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Mapping of the binding site for Fc?R in human IgM-Fc.

ABSTRACT: Fc?R is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fc?/?R and pIgR, but unlike these two other IgM receptors which also bind IgA, Fc?R exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/Fc?R interaction mainly involves the C?4 domains with possible contributions from either C?3 or C?2. To define the binding site more precisely, we generated three recombinant IgM-Fc proteins with specific mutations in the C?3 and C?4 domains, as well as a construct lacking the C?2 domains, and analyzed their interaction with the extracellular Ig-like domain of Fc?R using surface plasmon resonance analysis. There is a binding site for Fc?R in each IgM heavy chain. Neither the absence of the C?2 domains nor the quadruple mutant D340S/Q341G/D342S/T343S (in C?3 adjacent to C?2) affected Fc?R binding, whereas double mutant K361D/D416R (in C?3 at the C?4 interface) substantially decreased binding, and a single mutation Q510R (in C?4) completely abolished Fc?R binding. We conclude that glutamine at position 510 in C?4 is critical for IgM binding to Fc?R. This will facilitate discrimination between the distinct effects of Fc?R interactions with soluble IgM and with the IgM BCR.

SUBMITTER: Nyamboya RA

PROVIDER: S-EPMC6905151 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Mapping of the binding site for FcμR in human IgM-Fc.

Nyamboya Rosemary A RA Sutton Brian J BJ Calvert Rosaleen A RA

Biochimica et biophysica acta. Proteins and proteomics 20190823 1

FcμR is a high-affinity receptor for the Fc portion of human IgM. It participates in B cell activation, cell survival and proliferation, but the full range of its functions remains to be elucidated. The receptor has an extracellular immunoglobulin (Ig)-like domain homologous to those in Fcα/μR and pIgR, but unlike these two other IgM receptors which also bind IgA, FcμR exhibits a binding specificity for only IgM-Fc. Previous studies have suggested that the IgM/FcμR interaction mainly involves th ...[more]

PMID: 31449905

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Project description:Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against infections and for regulation of immune responses to self-antigens. The roles of its Fc receptor (FcµR) in these IgM effector functions have begun to be explored. In the present study, by taking advantage of the difference in IgM-ligand binding of FcµRs of human (constitutive binding) and mouse (transient binding), we replaced non-conserved amino acid residues of human FcµR with mouse equivalents before establishment of cell lines stably expressing mutant or wild-type (WT) receptors. The resultant eight-different mutant FcµR-bearing cells were compared with WT receptor-bearing cells for cell-surface expression and IgM-binding by flow cytometric assessments using receptor-specific mAbs and IgM paraproteins as ligands. Three sites Asn66, Lys79-Arg83, and Asn109, which are likely in the CDR2, DE loop and CDR3 of the human FcµR Ig-like domain, respectively, were responsible for constitutive IgM binding. Intriguingly, substitution of Glu41 and Met42 in the presumed CDR1 with the corresponding mouse residues Gln and Leu, either single or more prominently in combination, enhanced both the receptor expression and IgM binding. A four-aa stretch of Lys24-Gly27 in the predicted A ß-strand of human FcµR appeared to be essential for maintenance of its proper receptor conformation on plasma membranes because of reduction of both receptor expression and IgM-binding potential when these were mutated. Results from a computational structural modeling analysis were consistent with these mutational data and identified a possible mode of binding of FcµR with IgM involving the loops including Asn66, Arg83 and Asn109 of FcµR interacting principally with the Cµ4 domain including Gln510 and to a lesser extent Cµ3 domain including Glu398, of human IgM. To our knowledge, this is the first experimental report describing the identification of amino acid residues of human FcµR critical for binding to IgM Fc.

Dataset Information

Mapping of the binding site for Fc?R in human IgM-Fc.

Publications

Mapping of the binding site for FcμR in human IgM-Fc.

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