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. 1993 Dec 1;151(11):6123-34.

Third Component of Trout Complement. cDNA Cloning and Conservation of Functional Sites

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  • PMID: 8245455

Third Component of Trout Complement. cDNA Cloning and Conservation of Functional Sites

J D Lambris et al. J Immunol. .

Abstract

Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins. It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes. Structural and functional analysis of C3 from different species, in addition to phylogenetic information, provides insights into the structural elements mediating the various functions. This study describes the cDNA cloning of one of two isoforms of the third complement component, C3-1, of rainbow trout (Salmo gairdneri) and the analysis of its functional sites. By screening a trout liver lambda gt11 library with anti-trout C3 chain-specific antibodies and polymerase chain reaction we have determined the cDNA sequence of trout C3-1. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides, corresponding to different regions of trout C3-1. C3-1 consists of 1640 amino acids with a calculated molecular mass of 181,497 Da. The sequence contains two potential N-glycosylation sites, one on each chain of C3. The deduced protein sequence showed 44.1, 43.3, 44.2, 44.9, 43.1, 43.8, 45.9, 29.9, and 33.1% amino acid identities to human, mouse rat, guinea pig, rabbit, cobra, frog, hagfish, and lamprey C3, whereas the identities to human C4, C5, and alpha 2M are 30.4, 28, and 22.9%, respectively. The trout C3 amino acid sequence shows clusters of high and low similarity to C3 from other species. In the regions of high similarity belong the C3 domains that contain the thiolester site and the properdin binding sites, whereas the regions that correspond to regions of human C3 where CR1 and CR2 bind show low amino acid sequence similarity. The deduced amino acid sequence shows that the C3 convertase cleavage site (Arg-Ser) is conserved in trout C3, whereas the factor I cleavage sites are Arg-Ala and Arg-Thr instead of Arg-Ser, which is found in the C3 of other species. Protein sequencing of the trout C3 fragments fixed on zymosan during complement activation confirmed the cleavage of trout C3 by trout C3 convertase and factor I at Arg-Ser and Arg-Thr, respectively.

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