Differences between Human and Mouse IgM Fc Receptor (FcµR)

Abstract

Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since the bona fide IgM Fc receptor (FcµR) was identified in humans by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. In this short essay, we describe the differences between human and mouse FcµRs in terms of their identification processes, cellular distributions and ligand binding activities with emphasis on our recent findings from the mutational analysis of human FcµR. We have identified at least three sites of human FcµR, i.e., Asn66 in the CDR2, Lys79 to Arg83 in the DE loop and Asn109 in the CDR3, responsible for its constitutive IgM-ligand binding. Results of computational structural modeling analysis are consistent with these mutational data and a model of the ligand binding, Ig-like domain of human FcµR is proposed. Serendipitously, substitution of Glu41 and Met42 in the CDR1 of human FcµR with mouse equivalents Gln and Leu, either single or more prominently in combination, enhances both the receptor expression and IgM binding. These findings would help in the future development of preventive and therapeutic interventions targeting FcµR.

Keywords: 3D structure; FcR; FcµR; Fcα/µR; IgM binding; computational structural model; pIgR; species difference.

Figure 1

Schematic presentation of homology between human and mouse FcµRs. FcµR is depicted as a racquet-like shape consisting of N-terminal Ig-like domain (blue closed oval shape), stalk region (above the top line), transmembrane (between the two lines) and the cytoplasmic tail (below the bottom line). Hatch marks indicate exon boundaries and small red, green and yellow circles indicate a charged His residue in the transmembrane region and conserved five Ser and three Tyr residues in the cytoplasmic tail, respectively. Numbers on the left indicate percentage identity between human and mouse receptors in the overall or indicated regions. The position of aa addition (single letter code within frame) or gap (- within frame) in human (390-aa, left) and mouse (422-aa, right) FcµR are shown beside the cartoon.

Figure 2

Transient IgM binding by mouse FcµR-bearing transductant. AKR-derived thymoma line BW5147 cells stably expressing mouse FcµR and GFP (green) and WT BW5147 cells (black) were plated at 5 × 10⁴ cells/mL, cultured at 37 °C, and harvested after the indicated time periods. An equal mixture of FcµR⁺/GFP⁺ cells and WT cells was incubated with mouse IgG1κ mAb specific for mouse FcµR (MM3 clone; upper panel) or mouse IgMκ paraprotein (TEPC183; lower panel), washed, and developed by PE-labeled, rat IgG1κ anti-mouse κ mAb (187.1 clone) to determine the FcµR level and IgM binding, respectively. Stained cells were analyzed by flow cytometry. Note the IgM binding by FcµR⁺/GFP⁺ cells at early stages of culture (see arrows) compared to the minimal changes in cell surface receptor levels. GFP⁺ cells are circled with red dotted lines and a black line corresponds with mean fluorescence intensity of PE of FcµR⁺/GFP⁺ cells at 0 h for comparison.

Figure 3

IgM binding of human/mouse chimeric FcµRs. BW5147 cells stably expressing FcµR composed of Ig-like domain, stalk region and transmembrane/cytoplasmic tail (top left) of either human (H) or mouse (M) origin (left) were incubated with TEPC183 IgMκ for IgM binding (green lines), HM14 mAb to the stalk region of human FcµR (hu. Stalk; red lines) or MM3 mAb to the stalk region of mouse FcµR (mo. Stalk; blue lines), before developing with PE-labeled goat anti-mouse Ig antibodies and analyzing by flow cytometry. Note that IgM binding was only observed with chimeric receptor containing human-derived Ig-like domain.

Figure 4

Amino acid sequence alignment of the Ig-like domains of human and mouse FcµRs along with mutational results. The numbers on the top and bottom of the sequence indicate the amino acid (single letter code) position with regard to the first Met residue of human (Hu) and mouse (Mo) FcµR. Amino acid identity and gaps are indicated by dots (•) and dashes (-), respectively. Predicted ß-strands (blue lines) and complementary determining regions (CDRs; red lines) of human FcµR are indicated at the top of the sequence. The human aa residues highlighted in red, green or yellow colors were replaced with the corresponding mouse residues, and eight different human FcµR mutants are shown underneath with pale yellow highlights. Receptor level and IgM binding potential of each human FcµR mutant are indicated as arrows with decrease (↓), increase (↑) or no change (→). Accession numbers of human and mouse FcµRs in NCBI are NM_005449 and NM_026976, respectively.

Figure 5

Model structure of human FcµR Ig-like domain. Models of human FcµR domain (left) and after 180° horizontal rotation (right) are shown highlighting the aa residues mutated in this study: K24-G27 (red), E41/M42 (green), N66 (blue), K79-R83 (cyan), and N109 (magenta). The C-terminus (purple) and β strands are also indicated. Polymeric Ig receptor domain 1 (PDB 5D4K) was used as a template to create the human FcµR model.