title: Human complement factor I does not require cofactors for cleavage of synthetic substrates.
abstract: Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino-4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH(3)). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzenesulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, only Cr(2+) and Fe(3+) were found to inhibit both the proteolytic and amidolytic activity of fI.
authors.affiliation: Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. stefanos.tsiftsoglou@bioch.ox.ac.uk
authors.forename: Stefanos A
authors.initials: SA
authors.lastname: Tsiftsoglou
authors.forename: Robert B
authors.initials: RB
authors.lastname: Sim
mesh_terms: D017244:Complement Factor I / Q000037:antagonists & inhibitors / Q000502:physiology*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
chemical_list: D017244:Complement Factor I; D008274:Magnesium; D015032:Zinc