Mapping of amino acid residues in the C mu 3 domain of mouse IgM important in macromolecular assembly and complement-dependent cytolysis.
By analyzing the effects of single site mutations of a TNP-binding mouse IgM we have identified amino acid residues clustered in two regions in the C mu 3 domain that are important in the complement-dependent cytolytic activity of polymeric IgM. Some of the mutations also impaired IgM polymerization. For one of these clusters, D432G, P434A, and P436S, which lies on the fy2 and fy3 strands and their connecting loop, polymerization was little affected and the effect on the cytolytic activity of the polymer fraction was taken to imply direct involvement of the residue in C1 binding. The other cluster, involving residues D356A K361A and D417G, is situated at the other end of the C mu 3 domain closer to the center of the Fc mu disc. The D356A K361A and D417G mutations significantly impaired polymer formation, suggesting that these residues are necessary for proper folding or packing of the C mu 3 domains and may affect cytolysis only indirectly. Some other mutations had little or no effect on polymerization or cytolytic activity (E423A, E527G), whereas some mutations impaired only IgM polymerization without affecting cytolytic activity (D344A, K361A, K443A P544G). In others the defect in polymerization was so profound that only the monomer formed (H430A/N/Q and K438G). Our results also suggest that the C1 binding site of IgM is not strictly homologous to the C1 binding site of IgG. Although mutation of E318 of IgG has been shown to reduce its cytolytic activity, mutation of the homologous residue in IgM, E423, was without effect as were mutations of other flanking-charged residues. Proline at 436 in IgM and 331 in IgG may, however, be a common element.
Arya
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S
Department of Biochemistry, University of Toronto, Ontario, Canada.
Chen
F
F
Spycher
S
S
Isenman
D E
DE
Shulman
M J
MJ
Painter
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015922:Complement C1q / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D006461:Hemolysis; D007127:Immunoglobulin Constant Regions / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007148:Immunoglobulin mu-Chains / Q000737:chemistry*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014301:Trinitrobenzenes / Q000276:immunology
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007148:Immunoglobulin mu-Chains; D046911:Macromolecular Substances; D014301:Trinitrobenzenes; D015922:Complement C1q