Rat complement factor H: molecular cloning, sequencing and quantification with a newly established ELISA.

Abstract

Factor H (FH) is the predominant soluble regulatory protein of the complement system. With a concentration of 300-600 microg/ml in human plasma it acts as a cofactor for the FI-mediated cleavage of the component C3b to iC3b. Furthermore, it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex (i.e. it has decay accelerating activity). FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of nearly 60 amino acid residues. For the screening of a rat liver cDNA library, we used two hybridization probes which had been produced by polymerase chain reaction (PCR). The probes were generated using degenerated primers which corresponded to conserved parts of the human and the murine factor H nucleotide sequences. The entire rat sequence spanned 4240 nucleotides with an open reading frame of 3708 nucleotides. These were preceded by 23 nucleotides of the 5' untranslated region, followed by a stop codon and a 3' untranslated region of 478 nucleotides including the polyadenylation-signal up to the beginning of the poly A tail. Comparison of the rat cDNA-derived coding sequence revealed identities of 74% to the human and 87% to the mouse FH nucleotide sequence. The translation product of rat FH mRNA was 1236 aa in length (leader sequence included) with an identity of 63% to the human and 81.5% to the murine protein. The degree of glycosylation of rat FH-Mr is about 9.5%. To quantitate FH in rat serum and supernatants of primary cultures of rat hepatocytes (HC), a reliable and sensitive sandwich-enzyme-linked immunosorbent assay (ELISA) was established. The concentration of FH in rat serum was calculated to be 238 microg +/- 21 microg/ml (mean +/- SD). Its concentration in the culture supernatants of HC was upregulated about three-fold by interferon (IFN)-gamma (100 U/ml).