Affinity and Kinetic Analysis of Fcγ Receptor IIIa (CD16a) Binding to IgG Ligands

Ping Li,Ning Jiang,Shanmugam Nagarajan,Robert Wohlhueter,Periasamy Selvaraj,Cheng Zhu

doi:10.1074/jbc.m609064200

Abstract

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

Highlights

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc ␥ receptors (Fc␥Rs) triggers a wide variety of effector functions
We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble Fc␥RIIIa fused with Ig Fc using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 ؋ 103, 8.2 ؋ 103, 1.1 ؋ 104 and 1.8 ؋ 104 M؊1 s؊1, off-rates of 4.7 ؋ 10؊3, 5.7 ؋ 10؊3, 5.9 ؋ 10؊3, and 1.9 ؋ 10؊2 s؊1, and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 ␮M, respectively
Of the three Fc␥Rs, CD64 binds to monomeric IgG with high affinity (Kd ϳ tens of nM) (10), CD32 and CD16b are of low affinities (Kd, several and several tens of ␮M, respectively) (6, 11, 12), and CD16a is considered as an intermediate affinity receptor (Kd ϳ hundreds of ␮M) for monomeric IgG (6, 10, 12)

Summary

Introduction

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc ␥ receptors (Fc␥Rs) triggers a wide variety of effector functions. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-Fc␥R interactions. In addition to the above results, which were obtained by conventional assays using Fc␥R-expressing cells, there were two studies using the surface plasmon resonance (SPR) technology and soluble Fc␥Rs. Galon et al (13) reported half-lives of the order of 10 min and equilibrium dissociation constants of several micromolar for sCD16bNA2 binding to human (h) IgG1 and IgG3.

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Conclusion