Molecular cloning and functional analysis of carp complement regulatory factors : Molecular mechanism of C3 fragmentation.

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In mammals, biodefensive roles of the complement system depend largely upon several kind& of C3-derived fragments, such as C3b, iC3b and C3d. In fish, little is known how the, complement component C3 is fragmented in physiological conditions. The goal of the present study was to identify a complement regulatory molecule that mediates C3 fragmentation in the common carp, and to analyze its function at the protein level. Two homologous cDNA clones that encode isotypic variants of mammalian factor I orthologue was isolated from a carp hepatopancreas cDNA library. The deduced amino acid sequences are 85% identical. It is interesting to note that one of them contained a novel insertion at its N-terminus and is expressed mainly in the hepatopancrease. Another is expressed predominantly in the ovary, suggesting its function outside of complement-regulation., At the protein level, we have searched factors that inhibit the hemolytic activity of normal carp serum against rabbit erythrocytes. As a result, two distinct factors with molecular masses of 400 kDa and 65 kDa. The 65 kDa putative regulatory factor has been purified by combination of affinity chromatography on heparin-agarose and anion-exchange chromatogarphy on Mono-Q. The partially purified 65 kDa factor was heat-labile. In addition, the 65 kDa factor prevented siginificant C3-deposition on the rabbit erythrocyte membranes, suggesting that the 65 kDa factor inhibits complement activation before the activation cascade proceeds to the reaction step involving C3.