The involvement of PDGF/VEGF related factor in regulation of immune and neuroendocrine in Chinese mitten crab Eriocheir sinensis.

Members of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family have been implicated in cell proliferation, cell differentiation, and cell migration, vascular development, angiogenesis and neural development. In the present study, a novel PDGF/VEGF related factor gene was cloned and identified in Chinese mitten crab Eriocheir sinensis (designated as EsPVF1). The full-length cDNA of EsPVF1 was of 1173 bp, consisting a 5' untranslated region (UTR) of 54 bp, a 3' UTR of 1131 bp with a poly (A) tail, and an open reading frame (ORF) of 588 bp encoding 196 amino acid residues. A signal peptide of 20 amino acid residues, a PDGF/VEGF homology growth factor domain of 81 amino acids, and a typical cysteine knot motif (CXCXC) were identified in the deduced amino acid sequence of EsPVF1. By fluorescent quantitative real-time PCR, the EsPVF1 mRNA was detected ubiquitously in the select tissues of hemocytes, gonad, heart, muscle, hepatopancreas and gill, with the high abundance in hemocytes and gonad. The mRNA expression level of EsPVF1 was up-regulated and reached the highest at 24 h after Vibrio anguillarum challenge, while it was induced at 3 h, 6 h, 12 h, 24 h and 48 h compared with the untreated group after Pichia pastoris GS115 challenge. Tissue injury also induced the mRNA expression of EsPVF1 in hemocytes of crabs, and the expression level increased obviously at 8 h. The cDNA fragment encoding mature peptide of EsPVF1 was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. Biogenic amine in hemolymph pre-incubated with recombinant protein of EsPVF1 (rEsPVF1) was detected by fluorimetric method. Norepinephrine and dopamine in hemolymph incubated with rEsPVF1 were higher than that in the blank group. Therefore, EsPVF1 could significantly provoke the release of norepinephrine and dopamine. The results collectively indicated that EsPVF1 was involved in regulation of the immune response and neuroendocrine system in crabs.

Identification and characterization of a Cystatin gene from Chinese mitten crab Eriocheir sinensis.

Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes. In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Eriocheir sinensis (designated EsCystatin) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 5.48 and the predicted molecular weight of 13.39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin. Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin. But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystatin were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart. After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0.6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01) at 24 h. Afterwards, EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2.8-fold of that in blank (P < 0.01)). The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain. When the concentration of EsCystatin protein was of 300 microg mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms.