title: Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata.
abstract: Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin.
authors.affiliation: Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Dianchang
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Ma
authors.affiliation: Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China. Electronic address: jiangsg@21cn.con.
authors.forename: Shigui
authors.initials: S
authors.lastname: Jiang
mesh_terms: D000818:Animals; D000460:Chlorophyta / Q000276:immunology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000276:immunology*; D058087:Haptophyta / Q000276:immunology; D049911:Pinctada / Q000235:genetics* / Q000276:immunology*; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin
keywords: Inhibitory activity; Microbial challenge; Pinctada fucata; Quantitative real-time PCR; Serine proteinase inhibitor