Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein

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Abstract

Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 bp with an open reading frame of 438 bp coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P\Y)(Y\F)QIK, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 °C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action.

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Animals

The sea cucumber S. japonicus was provided by Dalian Zhangzidao Island Fishery Group Corporation, Dalian, P. R. China. The fresh sea cucumbers were washed with deionized water, and dissected immediately. Tissues such as body wall, intestine, and digestive tract were collected, quick frozen in liquid nitrogen and stored at − 80 °C until use.

Total RNA

The frozen samples from S. japonicus tissues were ground under liquid nitrogen with a mortar and pestle. Total RNA was extracted using TRIzol Reagent

Sequence analysis of S. japonicus lysozyme cDNA

To obtain cDNA encoding the lysozyme of the sea cucumber S. japonicus (SjLys), two degenerate primers were designed from the analysis and comparison of selected known lysozyme and lysozyme-like gene sequences of other echinoderms and invertebrates, such as starfish, sea urchin, nematode, etc. As a result, a 220 bp fragment was amplified, cloned, and sequenced. The BLAST search showed that this sequence was highly homologous to lysozyme sequences. The complete cDNA sequence was then determined

Discussion

In the present study, the first cDNA encoding an i-type lysozyme was identified from S. japonicus (SjLys) by RT-PCR and RACE techniques. Like i-type lysozymes found in bivalves, SjLys contained a hydrophobic signal peptide at the N-terminus and a hydrophilic tail at the C-terminus. A characteristic of the mature peptide of SjLys in the primary sequence is the high content (8%) of cysteine residues (10 out of 125 residues), and it predictedly forms four disulfide bonds. Disulfide bonds formed

Acknowledgements

We thank Professor José E. García-Ararrás, Department of Biology, University of Puerto Rico, USA, for revision and suggestion on the manuscript. We also thank Dr. Zongshen Zhang and other laboratory members for technical advice and helpful discussions. This work was financially supported by the National Natural Science Foundation of China (NSFC; No. 30571449) and National Basic Research Program of China (973 Program; No. 2006CB708210).

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