Inhibition of factor I by diisopropylfluorophosphate. Evidence of conformational changes in factor I induced by C3b and additional studies on the specificity of factor I.
The factor I-mediated cleavage of C3b, using factor H as a cofactor was completely inhibited by diisopropylfluorophosphate (DFP) when factor I and C3b were incubated with DFP before the addition of factor H. Inhibition, although to a lesser degree, was observed when factor H was present during DFP-exposure. No inhibition in factor I activity was seen when factor I and H were incubated with DFP either alone or together. It was also demonstrated that the 38-kDa subunit of factor I bound radiolabeled DFP when factor I and C3b together were exposed to DFP. These observations suggest that factor I interacts with C3b in a manner that exposes its catalytic site to DFP, an interaction that is independent of factor H. The inhibitory effect by DFP on factor I led us to further investigate the factor I cleavage products of iC3b, inasmuch as previous reports were ambiguous as to whether digestion occurs in the presence of DFP. Digestion of C3b bound to activated thiol Sepharose (ATS-C3b) in the presence of factor H at low pH and ionic strength and in serum by complement activation produced C3d,g-like fragments with apparent molecular mass of 41 and 43 kDa. These fragments were shown to have three different N-terminal and two different C-terminal ends. The major fragments had N-terminal sequences starting with Glu933, as shown by sequence determination. Traces of fragments extending beyond this point were also found, shown by Western blot analysis using a panel of mAb previously shown to bind to epitopes exposed within a region of C3 spanning residues 929 to 943, as well as a shorter fragment starting with Glu938. When digestion of C3b is carried out in the presence of DFP, the factor I level necessary for digestion is elevated and may explain how the first two cleavages producing iC3b but not the following giving C3d,g, can occur. The finding of several factor I cleavage sites in the C3d,g region of C3 demonstrates that factor I has a broad specificity, mainly for arginyl bonds. It has also been shown to digest a lysyl bond exposed in ATS-bound C3b.
Ekdahl
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Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden.
Nilsson
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UR
Nilsson
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B
D000595:Amino Acid Sequence; D003179:Complement C3b / Q000378:metabolism*; D003180:Complement C3b Inactivator Proteins / Q000378:metabolism; D017242:Complement Factor H; D017244:Complement Factor I; D006801:Humans; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors; D013379:Substrate Specificity
C493495:CFH protein, human; D003180:Complement C3b Inactivator Proteins; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; D003179:Complement C3b; D017242:Complement Factor H; D012697:Serine Endopeptidases; D017244:Complement Factor I