Abstract
CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.
Highlights
CD22 binds to several T cell glycoproteins, including CD45, and binding of soluble CD22 to CD45 attenuates the increase in intracellular calcium normally seen in T cells following stimulation with anti-CD3 [8, 12]
In a series of experiments utilizing both the full-length CD2213 molecule expressed in COS cells or a truncated three domain construct fused to the Fc portion of Ig (CD22Rg), we and others have demonstrated that its ability to bind to cells or lactose;SNA,Sambucus nigra agglutinin; ICso, concentration giving50% inhibition of binding; FGP, fibrinogen glycopeptides; RIC, relative inhibitory concentrations (ICso relative to a2-6-sLac); PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; ONP, o-nitrophenyl
These studies were performed both with the native transmembrane CD2213 molecule, transiently expressed in COS cells, as well as with a fusion protein termed CD22Rg
Summary
Vol 270, No 13, Issue of March 31, pp. 7523-7532, 1995 Printed in U.S.A. EVIDENCE FOR A MINIMAL STRUCTURAL RECOGNITION MOTIF AND THE POTENTIAL IMPORTANCE OF MULTISITE BINDlNG*. In a series of experiments utilizing both the full-length CD2213 molecule expressed in COS cells or a truncated three domain construct fused to the Fc portion of Ig (CD22Rg), we and others have demonstrated that its ability to bind to cells or lactose;SNA,Sambucus nigra agglutinin; ICso, concentration giving50% inhibition of binding; FGP, fibrinogen glycopeptides; RIC, relative inhibitory concentrations (ICso relative to a2-6-sLac); PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; ONP, o-nitrophenyl. These experiments, together with additional work in the accompanying papers [22, 23] examining the role of CD22 in cell-adhesion events, offer new insights into how this Sia-specific lectin may function in complex biological systems
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