title: PtPLC, a pacifastin-related inhibitor involved in antibacterial defense and prophenoloxidase cascade of the swimming crab Portunus trituberculatus.
abstract: Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus. The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli, and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa, but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro, and lead to 72.8% inhibition of PO activity at the concentration of 9.11 μM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, 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 266071, China; National & Local Joint Engineering Laboratory for Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, 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 266071, China; University of Chinese Academy of Sciences, Beijing 100049, 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 266071, China; University of Chinese Academy of Sciences, Beijing 100049, 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 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Shuangyan
authors.initials: S
authors.lastname: Wang
authors.affiliation: School of Marine Science, Ningbo University, Ningbo 315211, China.
authors.forename: Chunlin
authors.initials: C
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003386:Brachyura / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000382:microbiology*; D004156:Catechol Oxidase / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000502:physiology; D016982:Micrococcus luteus / Q000502:physiology; D010843:Pichia / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D044149:Vibrio alginolyticus / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D060829:Arthropod Proteins; D004792:Enzyme Precursors; D011506:Proteins; D011994:Recombinant Proteins; C106709:pacifastin; C038451:pro-phenoloxidase; D004156:Catechol Oxidase
keywords: Bacterial growth inhibition; Pacifastin; Portunus trituberculatus; Prophenoloxidase activating system; Serine protease inhibitor