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 (Val451-->Met, Phe455-->Ser, and Pro476-->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 III. 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 Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->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 Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->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 carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.
Eldering
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Unité d'Immunogénétique, Institut Pasteur, INSERM Unit 276, Paris, France.
Verpy
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Roem
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D
Meo
T
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Tosi
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M
D000799:Angioedema / Q000097:blood / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal; D002264:Carboxylic Acids / Q000737:chemistry*; D002460:Cell Line; D002850:Chromatography, Gel; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D011233:Precipitin Tests; D015843:Serpins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014357:Trypsin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002264:Carboxylic Acids; D015843:Serpins; D014357:Trypsin