Abstract
Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. Analysis has been performed using a novel multivalent platform comprising biotinylated sialosides bound to a streptavidin-alkaline phosphatase conjugate. Each siglec was found to have a unique specificity for binding 16 different sialoside-streptavidin-alkaline phosphatase probes. The relative affinities of monovalent sialosides were assessed for each siglec in competitive inhibition studies. The quantitative data obtained allows a detailed analysis of each siglec for the relative importance of sialic acid and the penultimate oligosaccharide sequence on binding affinity and specificity. Most remarkable was the finding that myelin-associated glycoprotein (Siglec-4) binds with 500-10,000-fold higher affinity to a series of mono- and di-sialylated derivatives of the O-linked T-antigen (Galbeta(1-3)-GalNAc(alpha)OThr) as compared with alpha-methyl-NeuAc.
Highlights
Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids
Comparison of the siglecs revealed striking differences in their overall affinities for binding the four reference compounds, with IC50 values ranging from ϳ0.15 mM for murine and human CD22 (Siglec-2) to 3.7 mM for CD33 (Siglec-3), and no inhibition at the highest concentration tested for hSiglec-6
Most have used multivalent assays based on binding of siglecs to sialosides attached to a polyacrylamide or other polymeric carrier (1, 34, 37, 49, 52–55, 57–59), to gangliosides adsorbed to microtiter plates (30, 36, 38) or to neo-glycoprotein conjugates (27, 28)
Summary
Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. The quantitative data obtained allows a detailed analysis of each siglec for the relative importance of sialic acid and the penultimate oligosaccharide sequence on binding affinity and specificity. CD22 is well established to be an important modulator of B cell receptor signaling through recruitment of a protein phosphatase (SHP-1) by the phosphorylated ITIM motifs in its cytoplasmic domain (5–7). There is ample evidence that the functions of siglecs are modulated by the interaction with sialic acid containing ligands, establishing detailed mechanisms remains a challenge due to the ubiquitous distribution and structural diversity of sialyloligosaccharides on glycoproteins and glycolipids (3, 7, 11, 21). Addition of a 9-O-acetyl group or truncation of the sialic acid to the C-7 analog with periodate abolishes binding of several siglecs examined (27, 29 –33)
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