title: Rock bream (Oplegnathus fasciatus) serpin, protease nexin-1: transcriptional analysis and characterization of its antiprotease and anticoagulant activities.
abstract: Protease nexin-1 (PN-1) is a serine protease inhibitor (SERPIN) protein with functional roles in growth, development, patho-physiology and injury. Here, we report our work to clone, analyze the expression profile and characterize the properties of the PN-1 gene in rock bream (Rb), Oplegnathus fasciatus. RbPN-1 encodes a peptide of 397 amino acids (AA) with a predicted molecular mass of 44 kDa and a 23 AA signal peptide. RbPN-1 protein was found to harbor a characteristic SERPIN domain comprised of a SERPIN signature and having sequence homology to vertebrate PN-1s. The greatest identity (85%) was observed with PN-1 from the three-spined stickleback fish, Gasterosteus aculeatus. The functional domains, including a heparin binding site and reactive centre loop were conserved between RbPN-1 and other fish PN-1s; in particular, they were found to correspond to components of the human plasminogen activator inhibitor 1, PAI-1. Phylogenetic analysis indicated that RbPN-1 was closer to homologues of green spotted pufferfish and Japanese pufferfish. Recombinant RbPN-1 demonstrated antiprotease activity against trypsin (48%) and thrombin (89%) in a dose-dependent manner, and its antithrombotic activity was potentiated by heparin. The anticoagulant function prolonged clotting time by 3.7-fold, as compared to the control in an activated partial thromboplastin time assay. Quantitative real-time PCR results indicated that RbPN-1 is transcribed in many endogenous tissues at different levels. Lipopolysaccharide (LPS) stimulated a prolonged transcriptional response in hematic cells, and Rb iridovirus up-regulated the RbPN-1 mRNA level in hematic cells to a maximum of 3.4-fold at 12 h post-infection. Interestingly, LPS and Edwardsiella tarda significantly induced the RbPN-1 transcription at the late phase of infection. In vivo studies indicated that injury response caused a temporal suppression in RbPN-1 transcription, in conjunction with that of another SERPIN, rock bream heparin cofactor II, RbHCII. Taken together, our findings suggest that PN-1 functions as an antiprotease and anticoagulant and that SERPINs (PN-1 and HCII) are likely to contribute to immunity and post-injury responses.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province 690-756, Republic of Korea.
authors.forename: Navaneethaiyer
authors.initials: N
authors.lastname: Umasuthan
authors.forename: Ilson
authors.initials: I
authors.lastname: Whang
authors.forename: Jong-Oh
authors.initials: JO
authors.lastname: Kim
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh
authors.forename: Sung-Ju
authors.initials: SJ
authors.lastname: Jung
authors.forename: Cheol Young
authors.initials: CY
authors.lastname: Choi
authors.forename: Sang-Yeob
authors.initials: SY
authors.lastname: Yeo
authors.forename: Jeong-Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Jae Koo
authors.initials: JK
authors.lastname: Noh
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004266:DNA Virus Infections / Q000235:genetics / Q000378:metabolism*; D020609:Edwardsiella tarda / Q000472:pathogenicity*; D004756:Enterobacteriaceae Infections / Q000235:genetics / Q000378:metabolism*; D005399:Fishes; D020869:Gene Expression Profiling; D006801:Humans; D017994:Iridovirus / Q000472:pathogenicity*; D010314:Partial Thromboplastin Time; D010802:Phylogeny; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017385:Sequence Homology; D058258:Serpin E2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D017395:Plasminogen Activator Inhibitor 1; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D058258:Serpin E2