COOH-terminal Substitutions in the Serpin C1 Inhibitor That Cause Loop Overinsertion and Subsequent Multimerization

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family, We have studied the structural consequences of three substitutions (Val(451) --> Met, Phe(455) --> Ser, and Pro(476) --> Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema, Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin m. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional, Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor, A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val(451) --> Met and Pro(476) --> Ser mutant proteins, and to a lesser extent Phe(455) --> Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val(451) --> Met and Pro(476) --> Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody, Investigation of plasmas of patients with the Val(451) --> Met or Pro(476) --> Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxyl-terminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in over-insertion of the loop into beta-sheet A, which subsequently leads to multimerization.