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. Glycoprobes carrying unique oligosaccharide structures such as GD3 (NeuAc␣2,8NeuAc␣2,3Gal␤1,4Glc) and LSTb (Gal␤1,3[NeuAc␣2,6]GlcNAc␤1,3Gal␤1,4Glc) oligosaccharides bound to Siglec-7 better than those carrying LSTc (NeuAc␣2,6Gal␤1,4GlcNAc␤1,3Gal␤1,4Glc) or GD1a (NeuAc␣2,3Gal␤1,3GalNAc␤1,4[NeuAc␣2,3]Gal-␤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.

Siglec-7 (p75/AIRM1) is an inhibitory receptor on human natural killer cells (NK cells) and monocytes. The cytoplasmic domain of Siglec-7 contains two signaling motifs: a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ile435-Gln-Tyr-Ala-Pro-Leu440) and a membrane-distal motif (Asn458-Glu-Tyr-Ser-Glu-Ile463). We report here that, upon pervanadate (PV) treatment, Siglec-7 recruited the protein tyrosine phosphatases Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase-1 (SHP-1) and SHP-2 less efficiently than did other inhibitory receptors such as Siglec-9 and leukocyte-associated Ig-like receptor (LAIR-1). Alignment of the amino acid sequences of the two Siglecs revealed only three amino acids difference in these motifs. To identify the amino acid(s) critical to recruitment efficiency, we prepared a series of Siglec-7-based mutants in which each of the three amino acids were replaced with the corresponding one of Siglec-9 (I435L, P439S, and N458T mutants). P439S and N458T mutants showed pronounced enhancement of SHP recruitment, but I435L mutant had little effect. A double mutant (P439S, N458T) or triple mutant (I435L, P439S, N458T) recruited SHPs as much as did Siglec-9, indicating that Pro439 in the proximal motif and Asn458 in the distal motif of Siglec-7 attenuate its ability to recruit phosphatases. These amino acids appeared to affect not only phosphatase recruitment but also the subsequent attenuation of Syk phosphorylation.

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