Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues

Abstract

The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free A-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength.

Sialoside Specificity of the Siglec Family Assessed Using Novel Multivalent Probes

“…The other siglecs exhibited little preference or more complex preferences for sialic acid type and linkage represented in the reference compounds. The observed results are in accord with previous reports that have evaluated the specificity of sialoadhesin, CD22, MAG, and Siglec-7 by competitive inhibition with sialosides in other assay systems (25,26,28,39,56).…”

“…Indeed, although human CD22 binds equally well to ␣2-6 sialosides containing either NeuAc or NeuGc, murine CD22 exhibits a strong preference for NeuGc (32,39,63). As shown here, the preferential affinity of mCD22 for NeuGc reflects a 10 -20-fold higher affinity for sialosides containing NeuGc over the same sequences containing NeuAc (12 and 13 compared with 19 and 20).…”

“…Subsequently, many of the siglecs have been evaluated to various extents for their linkage specificity, ␣(2-3), ␣(2-6), and ␣(2-8), and/or the ability to recognize the NeuAc and NeuGc forms of sialic acid using a variety of different assays (3,4). Several reports (25,26,28,39,40) have evaluated the specificity of one or several siglecs using competitive inhibition of multivalent binding assays by monovalent sialosides analogous to the studies reported here. However, most have used multivalent assays based on binding of siglecs to sialosides attached to a polyacrylamide or other polymeric carrier (1,34,37,49,(52)(53)(54)(55)(57)(58)(59), to gangliosides adsorbed to microtiter plates (30,36,38) or to neo-glycoprotein conjugates (27,28).…”

“…As shown here, the preferential affinity of mCD22 for NeuGc reflects a 10 -20-fold higher affinity for sialosides containing NeuGc over the same sequences containing NeuAc (12 and 13 compared with 19 and 20). Conversely, murine sialoadhesin and murine MAG have been demonstrated to exhibit negligible binding to NeuGc containing oligosaccharides in various multivalent assay systems (27,30,32,39). This preference for NeuAc is reflected in the increased affinity of these siglecs for NeuAc over NeuGc by 2-and 3-8-fold, respectively (Table II).…”

“…By their very nature, such multivalent assays typically yield qualitative information on the relative binding affinities of the sialoside sequences being studied. Several reports have evaluated sialoside specificity using competitive inhibition with monomeric sialosides yielding more quantitative comparisons of sialoside specificity, but only a few (1-3) of the siglecs have been compared in any given study (25,26,28,39,40). As a result, although important and useful information has been obtained, current information on the sialoside specificity of siglecs is somewhat fragmentary and does not readily yield to direct comparisons between members of the family.…”