New Aspects of Siglec Binding Specificities, Including the Significance of Fucosylation and of the Sialyl-Tn Epitope

Abstract

The siglecs (sialic acid-binding immunoglobulin superfamily lectins) are immunoglobulin superfamily members recognizing sialylated ligands. Most prior studies of siglec specificities focused on ␣2-3-and ␣2-6-sialyllactos(amin)es and on one or two of the siglecs at a time. Here, we explore several new aspects of specificities of the first six reported siglecs, using sialylated glycans presented in multivalent form, on synthetic polyacrylamide backbones, or on mucin polypeptides. First, we report that binding of siglec-1 (sialoadhesin), siglec-3 (CD33), siglec-4a (myelin-associated glycoprotein), and siglec-5 to ␣2-3 sialyllactosamine is affected markedly by the presence of an ␣1-3-linked fucose. Thus, while siglecs may not interfere with selectin-mediated recognition, fucosylation could negatively regulate siglec binding. Second, in contrast to earlier studies, we find that siglec-3 prefers ␣2-6-sialyllactose. Third, siglec-5 binds ␣2-8-linked sialic acid, making it the siglec least specific for linkage recognition. Fourth, siglecs-2 (CD22), -3, -5, and -6 (obesity-binding protein 1) showed significant binding to sialyl-Tn (Neu5Ac␣2-6-GalNAc), a tumor marker associated with poor prognosis. Fifth, siglec-6 is an exception among siglecs in not requiring the glycerol side chain of sialic acid for recognition. Sixth, all siglecs require the carboxyl group of sialic acid for binding. Finally, the presentation of the sialyl-Tn epitope and/or more extended structures that include this motif may be important for optimal recognition by the siglecs. This was concluded from studies using ovine, bovine, and porcine submaxillary mucins and Chinese hamster ovary cells transfected with ST6GalNAc-I and/or the mucin polypeptide MUC1.The siglecs (sialic acid-binding immunoglobulin superfamily lectins)1 are a class of Ig superfamily proteins (1) which show binding activity to specific glycan structures containing sialic acid (1-5). Sialic acids are acidic monosaccharides frequently found at the outer end of secreted and cell surface glycoconjugates (5-8), a good location for recognition by lectins such as the siglecs. To date, six different members of this family of lectins have been characterized (1, 2, 9 -14), and a recent paper describes a potential seventh member (15). The siglecs share overall structural characteristics, with an NH 2 -terminal V-set Ig domain followed by varying numbers of C2-set Ig domains. Siglec-1 (sialoadhesin, Sn) is the largest member, with 17 extracellular Ig domains, whereas siglec-3 (CD33) is the smallest, with only 2 of these domains. Each siglec has its own unique tissue distribution and specific phosphorylation sites on the cytosolic tail, indicating that each has specific functions mediated by the lectin activity. The latter suggestion is substantiated by the fact that each siglec seems to display distinctive specificities for recognition of sialic acid linkages. However, for most siglecs only a limited sampling of the wide array of sialylated glycans found in nature have been examined, primarily ...

Sialoside Specificity of the Siglec Family Assessed Using Novel Multivalent Probes

“…Conversely, high affinity binding of SLe X -SAAP (P) to sialoadhesin (Figs. 3 and 4) was not seen with the corresponding SLe X -polyacrylamide probe by Brinkman-Van der Linden et al (49). There is also little correlation between the intrinsic affinity of the sialosides (e.g.…”

“…3). Yet binding of the same sialosides attached to polyacrylamide showed equivalent binding of both linkages to Siglec-5 and Siglec-7 (34,49,55,58,59). Conversely, high affinity binding of SLe X -SAAP (P) to sialoadhesin (Figs.…”

“…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).…”

“…Development of ELISA-type Assay Using Sialoside-SAAP Probes-Commercially available multivalent sialoside conjugates consisting of a polyacrylamide polymer with pendent sialosides have proven to be useful probes of siglec function in both in vitro assays employing siglec-Fc chimeras and siglecs expressed on cell surfaces (49,51,52,54,55). Our initial attempts to use these conjugates in an ELISA-type assay to measure quantitatively probe binding to siglec-Fc chimeras in the presence and absence of free sialosides, however, proved to be highly variable between experiments and even within a single experiment.…”

Glycosylation defining cancer malignancy: New wine in an old bottle

“…A berrant glycosylation occurs in essentially all types of experimental and human cancers, as has been observed for over 35 years, and many glycosyl epitopes constitute tumor-associated antigens. A long-standing debate is whether aberrant glycosylation is a result or a cause of cancer.…”