Cg-TGF-β, a TGF-β/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection
Section snippets
Introduction.
In the economic context of controlling the reproduction and growth of bivalve molluscs, knowledge of physiological mechanisms is crucial. During culture of the Pacific oyster, Crassostrea gigas, the organisms are directly influenced by environmental factors (physical or chemical conditions and pathogens) and their physiology can be deregulated by potential xenobiotic components [1]. Consequently, a part of our work has focussed on the study of various physiological regulations including defence
Isolation and purification of gonadal cells
Gonadal tracts were extracted in January during the period of initiation of mitosis in gonadal cells. Cells were isolated from bladed tissues using a discontinuous percoll gradient and harvested by centrifugation.
Experimental stimulation by bacterial challenge and lipopolysaccharides (LPS)
To prevent the stress of manipulation and shipping, animals were bathed in seawater at 15 °C for 1 week prior to stimulation. To mimic an infection by Gram negative bacteria, a mixture of three Vibrio species (Vibrio metschnikovii, V. alginolyticus, V. harveyii) (108 bacteria/100 μL) or
Molecular cloning of Cg-TGF-β cDNA and characterisation of gene
The RT-PCR with the consensual degenerated primers performed on cDNA from C. gigas gonadal cells led to identification of a novel TGF-β member, named Cg-TGF-β-related factor. Subsequently, an entire transcript was obtained by RACE-PCR with gene specific primers. The complete full-length cDNA comprises 1956 nucleotides with a coding sequence of 1161 nucleotides preceded by an in-frame stop codon. Two potential non-canonical sites of polyadenylation are located at 1384 bp (AATTAA) and 1901 bp
Discussion
In the present study, we report the characterisation of the cDNA encoding Cg-TGF-β, a peptide related to TGF-β (including myostatin and myoglianin), and to maverick and activins in both vertebrates and Drosophila. It could be cleaved by a pro-convertase at a tetrabasic site to a mature peptide. More particularly, this proteolytic site (RVKR) corresponds to a typical recognition motif of furin (RX(K/R)R), required for activation of vertebrate TGF-β [26]. The mature domain of Cg-TGF-β has all the
Acknowledgements
This work was supported by the Conseil Régional de Basse-Normandie, Agence de l’eau Seine-Normandie and FEDER Presage n°4474. The authors thank Dr. Ian Probert, University of Caen for carefully editing the manuscript and Dr. Nils Klüver for supplementary phylogenetic analysis.
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