Gene Cloning And Functional Analysis Of Peptidoglycan Recognition Protein And Glutathione S-transferase In Hyriopsis Cumingii

Hyriopsis cumingii, as a kind of important economic freshwater pearl mussel species and aquatic environment monitoring animal, it is widely cultured in southern China. Because of its high degree of accumulation and tolerance on the environment pollutants, as well as one of the ways for ecological restorations of lakes, which undoubtedly pose some potential risks on aquatic ecosystems and human healthy. Therefore studies the immune defense and immune toxicity associated molecules is essential and meaningful. In the present study, subtractive cDNA library was constructed by suppression subtractive hybridization (SSH) based on hepatopancreas of mussel, the peptidoglycan recognition proteins (PGRPs) and Glutathione-S-transferases (GSTs) were cloned and characterized, the expression pattern and functional characterization was analyzed.Subtractive cDNA library was constructed by suppression subtractive hybridization (SSH), and approximately400positive clones were sequenced, from which98high quality sequences were obtained by BLAST analysis. The screening identified numerous genes involved in apoptosis, signal transduction, cytoskeletal remodel, innate immunity, material and energy metabolism, translation and transcription which were extensively discussed. The results of this study add large amount of information to the mussel genome data, and for the first time present the basic data on toxicity effect of MC-LR on mussel.In the present study, a short-form PGRP, designated as HcPGRPS1was identified from freshwater mussel Hyriopsis cumingi. The full cDNA sequences of HcPGRPS1was1137bp, with open reading frame (ORF) of708bp encodes a polypeptide of235amino acid (aa). Sequence analysis showed that HcPGRPS1have one C-terminal PGRP domain that is conserved through evolution, and these proteins are predicted to be amidases. Phylogenetic tree based on full-length amino acid sequences showed that HcPGRPS1were clustered closely with CgPGRPS3of Crassostrea gigas. Real-time PCR results showed that the mRNA transcripts of HcPGRPS1were constitutively expressed in all tested organs/tissues, with the highest level in hepatopancreas, then in gonad and intestinal. The expression in tissues (gonad, kidney, gill and foot) was up-regulated significantly after LPS or PGN stimulation. The recombinant protein of HcPGRPS1 exhibited binding activity and peptidoglycan-lytic amidase activity towards Lys-PGN from Staphylococcus aureus and DAP-PGN from Bacillus subtilis. Furthermore, recombinant HcPGRPS1displayed strong antibacterial activity to both Gram-negative bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas sobria and Gram-positive bacteria Staphyloccocus aureus in the presence of Zn2+. These results suggested that HcPGRPSl plays a multifunctional role in the defense and protection mechanisms of mussel innate immunity against infections.The full cDNA sequences of HcPGRPS2was973bp, with open reading frame (ORF) of657bp encodes a polypeptide of218amino acid (aa). Furthermore, the splicing patterns genes were described, and designated as HcPGRPS2a, with full-length of537bp encodes a polypeptide of151amino acid. Sequence analysis showed that HcPGRPS2have one C-terminal PGRP domain that is conserved through evolution, and these proteins are predicted to be amidases. Phylogenetic tree based on full-length amino acid sequences showed that HcPGRPS2were clustered closely with EsPGRP4of Euprymna scolopes. The results of Real-time PCR and Western blotting showed that the mRNA transcripts of HcPGRPS2were constitutively expressed in all tested organs/tissues, with the highest level in hepatopancreas, then in intestinal. The expression in tissues (hepatopancreas, foot or gill) was up-regulated significantly after LPS or PGN stimulation.In this study, a full-length cDNA of a novel sigma-like GST was identified from freshwater mussel Hyriopsis cumingi (HcGSTS). It was996bp containing an open reading frame of612bp, encoding203amino acid residues with a predicted protein molecular weight of23.3kDa and an estimated pI of5.46, which shared58%identity with GST from Ruditapes philippinarum. Sequence analysis showed that the predicted protein sequence of HcGSTS not only has the distinct highly conserved GSH binding site of N-terminal, but also the relatively diverse substrate binding site of C-terminal. Alignment analysis and phylogenetic relationship suggested that the HcGSTS of Hyriopsis cumingi belonged to sigma-class like with other known class sigma GSTs from different organisms. The mRNA of HcGSTS was constitutively expressed in a wide range of all tested tissues with different levels of normal mussels with highest expression level in hepatopancreas, followed by kidney and gonad. Expression pattern in tissues (hepatopancreas, gonad, kidney, gill and foot) of HcGSTS in response to LPS, PGN and MC-LR-challenged were also investigated. The mRNA expression of HcGSTS was significantly up-regulated (P<0.05) after mussels was stimulated by PGN and LPS in all detected tissues, but except in the gills challenged by PGN. In addition, the expression of HcGSTS mRNA in kidney and foot was also significantly up-regulation (P<0.05) induced by MC-LR. The specific activity of the recombinant pET32a-HcGSTS was4.54±0.08μmol/min/mg. Our results suggested that the sigma class glutathione S-transferase gene HcGSTS involve both the immune response against bacterial pathogens and the detoxification of exogenous toxic compounds.