Complete sequencing and expression of three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway of the complement system in rainbow trout Oncorhynchus mykiss

Abstract

Three complement components, C1r, C4 and C1 inhibitor, of the classical activation pathway have been fully sequenced and their expression investigated in rainbow trout (Oncorhynchus mykiss). Trout C1r cDNA encodes a 707-amino-acid (aa) protein with a theoretical M(r) of 77,200. The trout translation shows highest homology with carp C1r/s, and lower, equal homologies to mammalian C1r and C1s, and MASPs from other vertebrate species. However, phylogenetic analysis and structural features suggest that the trout sequence, together with the two carp sequences, are the orthologues of mammalian C1r. The trout C4 cDNA encodes a 1,724-aa protein with a theoretical M(r) of 192,600. The trout translation shows higher homologies to the carp C4B and medaka C4, but lower homologies to C4 from other species and the carp C4A. It has a predicted signal peptide of 22 aa, a alpha-chain of 773 aa, a beta-chain of 635 aa and a lambda-chain of 288 aa. Trout C1 inhibitor cDNA encodes a 611-aa protein with a theoretical M(r) of 68,700. The trout translation has a C-terminal serpin domain with high homologies with mammalian counterparts (~37% identities), and a longer N-terminus, with no significant homology to other serpins, which contains two Ig-like domains. A molecule containing two Ig-like domains followed by a serpin domain, has also been found in an EST clone from another bony fish, the Japanese flounder. This suggests a unique structural feature of C1 inhibitor in fish. The functional significance of the Ig domains is discussed. The liver is the major site of expression of the three trout complement components, C1r, C4 and C1 inhibitor, although their expression is also detectable in other tissues. The extra-hepatic expression of complement genes may be important for local protection and inflammatory responses. Low-level constitutive expression of the three components was also detectable in a trout monocyte/macrophage cell line RTS-11, but only the expression of C4 could be upregulated by LPS.

Molecular and Functional Characterization of IL-15 in Rainbow TroutOncorhynchus mykiss:A Potent Inducer of IFN-γ Expression in Spleen Leukocytes

“…We identified the first fish IL-15 sequence in rainbow trout (AJ555868) in 2003 by surveying SSH sequences prepared from trout tissues challenged by the pathogenic bacterium Aeromonas salmonicida (31). Several fish IL-15 sequences, as well as IL-15-like molecules, have also been described by in silico cloning (32-34), but so far no function has been assigned to any fish IL-15.…”

“…salmonicida, MT 423 (40), was used to challenge female rainbow trout of ϳ300 g. The bacterial preparation, challenge procedure, and tissue sampling were described previously (31). Gill tissues were collected and used for total RNA preparation.…”

“…Gill tissues were collected and used for total RNA preparation. The construction of SSH libraries and random sequence analysis of SSH clones were as described before (31).…”

Two Divergent Isotypes of the Fourth Complement Component from a Bony Fish, the Common Carp (Cyprinus carpio)

“…The expressed sequence tag (EST) sequence BI673185 in Table III homologous to carp C4-1 corresponds to a zebrafish genomic contig AL935181, which includes one of the two sequence segments encoding the zebrafish C4 sequence in a literature (9). In trout, the carp C4-2-like EST, CA362456, is 99% identical to the reported C4 mRNA sequence (10). In channel catfish, there is only one hit (BM438332) showing higher similarity to C4-2 than to C4-1 of carp.…”

Differential expression, modulation and bioactivity of distinct fish IL‐12 isoforms: Implication towards the evolution of Th1‐like immune responses

“…3 -and 5 -RACE was used to obtain full-length cDNA sequences from SMART cDNA as described previously [22,40]. Homology searching was conducted using the BLAST program [41].…”