title: A novel ubiquitin-protein ligase E3 functions as a modulator of immune response against lipopolysaccharide in Pacific oyster, Crassostrea gigas.
abstract: Ubiquitination is an important post-translational protein modification and plays a crucial role in various processes such as cell cycle, signal transduction, and transcriptional regulation. 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 of CgE3Rv1 gene was of 1455 bp encoding a polypeptide of 484 amino acids with the predicted molecular mass of 54.89 kDa. There were two transmembrane regions and a RING-variant (RINGv) domain identified in CgE3Rv1. CgE3Rv1 shared similar C4HC3 zinc-finger-like motif with those RINGv domain Ub-protein ligases E3s identified from vertebrates and invertebrates, and it was closely clustered with the membrane-associated RING-CH2 (MARCH2) Ub-protein ligases E3s in the phylogenetic tree. The mRNA transcript of CgE3Rv1 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 LPS, while the up-regulated mRNA expression was significantly decreased (p < 0.01) after acetylcholine stimulation. No significant changes of CgE3Rv1 expression were observed after peptidoglycan or mannan stimulation. Immunohistochemistry and in situ hybridization assays revealed that CgE3Rv1 protein and mRNA were dominantly distributed in the gonad. In the hemocytes, CgE3Rv1 was mainly located around the nucleus, and slightly distributed in the cytoplasm and on the cell membrane. Recombinant CgE3Rv1 RINGv domain protein (rCgE3Rv1-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, which was involved in the immune response against LPS and the interaction with cell surface signal molecules of neuroendocrine-immune system in oysters.
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China; National Engineering Research Center of Seafood, Dalian 116034, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Lusheng
authors.initials: L
authors.lastname: Xin
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Conghui
authors.initials: C
authors.lastname: Liu
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Jia
authors.affiliation: Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, China. Electronic address: lshsong@dlou.edu.cn.
authors.forename: Linsheng
authors.initials: L
authors.lastname: Song
authors.affiliation: School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China; National Engineering Research Center of Seafood, Dalian 116034, China. Electronic address: zhubeiwei@163.com.
authors.forename: Beiwei
authors.initials: B
authors.lastname: Zhu
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D049896:Crassostrea / Q000201:enzymology* / Q000276:immunology; D006066:Gonads / Q000201:enzymology; D006458:Hemolymph / Q000201:enzymology; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology; D009928:Organ Specificity; D010802:Phylogeny; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D054875:Ubiquitination
chemical_list: D008070:Lipopolysaccharides; D044767:Ubiquitin-Protein Ligases; D000109:Acetylcholine
keywords: Crassostrea gigas; Immune response; Lipopolysaccharide stimulation; Ub-protein ligase E3; Ubiquitination