A Small Region of the Natural Killer Cell Receptor, Siglec-7, Is Responsible for Its Preferred Binding to α2,8-Disialyl and Branched α2,6-Sialyl Residues: A COMPARISON WITH Siglec-9

Toshiyuki Yamaji,Takane Teranishi,Magnus S Alphey,Paul R Crocker,Yasuhiro Hashimoto

doi:10.1074/jbc.m110146200

Toshiyuki Yamaji, Takane Teranishi + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110146200

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Abstract

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells. Here we characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. Glyco-probes carrying unique oligosaccharide structures such as GD3 (NeuAc alpha 2,8NeuAc alpha 2,3Gal beta 1,4Glc) and LSTb (Gal beta 1,3[NeuAc alpha 2,6]GlcNAc beta 1,3Gal beta 1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal beta 1,4Glc) or GD1a (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4[NeuAc alpha 2,3]Gal beta 1,4Glc) oligosaccharides. In contrast, Siglec-9, which is 84% identical to Siglec-7, did not bind to the GD3 and LSTb probes but did bind to the LSTc and GD1a probes. To identify a region(s) responsible for their difference in binding specificity, we prepared a series of V-set domain chimeras between Siglecs-7 and -9. Substitution of a small region, Asn(70)-Lys(75), of Siglec-7 with the equivalent region of Siglec-9 resulted in loss of Siglec-7-like binding specificity and acquisition of Siglec-9-like binding properties. In comparison, a Siglec-9-based chimera, which contains Asn(70)-Lys(75) with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity. These results, combined with molecular modeling, suggest that the C-C' loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9.

Highlights

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells
Prior to Predicted C-CЈ Loop Determines Siglec-7 Binding Specificity the assay, Siglec-7-CHO cells were treated with sialidase, which removed endogenous sialic acids on the cell surface and enhanced probe binding without affecting binding specificity
The LSTc and GD1a-probes bound moderately to Siglec-7-CHO cells, much stronger binding was observed with the GD3- and LSTb-probes (Fig. 1A). These results suggest that the ␣2,8-disialyl residue (NeuAc␣2,8NeuAc␣2,3Gal) of the GD3-probe and the branched ␣2,6-sialyl residue (Gal␤1,3[NeuAc␣2,6]GlcNAc) of the LSTbprobe are more potent ligands than the terminal ␣2,6- or ␣2,3sialyl residues of the LSTc- or GD1a-probes

Summary

A COMPARISON WITH Siglec-9*

Siglec-7 is a sialic acid-binding lectin recently identified as an inhibitory receptor on natural killer cells. We characterize the sugar-binding specificity of Siglec-7 expressed on Chinese hamster ovary cells using polyvalent streptavidin-based glyco-probes. A Siglec-9-based chimera, which contains Asn70–Lys with additional amino acids derived from Siglec-7, exhibited Siglec-7-like specificity These results, combined with molecular modeling, suggest that the C-C؅ loop in the sugar-binding domain plays a major role in determining the binding specificities of Siglecs-7 and -9. CD33/Siglec-3 transiently expressed on COS cells appears to be masked by endogenous sialoglycoconjugates, because its sugar binding activity is detectable only after sialidase treatment, which removes endogenous sialic acids [10]. By preparing a series of V-set domain chimeric swaps between Siglecs-7 and –9, we have identified a small region in the sugar-binding domain that is important in determining the binding specificities of these proteins

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION