Biophysical Characterization of O-Glycosylated CD99 Recognition by Paired Ig-like Type 2 Receptors

Ikuo Shiratori,Daisuke Kohda,Kimiko Kuroki,Jing Wang,Hisashi Arase,Katsumi Maenaka,Shigekazu Tabata,Mizuho Kajikawa

doi:10.1074/jbc.m709793200

Abstract

Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRalpha) and activating (PILRbeta) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance (SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILRalpha can bind to CD99 with low affinity (K(d) approximately 2.2 microm), but activating PILRbeta binds with approximately 40 times lower affinity (K(d) approximately 85 microm). In addition to our previous mutagenesis study (Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. (2008) J. Immunol. 180, 1686-1693), the SPR analysis showed that PILRalpha can bind to each Ala mutant of the two CD99 O-glycosylated sites (Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILRalpha binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRbeta can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRalpha and PILRbeta. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRalpha-CD99 interaction is enthalpically driven with a large entropy loss (-TDeltaS = 8.9 kcal.mol(-1)), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.

Highlights

Tional responses of immune cells [1,2,3,4]
An immunoprecipitation experiment showed that the inhibitory Paired Ig-like type receptors (PILRs)␣ can bind to CD99 more strongly than the activating PILR␤ [7], suggesting the functional dominance of inhibitory PILR␣, as in other paired receptor families, such as KIRs and CD94/NKG2
According to Wang et al [8], the ectodomain of mouse CD99 fused with the Fc fragment of human IgG at the C-terminal site was expressed by a transient expression system, using 293T cells

Summary

Introduction

Tional responses of immune cells [1,2,3,4] Some of these receptor families have members with very similar extracellular regions responsible for ligand recognition but with different functions, activation and inhibition, due to the amino acid sequences of the transmembrane and intracellular regions. These receptor families are called “paired receptor families” and include human killer cell immunoglobulin (Ig)-like receptors (KIRs/ CD158), human Leukocyte Ig-like receptors (LILRs/LIRs/ILTs/ CD85), CD94/NKG2, and mouse Ly49. We compared the molecular mechanisms for the recognition of sugar-modified ligands by PILRs and selectins

Results

Discussion

Conclusion