grantor:
University of TorontoThe IgM and IgG classes of immunoglobulins share an important biological function that is distinct from that of other classes. When bound to antigen, both can initiate the classical complement pathway by interacting with C1q, a subunit of the first component of complement (C1). To study the C1q binding properties of IgM in a form comparable to that of IgG, a 'monomeric' form of mouse IgM was prepared and its function was compared to that of a mouse IgG with the same antigen binding site. Monomeric IgM was isolated from a mutant (IgM P544G). The monomeric form of this mutant was not able to bind C1q or initiate complement-mediated lysis (CML) on haptenated SRBC. Under the same conditions, the positive control (IgG2b) bound C1q and initiated CML in a dose-dependent manner. Polymeric IgM isolated from this mutant was able to bind C1q and initiate CML as well as wildtype IgM. To understand monomeric IgM inactivity, mouse IgM/IgG2b hybrid mutants were created and analyzed for their C1q binding properties. Cã2, the intrinsically active C1q binding domain of IgG2b, did not bind C1q or initiate CML when placed in the monomeric IgM background ([mu][mu]ã[mu]). This suggests that the activity of an intrinsically active C1q binding domain is hidden by one or both of the neighbouring C[mu]2 and C[mu]4 domains. C[mu]3, the putative C1q binding domain of IgM, remained inactive when it was placed in the non-inhibitory IgG2b background (ãã[mu]ã), demonstrating that a pre-formed C1q binding site is not found in the C[mu]3 domain. Collectively, the results suggest that if the C[mu]3 contains the entire C1q binding site for IgM, the site is unformed and hidden. To determine if one or both of the C[mu]2 and CU[mu]4 domains are responsible for the inactivity of the [mu][mu]ã[mu] hybrid monomer two hybrids ([mu]ãã[mu] and [mu][mu]ãã) were created in which these domains were individually replaced by their corresponding IgG2b domains (ã-hinge and Cã3). The results show that C[mu]4 interferes with the interaction between C1 and the Cã2 domain whereas C[mu]2 interferes with the subsequent activation of C1. C[mu]2 and C[mu]4 may therefore have similar inhibitory effects on the C[mu]3 domain in the IgM monomer. The transplantation of C[mu]3 together with C[mu]4 into the IgG background permitted polymer formation. This polymer was able to bind C1q, although both the monomer and the polymer forms were not able to initiate CML; conversely, all IgM polymers with a transplanted Cã2 domain were active in both C1q binding and CML and demonstrated apparent Kd values for C1q (1-2 * 10-9 M) that were similar to that of wild-type IgM. The findings reported in this thesis are discussed in relation to the activity of the IgM polymer and are consistent with the view that the 'star' to 'staple' conformational change resulting when IgM binds to antigen is necessary for the expression of the C1q binding site. (Abstract shortened by UMI.)Ph.D
