title: Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides.
abstract: Lysozyme acts as an innate immunity molecule against pathogen infection. In this study, a new G-type lysozyme gene with a typical G-type lysozyme domain (designated as Ec-lysG) was cloned and characterized from the orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysG cDNA contains 1419 bp and encodes a 256-residue protein containing a 25-residue signal peptide at the N-terminus. BLAST analysis reveals Ec-lysG shares 64% identity with Siniperca chuatsi, but 63% to another reported G-type lysozyme from orange-spotted grouper (OSG-lysG). The genomic DNA of Ec-lysG contains four exons and three introns, with a total length of 2062 bp. An amino acid sequence alignment showed that Ec-lysG shares the fundamental structural features of G-type lysozyme, including the catalytic residues, substrate binding sites, and soluble lytic transglycosylase domain. Quantitative PCR showed that Ec-lysG transcript is most abundant in the head kidney, and less abundant in the heart. The expression of Ec-lysG was differentially upregulated in the head kidney after stimulation with lipopolysaccharide, Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). A subcellular localization analysis showed that Ec-lysG is distributed predominantly in the cytoplasm. Recombinant Ec-lysG (rEc-lysG) has optimal activity at pH 7.5 and 35°C. rEc-lysG showed lytic activities against Gram-positive bacterium Streptococcus iniae, Staphylococcus aureus, and Micrococcus lysodeikticus, and the Gram-negative bacterium V. alginolyticus. Scanning electron microscopy (SEM) showed that rEc-lysG acts on M. lysodeikticus cell walls. The overexpression of Ec-lysG in grouper cells did not significantly delay the occurrence of the cytopathic effect (CPE) induced by SGIV, and did not inhibit viral gene transcription. In conclusion, Ec-lysG might be a potent antibacterial protein, with a role in innate immunity.
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Shina
authors.initials: S
authors.lastname: Wei
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Youhua
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Huang
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; College of Fishery, Guangdong Ocean University, Zhanjiang 524088, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Cai
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Jingguang
authors.initials: J
authors.lastname: Wei
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Li
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
authors.forename: Zhengliang
authors.initials: Z
authors.lastname: Ouyang
authors.affiliation: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China. Electronic address: qinqw@scsio.ac.cn.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Qin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D029941:Fish Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D060226:Head Kidney / Q000201:enzymology / Q000276:immunology; D017994:Iridovirus / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D009928:Organ Specificity; D010473:Perciformes / Q000235:genetics*; D010802:Phylogeny; D021381:Protein Transport; D044149:Vibrio alginolyticus / Q000187:drug effects; D014779:Virus Replication
chemical_list: D000900:Anti-Bacterial Agents; D029941:Fish Proteins; D008070:Lipopolysaccharides; D009113:Muramidase
keywords: Antimicrobial activity; Epinephelus coioides; G-type lysozyme; Innate immunity