title: Identification and characterization of a serine protease inhibitor Esserpin from the Chinese mitten crab Eriocheir sinensis.
abstract: Serine protease inhibitors (serpins) represent an expanding superfamily of endogenous inhibitors that regulate proteolytic events and involve in a variety of physiological processes. A serine protease inhibitor, namely Esserpin, was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) analysis. The full-length cDNA of Esserpin was of 2367 bp, including an open reading frame (ORF) of 1371 bp encoding a polypeptide of 456 amino acids with estimated molecular mass of 49.95 kDa and theoretical isoelectric point of 6.03. A putative signal peptide of 23 amino acids and a classical serpin domain were identified in Esserpin. The deduced amino acid sequence of Esserpin shared homology with serpins from Fenneropenaeus chinensis and Pacifastacus leniusculus. The mRNA transcripts of Esserpin could be detected in all the examined tissues including heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highest expression level was present in gonad. After the crabs were challenged by Vibrio anguillarum and Pichia pastoris, the expression levels of Esserpin transcripts in hemocytes were significantly up-regulated, and peaked at 24 h (5.18-fold of blank group, P < 0.05) and 3 h (2.87-fold of blank group, P < 0.05), respectively. The functional activity of Esserpin was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombinant Esserpin (rEsserpin) could inhibit trypsin activities in a dose-dependent manner, and it could lead to 100% inhibition of trypsin activities under the concentration of 873.76 nM, while there was no evident inhibition of chymotrypsin observed with rEsserpin. Moreover, rEsserpin inhibited the growth of E. coli at the final concentration of 1747.52 nM, and it also significantly depressed (P < 0.05) the phenoloxidase activity in the plasma at the final concentration of 873.76 nM. These results indicated that Esserpin was a homologue of serpin in crab and it could be induced after immune stimulation and mediate immune response possibly via the inhibition of bacterial growth and the regulation of prophenoloxidase-activating system.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. wanglingling@ms.qdio.ac.cn
authors.forename: Lingling
authors.initials: L
authors.lastname: Wang
authors.forename: Zhaopeng
authors.initials: Z
authors.lastname: Ma
authors.forename: Jialong
authors.initials: J
authors.lastname: Yang
authors.forename: Yunchao
authors.initials: Y
authors.lastname: Gai
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhou
authors.forename: Leilei
authors.initials: L
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Yue
authors.forename: Linsheng
authors.initials: L
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003386:Brachyura / Q000201:enzymology / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D007113:Immunity, Innate; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D010843:Pichia / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D057057:Serine Proteases / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014733:Vibrio / Q000502:physiology
chemical_list: D060829:Arthropod Proteins; D011994:Recombinant Proteins; D015843:Serpins; D057057:Serine Proteases