C1q binding properties of monomer and polymer forms of mouse IgM mu-chain variants. Pro544Gly and Pro434Ala.
The effect of replacing proline with alanine at position 434 in the C mu 3 domain (P434A) and with glycine at position 544 in the C mu 4 domain (P544G) of the mu-chain of mouse IgM has been studied. The P434A substitution results in the loss of measurable complement-mediated cytolytic activity (CML) and a decrease in the association rate constant at low ionic strength (mu = 0.06), that results in a diminished Ka for C1q binding to P434A IgM bound to haptenated cells (0.4 x 10(9) M-1). Binding of C1(qr2s2) could not be detected. In contrast, replacement of proline at 544 had no measurable effect on the cytolytic or C1q/C1 binding properties of the polymeric molecule, supporting the view that the C mu 3 domain is important in C1q binding and CML. The secreted monomeric subunit of P544G was not able to mediate CML. Also, whereas hapten-bound P544G polymer bound C1q with a functional affinity of 1.5 x 10(9) M-1 at low ionic strength (mu = 0.06), similar to that observed with wild-type polymer (1.7 x 10(9) M-1) and wild-type IgG monomer (4.7 x 10(9) M-1), no C1q binding was detected with the P544G IgM monomer. This could not be attributed to differences in glycosylation. Inasmuch as the P544G mutation per se had no effect on the C1q binding properties of the polymer, we conclude that unlike IgG, aggregation does not sufficiently enhance the avidity of IgM monomer to enable it to activate complement. Augmentation of the site must occur during polymerization or when the IgM binds to Ag.
Department of Biochemistry, University of Toronto, Ontario, Canada.
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B
Taylor
J F
JF
Wright
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Arya
D E
DE
Isenman
M J
MJ
Shulman
R H
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Painter
D000818:Animals; D001704:Biopolymers / Q000378:metabolism; D002460:Cell Line; D003167:Complement Activation; D015922:Complement C1q / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D006825:Hybridomas; D007075:Immunoglobulin M / Q000378:metabolism*; D051379:Mice; D009154:Mutation / Q000502:physiology; D011485:Protein Binding / Q000276:immunology; D011863:Radioimmunoassay; D013329:Structure-Activity Relationship
D001704:Biopolymers; D007075:Immunoglobulin M; D015922:Complement C1q