Abstract
Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a >> GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.
Highlights
Siglecs contain an N-terminal V-type Ig domain, a varying number of C-type Ig domains, a single transmembrane domain, and a short cytoplasmic tail [1, 2]
Siglecs (CD22, CD33, sialoadhesin, myelin-associated glycoprotein (MAG),1 Schwann cell myelin protein (SMP), and siglec 5) share a significant degree of sequence similarity among their V-type domains, which are required for sialic acid binding
Siglec Adhesion to ␣-Series Gangliosides—In agreement with our previous report [17], MAG and sialoadhesin adhered with moderate potency to the two major brain gangliosides bearing terminal “NeuAc␣2,3Gal1,3GalNAc” determinants, GD1a and GT1b (Fig. 2, A and C, and Table I), whereas SMP-mediated adhesion to these gangliosides was less avid (Fig. 2B; SMP-mediated adhesion to weakly supportive gangliosides added at concentrations above 50 pmol/well was often less than maximum levels, perhaps due to charge repulsion [16])
Summary
Siglecs (sialic acid-dependent immunoglobulin-like family member lectins) contain an N-terminal V-type Ig domain, a varying number of C-type Ig domains, a single transmembrane domain, and a short cytoplasmic tail [1, 2]. Siglec Adhesion to ␣-Series Gangliosides—In agreement with our previous report [17], MAG and sialoadhesin adhered with moderate potency to the two major brain gangliosides bearing terminal “NeuAc␣2,3Gal1,3GalNAc” determinants, GD1a and GT1b (Fig. 2, A and C, and Table I), whereas SMP-mediated adhesion to these gangliosides was less avid (Fig. 2B; SMP-mediated adhesion to weakly supportive gangliosides added at concentrations above 50 pmol/well was often less than maximum levels, perhaps due to charge repulsion [16]).
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