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• W2004207764 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 α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > 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. 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 α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > 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. Sialoadhesins (1Kelm S. Pelz A. Schauer R. Filbin M.T. Song T. de Bellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar) are a structurally and functionally related family consisting of five immunoglobulin superfamily lectins (I-type lectins) (2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) including myelin-associated glycoprotein (MAG), 1The abbreviations used are: MAG, myelin-associated glycoprotein; SMP, Schwann cell myelin protein; mAb, monoclonal antibody; GQ1bα, IV3NeuAc,III6NeuAc,II3(NeuAc)2-Gg4Cer; GT1β, IV3NeuAc,III6(NeuAc)2-Gg4Cer; GM1α, III6NeuAc-Gg4Cer; GM4, I3NeuAc-GalCer; KDN-GM4, 3-deoxy-d-glycero-d-galacto-2-nonulosyl(α2–3)galactosyl(β1–1′) ceramide. Ganglioside nomenclature is according to Svennerholm (53Svennerholm L. Prog. Brain Res. 1994; 101: xi-xivCrossref PubMed Scopus (108) Google Scholar). Schwann cell myelin protein (SMP), CD22, CD33, and sialoadhesin. MAG and SMP are found on oligodendroglia and Schwann cells in the nervous system (3Trapp B.D. Ann. N. Y. Acad. Sci. 1990; 605: 29-43Crossref PubMed Scopus (142) Google Scholar, 4Dulac C. Tropak M.B. Cameron-Curry P. Rossier J. Marshak D.R. Roder J. Le Douarin N.M. Neuron. 1992; 8: 323-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar), 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 (5Crocker P.R. Freeman S. Gordon S. Kelm S. J. Clin. Invest. 1995; 95: 635-643Crossref PubMed Google Scholar, 6Sgroi D. Varki A. Braesch-Andersen S. Stamenkovic I. J. Biol. Chem. 1993; 268: 7011-7018Abstract Full Text PDF PubMed Google Scholar, 7Poltorak M. Sadoul R. Keilhauer G. Landa C. Fahrig T. Schachner M. J. Cell Biol. 1987; 105: 1893-1899Crossref PubMed Scopus (290) Google Scholar). 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 (8Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar). 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) (2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 8Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar, 9Crocker P.R. Kelm S. Hartnell A. Freeman S. Nath D. Vinson M. Mucklow S. Biochem. Soc. Trans. 1996; 24: 150-156Crossref PubMed Scopus (69) Google Scholar). Each I-type lectin binds to carbohydrate structures bearing a nonreducing terminal sialic acid (1Kelm S. Pelz A. Schauer R. Filbin M.T. Song T. de Bellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Sgroi D. Varki A. Braesch-Andersen S. Stamenkovic I. J. Biol. Chem. 1993; 268: 7011-7018Abstract Full Text PDF PubMed Google Scholar, 10Freeman S.D. Kelm S. Barber E.K. Crocker P.R. Blood. 1995; 85: 2005-2012Crossref PubMed Google Scholar). 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 (11Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (485) Google Scholar). In certain sialic acid-dependent recognition systems, determinant stringency is low. For example, selectins bind to oligosaccharides bearing truncated sialic acids (12Tyrrell D. James P. Rao N. Foxall C. Abbas S. Dasgupta F. Nashed N. Hasegawa A. Kiso M. Asa D. Kidd J. Brandley B.K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10372-10376Crossref PubMed Scopus (278) Google Scholar) or appropriately placed anionic groups (sulfates, carboxylic acids) otherwise unrelated to the sialic acid structure (13Green P.J. Tamatani T. Watanabe T. Miyasaka M. Hasegawa A. Kiso M. Yuen C.T. Stoll M.S. Feizi T. Biochem. Biophys. Res. Commun. 1992; 188: 244-251Crossref PubMed Scopus (175) Google Scholar, 14Brandley B.K. Kiso M. Abbas S. Nikrad P. Srivasatava O. Foxall C. Oda Y. Hasegawa A. Glycobiology. 1993; 3: 633-639Crossref PubMed Scopus (208) Google Scholar, 15Rao B.N.N. Anderson M.B. Musser J.H. Gilbert J.H. Schaefer M.E. Foxall C. Brandley B.K. J. Biol. Chem. 1994; 269: 19663-19666Abstract Full Text PDF PubMed Google Scholar, 16Kogan T.P. Dupre B. Keller K.M. Scott I.L. Bui H. Market R.V. Beck P.J. Voytus J.A. Revelle B.M. Scott D. J. Med. Chem. 1995; 38: 4976-4984Crossref PubMed Scopus (101) Google Scholar). In contrast, sialoadhesins appear to have more stringent sialic acid specificities (see “Discussion”) (9Crocker P.R. Kelm S. Hartnell A. Freeman S. Nath D. Vinson M. Mucklow S. Biochem. Soc. Trans. 1996; 24: 150-156Crossref PubMed Scopus (69) Google Scholar). In this study, we used cells expressing different sialoadhesins to explore and compare the fine structural preferences of their binding to target sialylated glycoconjugates. 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. GQ1bα, GT1β, GM1α, GM4 and its derivatives, and GM3 derivatives were synthesized de novo using previously described methods (17Hotta K. Ishida H. Kiso M. Hasegawa A. J. Carbohydr. Chem. 1995; 14: 491-506Crossref Scopus (42) Google Scholar, 18Hotta K. Komba S. Ishida H. Kiso M. Hasegawa A. J. Carbohydr. Chem. 1994; 13: 665-677Crossref Scopus (24) Google Scholar, 19Kiso M. Hasegawa A. Methods Enzymol. 1994; 242: 173-183Crossref PubMed Scopus (27) Google Scholar). 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 (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). GD1a gangliosides bearing sialic acid ethyl esters, 1-amides, and 1-alcohols were prepared as described (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Products were analyzed by thin-layer chromatography and fast atom bombardment mass spectrometry at the Middle Atlantic Mass Spectrometry Laboratory (21Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (237) Google Scholar).Figure 3Structure-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 ‡, p < 0.01. §, this preparation of GQ1b contains a small amount of contaminating GT1b (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). NeuAc-NeuAc linkages are all α2,8; other NeuAc linkages are as noted in the key.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (8Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar), both the long (L-MAG) (22Yang L.J.-S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (267) Google Scholar) and short (S-MAG) splice variants of rat MAG, quail SMP (4Dulac C. Tropak M.B. Cameron-Curry P. Rossier J. Marshak D.R. Roder J. Le Douarin N.M. Neuron. 1992; 8: 323-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar), and human CD22 (seven-Ig-like domain variant, CD22β) (23Wilson G.L. Fox C.H. Fauci A.S. Kehrl J.H. J. Exp. Med. 1991; 173: 137-146Crossref PubMed Scopus (127) Google Scholar, 24Stamenkovic I. Sgroi D. Aruffo A. Sy M.S. Anderson T. Cell. 1991; 66: 1133-1144Abstract Full Text PDF PubMed Scopus (314) Google Scholar). 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) (25Seed B. Aruffo A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3365-3369Crossref PubMed Scopus (789) Google Scholar). 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) (4Dulac C. Tropak M.B. Cameron-Curry P. Rossier J. Marshak D.R. Roder J. Le Douarin N.M. Neuron. 1992; 8: 323-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar,7Poltorak M. Sadoul R. Keilhauer G. Landa C. Fahrig T. Schachner M. J. Cell Biol. 1987; 105: 1893-1899Crossref PubMed Scopus (290) Google Scholar), SER-4 (sialoadhesin) (26Crocker P.R. Gordon S. J. Exp. Med. 1989; 169: 1333-1346Crossref PubMed Scopus (198) Google Scholar), and Chemicon 2112 (CD22; Chemicon International, Inc., Temecula, CA). Adhesion was performed as reported previously (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 22Yang L.J.-S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (267) Google Scholar, 27Yang L.J.-S. Zeller C.B. Schnaar R.L. Anal. Biochem. 1996; 236: 161-167Crossref PubMed Scopus (19) Google Scholar). 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 (28Blackburn C.C. Schnaar R.L. J. Biol. Chem. 1983; 258: 1180-1188Abstract Full Text PDF PubMed Google Scholar, 29Blackburn C.C. Swank-Hill P. Schnaar R.L. J. Biol. Chem. 1986; 261: 2873-2881Abstract Full Text PDF PubMed Google Scholar), 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 (22Yang L.J.-S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (267) Google Scholar), 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 (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), 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 (27Yang L.J.-S. Zeller C.B. Schnaar R.L. Anal. Biochem. 1996; 236: 161-167Crossref PubMed Scopus (19) Google Scholar). 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 ≈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'st test. 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 GQ1bα). 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 GQ1bα) (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. 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 α2,3-linked sialic acid. All gangliosides that supported statistically significant adhesion of SMP contained the NeuAcα2,3Gal 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 NeuAcα2,3Gal terminus, GQ1b (which bears only α2,8-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 (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We conclude that MAG and SMP bind with similar rank-order potency to gangliosides terminated with NeuAcα2,3Gal (see Fig. 3). In contrast to MAG and SMP, sialoadhesin had a distinctly broader binding specificity. Several gangliosides with terminal NeuAcα2,3Gal structures (GD1a, GT1b, GM3, and GM4) as well as GD1b (which bears only a terminal NeuAcα2,8NeuAc structure) supported nearly equivalent sialoadhesin-mediated adhesion (Fig. 1 C). GD3and GQ1b, which also bear only NeuAcα2,8NeuAc 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 (22Yang L.J.-S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (267) Google Scholar). These minor brain gangliosides bear a sialic acid linked α2,6 to the GalNAc(III) of the gangliotetraose core (structures in Fig.3) (30Hirabayashi Y. Nakao T. Irie F. Whittaker V.P. Kon K. Ando S. J. Biol. Chem. 1992; 267: 12973-12978Abstract Full Text PDF PubMed Google Scholar). 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). GT1β was equipotent to GT1b in supporting MAG and SMP binding, whereas GQ1bα was 10-fold more potent. GM1α, which contains a single α2,6-linked sialic acid, failed to support adhesion of either lectin. Therefore, the terminal NeuAcα2,3Gal 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, GQ1bα 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 (4Dulac C. Tropak M.B. Cameron-Curry P. Rossier J. Marshak D.R. Roder J. Le Douarin N.M. Neuron. 1992; 8: 323-334Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 7Poltorak M. Sadoul R. Keilhauer G. Landa C. Fahrig T. Schachner M. J. Cell Biol. 1987; 105: 1893-1899Crossref PubMed Scopus (290) Google Scholar), shown previously to block MAG binding to neurons (31Sadoul R. Fahrig T. Bartsch U. Schachner M. J. Neurosci. Res. 1990; 25: 1-13Crossref PubMed Scopus (70) Google Scholar) and gangliosides (22Yang L.J.-S. Zeller C.B. Shaper N.L. Kiso M. Hasegawa A. Shapiro R.E. Schnaar R.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 814-818Crossref PubMed Scopus (267) Google Scholar), 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 (32Crocker P.R. Kelm S. DuBois C. Martin B. McWilliam A.S. Shotton D.M. Paulson J.C. Gordon S. EMBO J. 1991; 10: 1661-1669Crossref PubMed Scopus (240) Google Scholar) inhibited binding of sialoadhesin to GT1b (data not shown). 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. 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 (21Dell A. Reason A.J. Khoo K.-H. Panico M. McDowell R.A. Morris H.R. Methods Enzymol. 1994; 230: 108-132Crossref PubMed Scopus (237) Google Scholar). Since GM3 and GM4 (bearing a terminalN-acetylneuraminic acid) supported substantial adhesion mediated by both sialoadhesin and MAG, a series of synthetic analogs based on these structures (19Kiso M. Hasegawa A. Methods Enzymol. 1994; 242: 173-183Crossref PubMed Scopus (27) Google Scholar) 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 (20Collins B.E. Yang L.J.-S. Mukhopadhyay G. Filbin M.T. Kiso M. Hasegawa A. Schnaar R.L. J. Biol. Chem. 1997; 272: 1248-1255Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) or sialoadhesin (33Kelm S. Schauer R. Manuguerra J.-C. Gross H.-J. Crocker P.R. Glycoconjugate J. 1994; 11: 576-585Crossref PubMed Scopus (179) Google Scholar) binding. Sialoadhesins (1Kelm S. Pelz A. Schauer R. Filbin M.T. Song T. de Bellard M.E. Schnaar R.L. Mahoney J.A. Hartnell A. Bradfield P. Crocker P.R. Curr. Biol. 1994; 4: 965-972Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 8Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar, 9Crocker P.R. Kelm S. Hartnell A. Freeman S. Nath D. Vinson M. Mucklow S. Biochem. Soc. Trans. 1996; 24: 150-156Crossref PubMed Scopus (69) Google Scholar) are a functionally and structurally related subfamily of carbohydrate-binding immunoglobulin superfamily members (I-type lectins) (2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217" @default.
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