Identification of Amino Acid Residues in Human IgM Fc Receptor (FcµR) Critical for IgM Binding

Abstract

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.

Differences between Human and Mouse IgM Fc Receptor (FcµR)

“…(The numbers in IgM indicate the aa position from the first N-terminal aa residue in the Cµ1 domain, not from the first Met residue that was applied for FcµR, because of variable lengths of Ig VH regions.) On the other hand, our docking model of pentameric IgM and FcµR revealed that Gln387 in the Cµ4 and to a lesser extent, Glu275 in the Cµ3 of IgM and Asn66 in the CDR2, Arg83 in the DE loop and Asn109 in the CDR3 of FcµR were all at the interface, suggesting their potential involvement in IgM binding [34]. This model was thus consistent with substantial loss of IgM binding in mutants involving removal of Asn66 or substitution of Lys79-Arg83 or Asn109 with the mouse equivalents and with the involvement of both Cµ4 and Cµ3 domains in FcµR binding [9,[37][38][39][40].…”

“…It was noteworthy that none of the above replacements caused significant alterations in the main-chain conformation of the receptor as determined by modeling of the substitutions or changes in the melting temperature assessed by molecular dynamics simulations. The results of receptor levels and IgM binding potential are summarized in a cartoon fashion in Figure 4, and the detailed flow cytometric data have been reported elsewhere [34].…”

Soluble Fc Receptor for IgM in Sera From Subsets of Patients With Chronic Lymphocytic Leukemia as Determined by a New Mouse Monoclonal Antibody

“…In some experiments for transductants, BD Perm/Wash Buffer was used for intracellular staining. Stained cells were examined by Becton Dickinson (BD) LSRFortessa flow cytometer along with FACSDiva software (BD Bioscience), and flow cytometric data were analyzed with Flowjo software (BD) ( 14 ). In some experiments, PBMCs isolated from healthy individuals were examined for cell surface and intracellular staining with HMD22 mAb as described ( 5 ).…”

B-Lymphozyten und Plasmazellen als Treiber rheumatischer Erkrankungen

“…Die Ergebnisse der rechnergestützten Strukturmodellierungsanalyse stimmen mit diesen Mutationsdaten überein (Abb. 1 ; [ 30 ]). Diese Erkenntnisse könnten bei der künftigen Entwicklung von präventiven und therapeutischen Maßnahmen, die auf den FcµR abzielen, hilfreich sein.…”