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 dom ain (P544G) of the mu-chain of mouse IgM has been studied. The P434A s ubstitution results in the loss of measurable complement-mediated cyto lytic activity (CML) and a decrease in the association rate constant a t low ionic strength (mu = 0.06), that results in a diminished K-a for C1q binding to P434A IgM bound to haptenated cells (0.4 x 10(9) M(-1) ). Binding of C1(qr(2)s(2)) could not be detected. In contrast, replac ement of proline at 544 had no measurable effect on the cytolytic or C 1q/C1 binding properties of the polymeric molecule, supporting the vie w that the C mu 3 domain is important in C1q binding and CML. The secr eted monomeric subunit of P544G was not able to mediate CML. Also, whe reas hapten-bound P544G polymer bound C1q with a functional affinity o f 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 P54 4G IgM monomer. This could not be attributed to differences in glycosy lation. Inasmuch as the P544G mutation per se had no effect on the C1q binding properties of the polymer, we conclude that unlike IgG, aggre gation does not sufficiently enhance the avidity of IgM monomer to ena ble it to activate complement. Augmentation of the site must occur dur ing polymerization or when the IgM binds to Ag.