supplemented with 20 µg/ml mAb 513 (filled bars), or in medium supplemented with control isotype-matched mAb (light stippled bars). Cell suspensions were incubated for 1 h at 0 °C to allow antibody binding, and then aliquots (300 µl) were added to preblocked microwells that had been adsorbed with phosphatidylcholine and cholesterol alone (control; open bars) or with ganglioside GT1b (stippled and closed bars). Cells were incubated, and adhesion was quantitated as described under "Experimental Procedures." Adhesion is expressed relative to the total number of cells added to each well and represents the mean ± S.D. of duplicate or triplicate determinations.
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Sialic Acid Substructure Binding Specificities of I-type Lectins

Sialic acid is a complex monosaccharide, with a carboxylic acid, an N-acyl group, and a glycerol side chain within its structure (see Fig. 7). Chemically modified and synthetic gangliosides were used to determine which sialic acid substituent groups are required for binding by sialoadhesin family members.


Fig. 7. Sialic acid substructural determinants required for sialoadhesin and MAG binding. NeuAc is shown in its low energy conformation, with the carboxylic acid, 8- and 9-hydroxyl groups, acetamido nitrogen, and acetamido methyl groups on the top face and the 7-hydroxyl group and acetamido carbonyl on the bottom face. Structural specificities indicate that sialoadhesin interacts with the top face, perhaps via a binding surface on the V-set Ig-like domain (37), whereas MAG binding is more complex, requiring key functional groups on both the top and bottom faces for binding to one or more as yet unspecified polypeptide domains.
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Since GD1a supports highly significant adhesion of MAG, SMP, and sialoadhesin (Fig. 1), it was used as a basis for testing sialic acid chemical modifications. GD1a was selectively oxidized with periodate under conditions that cleave exclusively between C-7-C-8 and C-8-C-9 on the sialic acid glycerol side chain. Mass spectrometry indicated equal conversion of GD1a sialic acids to their corresponding 7- and 8-carbon aldehydes (data not shown). A portion of the resulting GD1a aldehydes was reduced with sodium borohydride, resulting in conversion to the corresponding 7- and 8-carbon alcohols. As shown in Fig. 5, neither the 7/8-aldehyde nor 7/8-alcohol sialic acid derivatives of GD1a supported binding of any of the I-type lectins tested. Similarly, modifications of the carboxylic acids on GD1a abrogated binding. Conversion of both sialic acids on GD1a to the corresponding 1-ethyl esters, 1-amides, or 1-alcohols completely eliminated binding of MAG-, SMP-, and sialoadhesin-transfected COS cells (Fig. 5). The structures of all GD1a derivatives were confirmed by thin-layer chromatography, DEAE-Sepharose chromatography (of carboxylate derivatives), and fast atom bombardment mass spectrometry at the Middle Atlantic Mass Spectrometry Laboratory (21).


Fig. 5. Adhesion of COS cells expressing sialoadhesins to GD1a with chemically modified sialic acids. Adhesion of COS cells transiently transfected to express MAG (A), SMP (B), or sialoadhesin (C) to microwells adsorbed with phosphatidylcholine, cholesterol, and the indicated concentrations of GD1a or its chemical derivatives was determined as described under "Experimental Procedures" and in the legend to Fig. 1. open circle , GD1a; diamond , 7/8-aldehydes; black-down-triangle , 7/8-alcohols; bullet , 1-ethyl ester; black-square, 1-amide; triangle , 1-alcohol.
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Since GM3 and GM4 (bearing a terminal N-acetylneuraminic acid) supported substantial adhesion mediated by both sialoadhesin and MAG, a series of synthetic analogs based on these structures (19) was used to determine the role of each sialic acid hydroxyl group and the sialic acid N-acyl group on adhesion (binding of SMP to GM3 and GM4 was insufficient to allow valid comparisons). Consistent with chemical modification studies, the 8-deoxy and 9-methoxy forms of GM3 failed to support adhesion mediated by either MAG or sialoadhesin (Fig. 6). In contrast, the 4-deoxy and 7-deoxy forms of GM3 were comparable to GM3 in supporting sialoadhesin-mediated adhesion, but failed to support substantial MAG-mediated adhesion. Furthermore, the sialic acid acetamido group appears to be involved in lectin binding. GM4 supported sialoadhesin and MAG binding, whereas a derivative bearing a 5-deaminated analog of neuraminic acid (KDN-GM4) failed to support binding by either lectin (Fig. 6). These data are consistent with the prior published observations that glycoconjugates bearing N-glycolylneuraminic acid fail to support MAG (20) or sialoadhesin (33) binding.


Fig. 6. Adhesion of COS cells transfected with sialoadhesins to synthetic gangliosides containing modified sialic acids. Adhesion of COS cells transiently transfected to express MAG (A) or sialoadhesin (B) to microwells adsorbed with phosphatidylcholine, cholesterol, and the indicated gangliosides was determined as described under "Experimental Procedures" and in the legend to Fig. 1. Synthetic GM3 analogs are identified by the chemical modifications to their N-acetylneuraminic acid moiety. open circle , GM3; black-square, 4-deoxy; triangle , 7-deoxy; black-down-triangle , 8-deoxy; diamond , 4,8-dideoxy; bullet , 4-methoxy; square , 9-methoxy; black-triangle, GM4; down-triangle, KDN-GM4.
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DISCUSSION

Sialoadhesins (1, 8, 9) are a functionally and structurally related subfamily of carbohydrate-binding immunoglobulin superfamily members (I-type lectins) (2). The sialoadhesin family consists of the eponymous member (sialoadhesin), MAG, SMP, CD22, and CD33. MAG and SMP are expressed on myelinating cells in the nervous system, sialoadhesin on a subset of macrophages, CD22 on certain B lymphocytes, and CD33 on cells of myelomonocytic lineage (9). Sialoadhesins mediate cell-cell interactions by binding to target sialylated glycoconjugates (1, 6, 10, 32). They share the same general polypeptide domain structure: an amino-terminal V-set Ig-like domain followed by one or more C2-set Ig-like domains, a transmembrane domain, and a short cytoplasmic tail. The ligand recognition site has been localized to the amino-terminal V-set domain (sialoadhesin) (34) or the V-set domain with contributions from the adjacent C2-set domain (CD22) (34, 35). Additionally, sialoadhesins have extensive sequence similarity. The first two amino-terminal Ig-like domains of MAG and SMP are 56% identical (72% similar, including conservative amino acid replacements), and other sialoadhesins range from 32 to 43% sequence similarity in pairwise comparisons. Site-directed mutagenesis (36, 37) indicates that sialoadhesin and CD22 bind to sialylated glycoconjugates via amino acids on one surface of the V-set domain. This is consistent with sialoadhesin's sialic acid substituent group binding specificity (see below), whereas MAG's specificity indicates a more complex binding site.

Target ligands for the sialoadhesins are glycoconjugates in which a terminal sialic acid is essential for binding (1, 2, 6, 9, 10, 38). In previous studies, CD22 bound only to alpha 2,6-linked sialic acids (1, 39), whereas MAG and CD33 bound only to alpha 2,3-linked sialic acids (1, 10, 22). Sialoadhesin bound predominantly to terminal alpha 2,3-linked sialic acids (1), although weaker binding to alpha 2,8-linked sialic acids was demonstrated (32). Among structures with alpha 2,3-linked sialic acids, MAG bound preferentially to "3-O" structures (NeuAcalpha 2,3Galbeta 1,3GalNAc), which are common termini on gangliosides (the major sialoglycoconjugates of the nervous system) (40) and O-linked glycoproteins (1). Sialoadhesin and CD33 bound similarly to "3-O" and "3-N" (NeuAcalpha 2,3Galbeta 1,4GlcNAc) structures (1, 10).

Our prior studies demonstrated that (i) MAG bound to gangliosides with the specificity GQ1balpha  > GT1b = GD1a > GM3 >>  GM1, GD1b, GQ1b, the latter of which did not support adhesion; and (ii) modification of the glycerol side chain, carboxylic acid, or N-acyl group abrogated MAG-mediated adhesion (20, 22). This study confirms and extends those findings. MAG-mediated adhesion was repeated to the above gangliosides as well as to GM4, KDN-GM4, six synthetic derivatives of GM3, and various gangliosides with alpha 2,6-sialic acids linked to the GalNAc(III) of the gangliotetraose core. These new data were compared directly with adhesion of COS cells expressing CD22, sialoadhesin, SMP, and the short isoform of MAG.

CD22 failed to bind to alpha 2,3- or alpha 2,8-linked sialic acid termini on gangliosides (data not shown). In contrast (Figs. 1 and 2), sialoadhesin bound to most of the gangliosides tested, including many with terminal alpha 2,3- or alpha 2,8-sialic acids. Nine of 12 gangliosides tested supported sialoadhesin binding within the same approx 10-fold concentration range, indicating that sialoadhesin does not markedly distinguish the sialic acid linkage (alpha 2,3 versus alpha 2,8) or the neutral core (e.g. compare GD1b and GM4 in Fig. 3). A previous study of detergent-solubilized 125I-sialoadhesin binding to gangliosides using thin-layer chromatography plate overlay (32) also reported a broad specificity.

Consistent with their extensive sequence similarity, the two neural sialoadhesins, SMP and MAG, were remarkably similar in their ganglioside binding (Figs. 1, 2, 3). Both bound only to structures bearing terminal alpha 2,3-linked sialic acids (e.g. GT1b and GD1a) and failed to bind to those terminated with alpha 2,8-linked structures (e.g. GD1b and GD3). Among glycoconjugates with alpha 2,3-linked sialic acid termini, SMP and MAG distinguished sialic acid linkage patterns and neutral core variations. In contrast to sialoadhesin, di- and trisialogangliosides with the gangliotetraose core (GD1a and GT1b) supported adhesion of SMP and MAG approx 10-fold better than did monosialogangliosides (GM3 and GM4). Furthermore, the Chol-1 ganglioside, GQ1balpha , was 10-fold more potent than any other ganglioside tested (Figs. 1, 2, 3). Chol-1 gangliosides are quantitatively minor structures that are expressed exclusively on cholinergic neurons (30, 41). The functional significance of their preferential binding to the neural sialoadhesins is not known. Although the terminal tetrasaccharide on GQ1balpha is also found on O-linked glycoproteins (42, 43), polyclonal antibodies against Chol-1 gangliosides do not cross-react with any glycoprotein (44), suggesting that the oligosaccharide on GQ1balpha adopts a unique conformation that fits particularly well in the SMP and MAG binding pockets. In addition to having similar carbohydrate recognition specificities, the observation that both SMP- and MAG-mediated adhesion to gangliosides is inhibited by the same conformationally restricted monoclonal antibody (mAb 513) (45) confirms the similarity of their binding sites.

Sialic acids are unusual among monosaccharides in their complexity and diversity (11). They carry a carboxylic acid (C-1), an N-acyl group attached to C-5, and a glycerol side chain attached to C-6 (Fig. 7), each of which is involved in molecular recognition by certain sialoadhesins. Blocking the carboxylic acid abrogates binding (Fig. 5), as does replacement of the acetamido group with a hydroxyl (compare GM4 with KDN-GM4 in Fig. 6) or truncation of the glycerol side chain (Fig. 5). These data are consistent with prior studies on the sensitivity of sialoadhesin and CD22 binding to modifications of the sialic acid residue (6, 33, 46-48) and contrast with studies on selectins, in which extensive modifications of sialic acids have no effect (12, 14, 49). In fact, substitution of the entire sialic acid (e.g. on sialyl-LeX or sialyl-Lea) with a sulfate ester results in retention of ligand binding by all selectins (13, 14), but abrogates binding by CD22 (47).

The sialic acid substructural binding specificities of sialoadhesin and MAG have implications for ligand docking on the proteins. For sialoadhesin, modification of the C-8 or C-9 hydroxyl, the acetamido nitrogen or methyl group (33), or the C-1 carboxylic acid eliminated binding (Figs. 5 and 6), whereas removal of the C-4 or C-7 hydroxyl was without effect. This pattern is consistent with binding primarily to a single face of the sialic acid (top face in Fig. 7). Sialic acid binding to sialoadhesin can be compared with x-ray crystallography of sialic acid binding to the influenza virus hemagglutinin (50), in which a carboxylate oxygen, the acetamido nitrogen, and the 8- and 9- hydroxyls face into a depression on the hemagglutinin surface, whereas the 7-hydroxyl faces the solvent. This model is consistent with Ig-domain studies and site-directed mutagenesis (34, 37), which place the ligand-binding site of sialoadhesin on a contiguous cluster of residues on the surface of the GFCC'C" beta -sheet of the V-set Ig-like domain.

Sialic acid modifications that block sialoadhesin binding also block MAG binding. In addition, removal of either the 4- or 7-hydroxyl inhibits MAG binding (Fig. 6). Since the 7-hydroxyl and 8/9-hydroxyls extend in opposite directions (Fig. 7), a more complex model of MAG binding is implicated. One possibility is that the MAG binding site consists of a deep pocket or apposing polypeptide sheets. Alternatively, the 7-hydroxyl group may stabilize a conformation of the oligosaccharide that is preferentially bound by MAG at a single protein surface. To date, no direct evidence addresses whether one or more than one protein surface on MAG is responsible for sialic acid binding, although biophysical and electron microscopic studies suggest that MAG may have a bent rod configuration with apposed Ig-like domains (51, 52). Studies using chimeric molecules indicate that the first three Ig domains of MAG are necessary and sufficient for binding to neurons (45) and sialoglycoconjugates (1), although the sialic acid substructure specificities of truncated forms of MAG have not been reported. Further protein structural and functional studies will be needed to establish the sialoglycoconjugate-binding site on MAG (and on SMP) and to determine the precise role each sialic acid hydroxyl group plays in protein binding.


FOOTNOTES

*   This work was supported by National Science Foundation Grant IBN-9631745, and by grants from the National Multiple Sclerosis Society and the Medical Research Council (to M. B. T. and J. C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is dedicated to the vision and collaborative spirit of our friend and colleague, Dr. Akira Hasegawa, who passed away October 10, 1996.


§   Supported in part by National Institutes of Health Training Grant GM07626.
Dagger Dagger    To whom correspondence should be addressed: Dept. of Pharmacology, The Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-8392; Fax: 410-955-3023; E-mail: rschnaar{at}welchlink.welch.jhu.edu.
1   The abbreviations used are: MAG, myelin-associated glycoprotein; SMP, Schwann cell myelin protein; mAb, monoclonal antibody; GQ1balpha , IV3NeuAc,III6NeuAc,II3(NeuAc)2-Gg4Cer; GT1beta , IV3NeuAc,III6(NeuAc)2-Gg4Cer; GM1alpha , III6NeuAc-Gg4Cer; GM4, I3NeuAc-GalCer; KDN-GM4, 3-deoxy-D-glycero-D-galacto-2-nonulosyl(alpha 2-3)galactosyl(beta 1-1') ceramide. Ganglioside nomenclature is according to Svennerholm (53).

ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Melitta Schachner for mAb 513 and Dr. Ivan Stamenkovic for the plasmid carrying CD22beta .


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