Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors

Paired Ig-like type 2 receptors (PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory (PILRα) and activating (PILRβ) 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 PILRα can bind to CD99 with low affinity (K_d ∼ 2.2 μM), but activating PILRβ binds with ∼40 times lower affinity (K_d ∼ 85 μM). 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 PILRα 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 PILRα binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILRβ can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILRα and PILRβ. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILRα-CD99 interaction is enthalpically driven with a large entropy loss (-TΔS =- 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.