title: PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity.
abstract: Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 μM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Graduate University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Guohui
authors.initials: G
authors.lastname: Shi
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; National & Local Joint Engineering Laboratory for Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China. Electronic address: zhxcui@qdio.ac.cn.
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Graduate University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Danli
authors.initials: D
authors.lastname: Luo
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Graduate University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Chengwen
authors.initials: C
authors.lastname: Song
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Graduate University of the Chinese Academy of Sciences, Beijing, China.
authors.forename: Xihong
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Min
authors.initials: M
authors.lastname: Hui
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
authors.forename: Yingdong
authors.initials: Y
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D003386:Brachyura / Q000737:chemistry* / Q000276:immunology; D004156:Catechol Oxidase / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects*; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli; D040461:Gene Components; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D015843:Serpins / Q000032:analysis / Q000235:genetics / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology*; D044149:Vibrio alginolyticus / Q000187:drug effects / Q000254:growth & development
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004792:Enzyme Precursors; D015843:Serpins; D014361:Trypsin Inhibitors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase
keywords: Bacterial growth inhibition; Portunus trituberculatus; Prophenoloxidase activating system; Serpin