title: The catalytically active serine protease domain of human complement factor I.
abstract: Factor I (fI) is a major regulator of complement. As a protease it has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH). Cleavage of C3b by fI yields iC3b, a major opsonin. The cleavage occurs through the formation of a ternary complex between the enzyme, the substrate, and the cofactor. The catalytic subunit of fI, the SP domain, accommodates substrate recognition and cleavage. The role of the fI heavy chain within the catalysis complex is unknown. Using partial proteolysis and affinity chromatography an intact form of the SP domain was generated and isolated from fI in high yield. fI and the SP domain were found to have similar amidolytic activities but strikingly different proteolytic activities on C3(NH(3)). fI did not cleave C3(NH(3)) in the absence of fH, while in its presence it cleaved C3(NH(3)) rapidly at two sites. The SP domain, however, slowly cleaved C3(NH(3)) in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. Pefabloc SC and antipain inhibited the proteolytic activity of both fI and the SP domain, but suramin inhibited only fI and not the SP domain. The contrast in the proteolytic activities suggests that the heavy chain domains and the cofactor must have roles in orienting the natural substrates and restricting cleavage to the two sites which yield iC3b through a highly specific catalysis.
authors.affiliation: MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England, UK. stefanos.tsiftsoglou@bioch.ox.ac.uk
authors.forename: Stefanos A
authors.initials: SA
authors.lastname: Tsiftsoglou
authors.forename: Antony C
authors.initials: AC
authors.lastname: Willis
authors.forename: Pengyun
authors.initials: P
authors.lastname: Li
authors.forename: Xuehui
authors.initials: X
authors.lastname: Chen
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Mitchell
authors.forename: Zihe
authors.initials: Z
authors.lastname: Rao
authors.forename: Robert B
authors.initials: RB
authors.lastname: Sim
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003179:Complement C3b / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005341:Fibrinolysin; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D003179:Complement C3b; D005340:Fibrinogen; D012697:Serine Endopeptidases; D005341:Fibrinolysin