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Fc?RI binding at the IgA1 CH2-CH3 interface induces long-range conformational changes that are transmitted to the hinge region.

ABSTRACT: IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, Fc?RI, by IgA immune complexes. Fc?RI binds to the IgA1-Fc domain (Fc?) at the CH2-CH3 junction and, except for CH2 L257 and L258, all side-chain contacts are contributed by the CH3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of Fc?RI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the Fc?RI:Fc? interaction. Surprisingly, individual mutation of CH2 residues L257 and L258, found at the periphery of the Fc?RI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the CH2-CH3 junction (or its equivalent). Given the importance of residues near the CH2-CH3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fc?. Our simulations indicate that Fc?RI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fc?, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that Fc?RI binding to the Fc? CH2-CH3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy.

SUBMITTER: Posgai MT

PROVIDER: S-EPMC6156678 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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FcαRI binding at the IgA1 CH2-CH3 interface induces long-range conformational changes that are transmitted to the hinge region.

Posgai Monica T MT Tonddast-Navaei Sam S Jayasinghe Manori M Ibrahim George M GM Stan George G Herr Andrew B AB

Proceedings of the National Academy of Sciences of the United States of America 20180904 38

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1-Fc domain (Fcα) at the CH2-CH3 junction and, except for CH2 L257 and L258, all side-chain contacts are contributed by the CH3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The en ...[more]

PMID: 30181292

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Project description:BackgroundDuring infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2-CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo.MethodsReplication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2-CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion.ResultsWe confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2-CH3 domain. Importantly, we further show that mutations in the CH2-CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV.ConclusionsOur findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs.

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Fc?RI binding at the IgA1 CH2-CH3 interface induces long-range conformational changes that are transmitted to the hinge region.

Publications

FcαRI binding at the IgA1 C<sub>H</sub>2-C<sub>H</sub>3 interface induces long-range conformational changes that are transmitted to the hinge region.

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