Molecular characteristics of lysozyme G in Euryglossa orientalis; cDNA cloning, phylogenic analysis, physicochemical properties and tissue gene expression.

Abstract

Lysozymes are the key molecules of innate immune system against bacterial infections. In the present study, we identified the molecular characteristics, physicochemical properties, antibacterial activity, evolutionary relationship and tissue expression pattern of g-type lysozyme in Euryglossa orientalis (EuOr LysG). The full-length EuOr LysG cDNA is composed of 588 nucleotides and an open reading frame encoding a protein with 195 amino acids with more than 65% identity to g-type lysozyme of Solea senegalensis (73%) and Scophthalmus rhombus (64%). Amino acid sequence alignment showed that EuOr LysG protein possessed a conserved catalytic motif (Glu71-Asp84-Asp101) and one predicted disulfide bond between Cys32 and Cys122. Phylogenetic analysis based on the g-type lysozyme sequences indicated that E. orientalis and other fish of Pleuronectiformes were diverged together in the evolutionary history. The K m and V max values of the recombinant EuOr LysG were 0.266mg/ml of Micrococcus lysodeikticus as substrate and 667U/mg of protein, respectively. The optimum temperature and pH of recombinant EuOr LysG were 45 and 6°C, respectively. Real-time PCR analysis showed that EuOr LysG transcript was most abundant in head kidney and gill and less in muscle. We also showed that the EuOr LysG had potent lytic activity against major fish bacterial pathogens with the highest activity against Bacillus cereus and Aeromonas hydrophila. Bacterial challenge with Vibrio parahaemolyticus could upregulate LysG in immune-related tissues. Our results help to understand the molecular and physicochemical characteristics of g-type lysozyme in E. orientalis which might play an important role in host defense against the bacterial infection.