The Immunomodulation Of Ubiquitination Enzyme CgE3Rv1 In Oyster Crassostrea Gigas

Ubiquitin-proteasome pathway is an important post-translational protein modification and plays a crucial role in various processes such as cell cycle, signal transduction, transcriptional regulation, DNA repair, and morphogenesis. Compared with vertebrate, there were only a few researches of ubiquitin to be studied in recent years. For mollusc, the protein ubiquitination has been discovered, while ubiquitin molecules and their mechanisms were still not well known. In the present study, a novel ubiquitin(Ub)-protein ligase E3(designed as CgE3Rv1) was identified from Crassostrea gigas, and its ubiquitination regulation in the immune response against lipopolysaccharide(LPS) stimulation was investigated.The open reading frame(ORF) of Cg E3Rv1 gene was of 1455 bp encoding a polypeptide of 484 amino acids with the predicted molecular mass of 54.89 k Da. There were two transmembrane regions and a RIN G-variant(RINGv) domain identified in CgE3Rv1. CgE3Rv1 shared similar C4HC3 zinc- finger- like motif with those RINGv domain Ub-protein ligases E3 s previously identified from vertebrates and invertebrates, and it was closely clustered with the membrane-associated RING-CH2(MARC H2) Ub-protein ligases E3 s in the phylogenetic tree. The mRN A transcript of Cg E3Rv1 was highest expressed in gonads and hemolymph(p <0.05), and its mRNA expression level in hemocytes was significantly increased at 6 h(p <0.01) after the stimulation of lipopolysaccharide(LPS), while the up-regulated mRNA expression was significantly decreased(p <0.01) after acetylcholine(AC h) stimulation. No significant changes of CgE3Rv1 expression were observed after peptidoglycan(PGN) or mannan(MAN) stimulation. Immunohistochemistry and in situ hybridization assays revealed that CgE3Rv1 protein and mRNA were dominantly distributed in the gonad. In the hemocytes, Cg E3Rv1 was mainly located around the nucleus, and slightly distributed in the cytoplasm and on the cell membrane. Recombinant CgE3Rv1 RINGv domain protein(rCg E3Rv1-RINGv) was confirmed to activate the Ub reaction system in vitro with the aid of Ub-activating enzyme E1 and Ub-conjugating enzyme E2. These results demonstrated that CgE3Rv1 was an Ub-protein ligase E3 and played important roles in the regulation of immune recognition in oysters.