title: Functional and structural domains of the sixth component (C6) of human complement.
abstract: The effects of serine protease inhibitors, diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), on hemolytic activity of C6 were reinvestigated. C6 was inactivated in a range of 1-10 mM by both of the inhibitors as previously reported. Limited proteolytic digestion was also studied to elucidate the functional and structural domains of C6. The major fragments produced by trypsin, plasmin, or lysyl endopeptidase could not be separated unless disulfide bonds were disrupted, but Staphylococcus aureus V8 protease yielded several fragments, each of which was not linked by disulfide bond. When C6 labeled with [3H]DFP was subjected to limited digestion with V8 protease, a fragment with a molecular weight of 38 kilodaltons (kDa) was mainly labeled and other fragments of 53 kDa and 26.4 kDa were also faintly labeled, while fragment 35 kDa wasn't labeled, indicating specific domains reactive with DFP. On the other hand, when C6 with or without DFP treatment was digested with V8 protease and those fragments were incubated with C5 and subjected to sucrose density ultracentrifugation, fragments 53, 38, 35 and 27.5 kDa interacted with C5 in both cases. These results suggest that C6 modified by DFP can interact with C5, and the amino-terminal sequences of fragment 38 and 35 kDa suggest the binding domain of C6 with C5 takes place within the two short consensus repeats.
authors.affiliation: School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nakano
authors.forename: M
authors.initials: M
authors.lastname: Hashimoto
authors.forename: N H
authors.initials: NH
authors.lastname: Choi
authors.forename: Y
authors.initials: Y
authors.lastname: Sugita
authors.forename: T
authors.initials: T
authors.lastname: Tobe
authors.forename: T
authors.initials: T
authors.lastname: Mazda
authors.forename: M
authors.initials: M
authors.lastname: Tomita
mesh_terms: D003183:Complement C6 / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D012697:Serine Endopeptidases
chemical_list: D003183:Complement C6; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase