Functional mapping of the FcγRII binding site on human IgG1 by synthetic peptides

Abstract

Receptors specific for the Fc part of IgG (Fc + R) are expressed by several cell types and play diverse roles in immune responses. Impaired function of the activating and inhibitory Fc + R may result in autoimmunity. Thus, the modulation of IgG-Fc + R interaction can be a target for the development of treatments for some autoimmune and inflammatory diseases. This study addresses the localization and functional characterization of linear sequences in human IgG1 which bind to Fc + RII. Peptides with overlapping sequences derived from the CH2 domain of human IgG1 between P 234 and S 298 were synthesized and used in binding and functional experiments. Binding of the peptides to Fc + R was assayed in vitro and ex vivo, and peptides found to interact were functionally tested. The shortest effective peptide was T 256-P 271 complexed by avidin exhibited functional activity; they induced Fc + RIIb-mediated inhibition of the BCR-triggered Ca 2+ response of human Burkitt lymphoma cells, and inflammatory cytokine production (TNF- § and IL-6) by the human monocyte cell line MonoMac. In conclusion, our results suggest that the selected peptides functionally represent the Fc + RII-binding part of IgG1.

IgG2m4, an engineered antibody isotype with reduced Fc function

“…Previous work suggested different functions for residue H268. In the work reported by Medgyesi et al 13 H268 was shown to be important in FcγRIIb binding while in another study by Brekke et al…”

“…Further studies have identified individual amino acid residues that interact with Fcγ receptors and complement C1q protein. For example, residues H268, 13,14 A330 and P331 were shown to be critical for Fcγ receptors and C1q bindings. [15][16][17][18][19][20] IgG1, IgG2 and IgG4 isotypes are the choices for therapeutic antibodies currently in clinical use.…”

Determination of ligand binding capacity of soluble FcγRII and FcγRIII in sera of patients with SLE

“…At the same time, mAbs used as capture antibodies recognize other epitopes related to IC binding sites, in contrast to monomeric IgG [8]. Our new ELISAs were not capable of distinguishing between "a" and "b" isoforms of sFcgRII and sFcgRIII, [1,10] however, a possibility of separating them on the basis of their specific binding capacity exists [2,9].…”

“…The absorbance was measured with a Labsystem Multiscan MS ELISA reader at 492 nm. Assuming one IgG molecule occupied one receptor site [9], the levels of sfFcgRII and sfFcgRIII were expressed as ng of equivalent rabbit IgG per ml serum on the basis of an IgG calibration curve.…”

Identification of a High Affinity FcγRIIA-binding Peptide That Distinguishes FcγRIIA from FcγRIIB and Exploits FcγRIIA-mediated Phagocytosis and Degradation

“…Because the extracellular domains of Fc␥RIIA and Fc␥RIIB are closely related in structure, having 93% amino acid identity in their extracellular domains, it has been challenging to design modified IgGs with mutations in the Fc region that distinguish between the two receptors (21). Strategies have been utilized to identify smaller molecules that target Fc␥ receptors, including design of IgG-derived peptide sequences that mimic the part of IgG-Fc that binds Fc␥ receptors (22)(23)(24)(25), but still neither of these discriminates between the two receptors.…”