One active C1r subunit is sufficient for the activity of the complement C1 complex: Stabilization of C1r in the zymogen form by point mutations

József Dobó, Péter Gál, Katalin Szilágyi, Sándor Cseh, Zsolt Lörincz, Verne N. Schumaker, Péter Závodszky

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15 Citations (Scopus)

Abstract

The binding of C1 (the first component of complement) to immune complexes leads to the autoactivation of C1r through the cleavage of the Arg463-Ile464 bond in the catalytic domain. Spontaneous activation of C1r (and C1) also occurs in the fluid phase, preventing the characterization of the zymogen form of C1r. To overcome this difficulty, the zymogen form of human C1r was stabilized by mutating the Arg in the Arg463-Ile464 bond to Gln. This mutant was designated as mutant QI. Recombinant C1r (wild type (wt) or mutant) was expressed in insect cells using serum-free medium in functionally pure form; therefore, the cell culture supernatant was suitable to reconstruct C1 for the hemolytic assay. Mutant QI was a stable, nonactivable zymogen and showed no hemolytic activity in reconstituted C1. However, this stable zymogen C1r mutant could form an active mixed dimer with the wt C1r, indicating that one active C1r subunit in the C1 complex is sufficient for the full activity of the entire complex. Our experiments also showed that the exchange of C1r monomers between the C1r dimers is completed in less than 16 h even at pH 7 and 4°C. Two other mutants were also constructed by changing Arg463 to Lys, or Ile464 to Phe, and were designated as mutants KI and RF, respectively. Although these substitutions did increase the stability of the proenzyme in the cell culture supernatant, the mutant proteins retained their ability to autoactivate, and both had a wt-like hemolytic activity.

Original languageEnglish
Pages (from-to)1108-1112
Number of pages5
JournalJournal of Immunology
Volume162
Issue number2
Publication statusPublished - Jan 15 1999

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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