Cloning and Expression of β-Defensin from Soiny Mullet (Liza haematocheila), with Insights of its Antibacterial Mechanism.
Beta-defensins are important part of innate immunity of fish, which are the first defense line against invading pathogens. In this study, the β-defensin (Lhβ-defensin) gene was cloned from spleen tissue of soiny mullet (Liza haematocheila). Lhβ-defensin cDNA was 747 bp in length, encoding 63 amino acids. Sequence alignment revealed that Lhβ-defensin contained six conserved cysteine residues and shared 97.5% sequence identities with grouper (Epinephelus coioides) β-defensin. Realtime PCR revealed that Lhβ-defensin was highest expressed in the immune related organs, such as spleen, kidney and gut of healthy fish. Following Streptococcus dysgalactiae infection, Lhβ-defensin was up-regulated in immune related organs, e.g. 17.6-fold in spleen and 10.87-fold in gut at 24 h post infection (hpi). Lhβ-defensin possessed a monomeric structure of a three-stranded anti-parallel β-sheet and an α-helix stabilized by three disulfide bonds formed by Cys30-Cys58, Cys36-Cys52, and Cys40-Cys59. In addition to the experimental work, computer simulation was also carried out to determine the possible conformation of β-defensin and its interaction with palmitoyloleoylphosphatidylglycerol (POPG), a model of bacteria membrane. The Lhβ-defensin was found to form dimeric structure stabilized by the van der Waals contacts of Leu35 and Cys37 in two anti-parallel β1-strands and the cation-π interaction between Tyr32 and Arg54 respectively in the two β1-strands. The most important interactions between β-defensin and membrane are the electrostatic interactions between Arg residues in β-defensin and head group of POPG bilayer as well as hydrogen bond interactions between them. Our results were useful for further understanding the potential mechanism of antimicrobial property of fish β-defensins.
Key Laboratory of Biochemistry and Biotechnology of Marine Wetland of Jiangsu Province, Yancheng Institute of Technology, Yancheng, 224051, Jiangsu, China.|Key Laboratory of Aquaculture and Ecology of Coastal Pool in Jiangsu Province, Yancheng Institute of Technology, Yancheng, 224051, Jiangsu, China.
Zhitao
Z
Qi
Key Laboratory of Biochemistry and Biotechnology of Marine Wetland of Jiangsu Province, Yancheng Institute of Technology, Yancheng, 224051, Jiangsu, China.
Wei
W
Xu
Tianjin Institute of Pharmaceutical Research, Tianjin, 300193, China.
Fancui
F
Meng
Key Laboratory of Biochemistry and Biotechnology of Marine Wetland of Jiangsu Province, Yancheng Institute of Technology, Yancheng, 224051, Jiangsu, China.
Qihuan
Q
Zhang
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, 80424, Taiwan, ROC.
Chenglung
C
Chen
Key Laboratory of Biochemistry and Biotechnology of Marine Wetland of Jiangsu Province, Yancheng Institute of Technology, Yancheng, 224051, Jiangsu, China.
Rong
R
Shao
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D009928:Organ Specificity / Q000235:genetics; D010802:Phylogeny; D055503:Protein Multimerization; D016415:Sequence Alignment; D023701:Smegmamorpha / Q000235:genetics* / Q000382:microbiology; D013290:Streptococcal Infections / Q000473:pathology; D013291:Streptococcus / Q000502:physiology; D014867:Water / Q000378:metabolism; D023083:beta-Defensins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D023083:beta-Defensins; D014867:Water