Impact of a Three Amino Acid Deletion in the CH2 Domain of Murine IgG1 on Fc-Associated Effector Functions

Abstract

Four murine IgG subclasses display markedly different Fc-associated effector functions because of their differential binding to three activating IgG Fc receptors (FcγRI, FcγRIII, and FcγRIV) and C1q. Previous analysis of IgG subclass switch variants of 34-3C anti-RBC monoclonal autoantibodies revealed that the IgG1 subclass, which binds only to FcγRIII and fails to activate complement, displayed the poorest pathogenic potential. This could be related to the presence of a three amino acid deletion at positions 233–235 in the CH2 domain uniquely found in this subclass. To address this question, IgG1 insertion and IgG2b deletion mutants at positions 233–235 of 34-3C anti-RBC Abs were generated, and their ability to initiate effector functions and their pathogenicity were compared with those of the respective wild-type Abs. The insertion of amino acid residues at positions 233–235 enabled the IgG1 subclass to bind FcγRIV but did not improve the binding to C1q. Accordingly, its pathogenicity was enhanced but still inferior to that of IgG2b. In contrast, the IgG2b deletion mutant lost its ability to bind to FcγRIV and activate complement. Consequently, its pathogenicity was markedly diminished to a level comparable to that of IgG1. Our results demonstrated that the initiation of FcγR- and complement-mediated effector functions of IgG2b was profoundly affected by the three amino acid deletion at positions 233–235, but that this natural three amino acid deletion could only partially explain the poor binding of IgG1 to FcγRIV and C1q. This indicates the lack in the IgG1 subclass of as yet unknown motifs promoting efficient interaction with FcγRIV and C1q.

Molecular and Cellular Pathways of Immunoglobulin G Activity In Vivo

“…As expected, IgG1 autoantibodies were solely dependent on the activating Fc RIII [74,76]. The activity of IgG2a and IgG2b, however, was dependent on both Fc RIII and Fc RIV [77,78]. With respect to the high affinity Fc RI an involvement only at high doses of the IgG2a autoantibody was noted, consistent with the fact that this activating Fc R is saturated with serum IgG in the steady state and that high amounts of immune complexes are required to trigger this receptor despite its high affinity for IgG2a [78].…”

“…The activity of IgG2a and IgG2b, however, was dependent on both Fc RIII and Fc RIV [77,78]. With respect to the high affinity Fc RI an involvement only at high doses of the IgG2a autoantibody was noted, consistent with the fact that this activating Fc R is saturated with serum IgG in the steady state and that high amounts of immune complexes are required to trigger this receptor despite its high affinity for IgG2a [78]. Due to the different contribution of the individual activating Fc Rs to AIHA, one might expect a different effector cell population to be involved in red blood cell removal.…”

Affinity of human IgG subclasses to mouse Fc gamma receptors

“…30 The affinities we obtained for mouse IgG for mouse FcgR and human IgG for human FcgR are in agreement with those previously reported. 12,14,16,21,[31][32][33][34] We have compared the mouse data obtained in this research with that found in literature in a summarizing table (Table S1). Some minor differences were noted, with our data falling well within the range of the affinities found by other groups.…”

Lack of Galactosylation Enhances the Pathogenic Activity of IgG1 but Not IgG2a Anti-Erythrocyte Autoantibodies

“…The generation of a 34-3C IgG1 classswitch variant and a 34-3C IgG2a F243A mutant carrying phenylalanine instead of alanine at position 243 in the CH2 domain was previously described (33,40). All the hybridoma and transfectoma cells were grown in DMEM supplemented with 1% Ultroser HY (PALL Life Sciences, Cergy, France), and IgG mAbs were purified from culture supernatants by protein G column chromatography.…”

“…Purification of N-glycans from different IgG mAbs was performed based on chemoselective glycoblotting technique, as described previously (40)(41)(42). In brief, IgG samples were reductively alkylated under the presence of detergent and then digested with trypsin and peptide N-glycosidase F (Roche Diagnostics, Penzberg, Germany) (43).…”