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

• Published in: The Journal of immunology (1950) Vol. 144; no. 11; pp. 4269 - 4274
• Main Authors: Ekdahl, KN, Nilsson, UR, Nilsson, B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Am Assoc Immnol 01.06.1990; American Association of Immunologists
• Subjects: Amino Acid Sequence; Biological and medical sciences; Complement; Complement C3b - metabolism; Complement C3b Inactivator Proteins - metabolism; Complement Factor H; Complement Factor I; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunologi; Immunology; In Vitro Techniques; Isoflurophate - pharmacology; MEDICIN; MEDICINE; Microbiology, immunology, infectious diseases; Mikrobiologi, immunologi, infektionssjukdomar; Molecular immunology; Molecular Sequence Data; Molecular Weight; Peptide Fragments - analysis; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors; Substrate Specificity; Human; Specificity; Fibrinogen; Cleavage; Inhibition; Complement C3b; Aminoacid sequence; Conformation
• ISSN: 0022-1767; 1550-6606; 1550-6606
• DOI: 10.4049/jimmunol.144.11.4269

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.