The Amino-terminal Immunoglobulin-like Domain of Sialoadhesin Contains the Sialic Acid Binding Site

doi:10.1074/jbc.270.44.26184

The Amino-terminal Immunoglobulin-like Domain of Sialoadhesin Contains the Sialic Acid Binding Site

COMPARISON WITH CD22 (*)

From the (1) Imperial Cancer Research Fund Laboratories, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom,
the (2) Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom, and
the (3) Biochemisches Institut der Universität Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

^§ To whom correspondence should be addressed. Tel.: 44-1865-222-355; Fax: 44-1865-222-418.

Abstract

Sialoadhesin and CD22 are members of a recently characterized family of sialic acid-dependent adhesion molecules belonging to the immunoglobulin superfamily. Sialoadhesin is a macrophage-restricted receptor containing 17 extracellular Ig-like domains which recognizes oligosaccharides terminating in NeuAcα2-3Gal in N- and O-linked glycans. CD22 is a B cell-restricted receptor with seven Ig-like domains which selectively recognizes oligosaccharides terminating in NeuAcα2-6Gal in N-glycans. Sequence similarity between these proteins is highest within their first four amino-terminal Ig-like domains. Here we identify the domain(s) containing the binding sites of both molecules by generating a series of extracellular domain deletion mutants fused to the Fc portion of human IgG1. Binding activity was analyzed by solid phase cell adhesion assays and also by surface plasmon resonance using purified glycophorin and CD45 as ligands for sialoadhesin and CD22, respectively. For sialoadhesin, the amino-terminal V-set Ig-like domain was both necessary and sufficient to mediate sialic acid-dependent adhesion of the correct specificity. In contrast, for murine CD22, only constructs containing both the V-set domain and the adjacent C2-set domain were able to mediate sialic acid-dependent binding. These results are consistent with the sialic acid binding site for both proteins residing in the membrane distal V-set domain, but for CD22 a direct contribution in binding from the neighboring C2-set domain cannot be excluded.

Footnotes

↵* This work was supported by the Imperial Cancer Research Fund and the Mizutani Foundation for Glycosciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MAG

myelin-associated glycoprotein

6-N

NeuAcα2-6Galβ1-4GlcNAc

3-O

NeuAcα2-3Galβ1-3GalNAc

3-N

NeuAcα2-3Galβ1-3(4)GlcNAc

BCECF-AM

2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester

mAb

monoclonal antibody

Sn(d1)Fc

sialoadhesin-immunoglobulin chimera containing the first domain of sialoadhesin fused to the Fc portion of human IgG1

Sn(d1-3L)Fc

sialoadhesin-immunoglobulin chimera containing domains 1+2+3 of sialoadhesin fused to the Fc portion of human IgG1 and expressed with the leader peptide of CD33

Sn(d1)

a soluble form of sialoadhesin consisting of the isolated V-set domain

PCR

polymerase chain reaction

PBS

phosphate-buffered saline

BSA

bovine serum albumin

NCAM

neural cell adhesion molecule

ICAM

intercellular adhesion molecule.
↵² P. R. Crocker, unpublished observations.
↵³ M. Vinson and P. A. van der Merwe, unpublished observations.
↵⁴ S. D. Freeman, unpublished observations.
- Received July 5, 1995.