One active C1r subunit is sufficient for the activity of the complement C1complex: Stabilization of C1r in the zymogen form by point mutations

The binding of CI (the first component of complement) to immune complexes l eads to the autoactivation of Clr through the cleavage of the Arg(463)-Ile( 464) bond in the catalytic domain. Spontaneous activation of Clr (and CI) a lso occurs in the fluid phase, preventing the characterization of the zymog en form of Clr, To overcome this difficulty, the zymogen form of human Clr was stabilized by mutating the Arg in the Arg(463)-Ile(464) bond to Gin. Th is mutant was designated as mutant QI, Recombinant Clr (wild type (wt) or m utant) was expressed in insect cells using serum-free medium in functionall y pure form; therefore, the cell culture supernatant was suitable to recons truct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymo gen and showed no hemolytic activity in reconstituted C1, However, this sta ble zymogen Clr mutant could form an active mix-ed dimer with the wt Clr, i ndicating that one active Clr subunit in the C1 complex is sufficient for t he full activity of the entire complex, Our experiments: also showed that t he exchange of Clr monomers between the Clr dimers is completed in less tha n 16 h even at pH 7 and 4 degrees C, Two other mutants were also constructe d by changing Arg(463) to Lys, or lle(464) to Phe, and were designated as m utants KI and RF, respectively. Although these substitutions did increase t he stability of the proenzyme in the cell culture supernatant, the mutant p roteins retained their ability to autoactivate, and both had a mt-like hemo lytic activity.