Investigation of the Mechanism of Complement Activation by Immunoglobulin G

Citation

Sand, Irving D. (1982) Investigation of the Mechanism of Complement Activation by Immunoglobulin G. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/p8jz-sj81. https://resolver.caltech.edu/CaltechTHESIS:05162018-161819743

Abstract

The synthesis of a tryptophan specific reagent, dimethyl (2-hydroxy-3-methyl-5-nitrobenzyl) sulfoniurn bromide, is described. The synthetic route incorporated ³H and ¹⁴C. The reagent was shown to specifically benzylate tryptophan which was either in solution as a free amino acid or in a protein. Physicochemical properties of the reagent which may be related to its specificity were examined.

Evidence is presented that a conformational change occurs in mouse IgG_2a when antigen binds and that this change is a necessary condition for the activation of the complement cascade. A ³H/¹⁴C-labeled, tryptophan-specific reagent was used to monitor the reactivity of tryptophans in the Fc region of the immunoglobulin. This reactivity correlated with the ability of the antibody to fix complement. Thus binding monovalent and multivalent antigens caused decreases in the reactivity of Fc tryptophan residues by 15% and 22%, respectively. Hapten had no effect on the reactivity of these residues. After reduction and alkylation of disulfide bonds in the IgG to an extent which abolished the antibody's ability to fix complement, the reactivity of Fc tryptophan did not change when monovalent antigen was bound. The Fc tryptophan reactivity of similarly reduced and alkylated IgG increased by 25% when multivalent antigen was bound.

A combined classical-alternate pathway assay was developed which distinguishes effects on those components preceeding C3 in the complement cascade from effects on C3 and later components. When antigen-antibody aggregates were assayed by this method, fixation was observed in both the classical and alternate pathways. However, when oligomers of mouse IgG_2a produced by chemical crosslinking were assayed, no fixation of the components of the alternate pathway was observed. The time course of classical pathway fixation by these synthetic oligomers indicates that some of the components preceding C3 in the cascade were being enzymatically degraded.

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