Localization of the complement-component-C3b-binding site and the cofactor activity for factor I in the 38kDa tryptic fragment of factor H

Abstract

Trypsin treatment of human factor H (H160) [enzyme/substrate ratio 1:100 (w/w), 30 min, 37 degrees C] generated a 38 kDa (H38) and a 142 kDa (H142) fragment linked by disulphide bonds (H38/142). The fragments were purified by reduction with 2-mercapto-ethanol, gel filtration on a Sephadex G-200 column and affinity chromatography with monoclonal anti-(factor H) antibody coupled to Sepharose 4B. This monoclonal antibody bound to a site in the 38 kDa fragment. To localize the C3b binding site in factor H we used two enzyme-linked immunosorbent assays (e.l.i.s.a.). For the first test, e.l.i.s.a. plates were coated with C3b; H160, H38/142, H38 and H142 were added, and their binding was monitored by goat anti-(factor H) and peroxidase-labelled rabbit anti-goat antibodies. Only intact factor H bound to the C3b-coated plates. For the second test, e.l.i.s.a. plates were coated with comparable amounts of factor H or its fragments, and C3b was offered at several dilutions. In contrast with the results from the first assay, C3b bound to intact factor H, H38/142 and H38 but not to H142, thus characterizing H38 as the fragment carrying the C3b-binding site. To identify the fragment responsible for the cofactor activity of factor H (cleavage of fluid-phase C3b by factor I), 125I-C3b was incubated with either H38 or H142 and factor I. H142 had no cofactor activity, whereas H38 had the same cofactor function as intact H. To further investigate the relationship between the C3b-binding site and the site of factor H essential for its cofactor activity, we made use of monoclonal antibodies directed against the H38. Those antibodies inhibiting the binding of C3b to H160 also inhibited the cofactor function, whereas those without effect on the C3b binding also did not interfere with the cofactor activity. This suggests that the C3b-binding site and the site essential for the cofactor activity of factor H are both localized in the 38 kDa tryptic fragment of factor H in close proximity or are identical.

Human Factor H-related Protein 5 (FHR-5)

“…SCRs 1-10 and 16 -20 of Factor H are believed to contain the binding site(s) for C3b, and decay-accelerating and cofactor activities have been localized to SCR 1-5 ( Fig. 9) (20,(23)(24)(25)(26)(27)(28).…”

Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b

“…It has been suggested that a total of three C3b-binding sites exist on fH. One has been located to the amino-terminal tryptic fragment (SCR1-6a) (33,34) and further mapped by functional assays to SCRs 1-4 (35)(36)(37). The other two binding sites have not yet been well characterized.…”

The C-terminus of complement factor H is essential for host cell protection

“…This 150 kDa glycoprotein is composed of 20 complement control protein (CCP) domains. The N-terminal part of the molecule (CCPs 1-4) is responsible for its complement regulatory activity (Alsenz et al, 1984;Kühn et al, 1995). FH has multiple binding sites for C3b, located within CCPs 1-4, CCPs 12-15 and CCPs 19-20 (Sharma and Pangburn, 1996;Jokiranta et al, 2000), and for heparin, located in CCP7, CCP9, CCPs 12-14, and CCPs 19-20 (Pangburn et al, 1991;Blackmore et al, 1996Blackmore et al, , 1998Ormsby et al, 2006).…”