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.

Li F ，Gai X ，Wang L ，Song L ，Zhang H ，Qiu L ，Wang M ，Siva VS ... -  《-》

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis.

NF-κB is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-κB-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor κB (IκB)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-κBs and phylogenetic analysis suggested that EsRelish was a member of the NF-κB family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Li F ，Wang L ，Zhang H ，Zheng P ，Zhao J ，Qiu L ，Zhang Y ，Song L ... -  《-》

Two novel secreted ferritins involved in immune defense of Chinese mitten crab Eriocheir sinensis.

As a principal extracellular iron storage molecule, secreted ferritin plays an important role in the iron-withholding strategy of innate immunity. In this study, two novel secreted ferritins were identified from Chinese mitten crab Eriocheir sinensis (designated as EsFer-1 and EsFer-2) by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsFer-1 and EsFer-2 were of 1278 and 1595 bp, respectively, both containing a putative iron response element (IRE) in their 5' UTRs and multiple A + U-destabilizing elements (TATT or ATTTA) in their 3' UTRs. The ORFs of these two crab ferritin cDNAs were of 639 and 663 bp, respectively, encoding two peptides of 212 and 220 amino acid residues each with a signal peptide and typical structures of ferritins such as four long alpha-helices, one short alpha-helix and an L-loop. EsFer-2 exhibited higher similarity with the H-ferritins from both invertebrates and vertebrates, while EsFer-1 was closer matched to L-ferritins. The eight amino acid residues identified as metal binding sites in vertebrate H-ferritins were conserved in EsFer-2 (Glu53, Tyr60, Glu87, Glu88, His91, Glu146, Glu177 and Gln178), but none of them was observed in EsFer-1. By fluorescent quantitative real-time PCR, mRNA transcripts of EsFer-1 and EsFer-2 were mainly detected in muscle, hepatopancreas and gill, and also marginally detectable in gonad, heart and hemocytes. After the crabs were challenged by bacteria Listonella anguillarum, the transcriptional levels of both EsFer-1 and EsFer-2 in hemocytes were up-regulated twice. In the first up-regulation, the mRNA relative expression levels of both EsFer-1 and EsFer-2 reached peak at 3 h post-challenge, while in the second up-regulation, they did not reach the highest point within the experiment duration. After the fungi Pichia pastoris GS115 challenge, there was only one transcriptional level peak of both the two ferritins, appearing at 6 h post-challenge. These results suggest that secreted EsFer-1 and EsFer-2 are crucial proteins in the iron-withholding defense system, and play important roles in the innate immune responses in crabs.

Kong P ，Wang L ，Zhang H ，Zhou Z ，Qiu L ，Gai Y ，Song L ... -  《-》

A γ-aminobutyrate type A receptor-associated protein involved in the immune response of Eriocheir sinensis.

The γ-aminobutyrate type A receptor-associated protein (GABARAP) is a ubiquitin-like modifier, which is implicated in membrane trafficking and fusion events of γ-aminobutyrate type A receptor, autophagy and apoptosis. In the present study, the gene encoding GABARAP (designated EsGABARAP) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends approach and expression sequence tag (EST) analysis. The full-length cDNA of EsGABARAP was of 457 bp, containing a 5' untranslated region (UTR) of 77 bp, a 3' UTR of 32 bp with a poly(A) tail and an open reading frame (ORF) of 348 bp encoding a polypeptide of 116 amino acids with the predicted molecular weight of 13.81 kDa and theoretical isoelectric point of 8.73. The deduced amino acid sequence of EsGABARAP shared higher similarity (91.8-97.4%) with those of other GABARAPs, and it contained a conserved MAP1_LC3 domain. In the phylogenetic tree, EsGABARAP was firstly clustered with GABARAPs from other animals and then gathered together with the same family proteins of GABARAP. The mRNA expression level of EsGABARAP in six tissues and its temporal expression level in haemocytes of crabs challenged with Listonella anguillarum were determined by quantitative real-time RT-PCR. The mRNA transcripts of EsGABARAP could be detected ubiquitously in the examined tissues, including haemocytes, hepatopancreas, muscle, gill, heart and gonad, with the highest expression level in hepatopancreas. The expression level of EsGABARAP mRNA in haemocytes was up-regulated after L. anguillarum challenge and reached 6.58-fold of that in blank group at 24 h (P < 0.05) and 7.52-fold at 48 h (P < 0.05). The increasing transcripts in haemocytes after L. anguillarum challenge gave the preliminary evidence for the involvement of EsGABARAP as a part of immune response against bacteria challenge in crabs.

Zhou Z ，Wang L ，Kong P ，Qiu L ，Zhang H ，Gao Y ，Yang J ，Song L ... -  《-》

A double WAP domain-containing protein from Chinese mitten crab Eriocheir sinensis with antimicrobial activities against Gram-negative bacteria and yeast.

The whey acidic protein (WAP) domain is characterized by a 'four-disulfide-core' (4-DSC) motif comprising of approximately 50 amino acids with eight highly conserved cysteine residues. Previous research indicated that WAP domain-containing proteins played an important role in the innate immunity of crustaceans. In the present study, a novel double WAP domain (DWD)-containing protein gene was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD was of 593 bp, consisting of a 5'-terminal untranslated region (UTR) of 71 bp, a 3' UTR of 120 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 402 bp. The ORF encoded a polypeptide of 133 amino acids with the predicted molecular weight of 14.4 kDa and the theoretical isoelectric point of 8.14, including a signal peptide of 22 amino acids and two WAP domains. The EsDWD mRNA transcripts were ubiquitously expressed in all the tested tissues, and its expression level in gill was significantly higher than that in other tissues. The mRNA expression of EsDWD in haemocytes was up-regulated after challenge of Vibrio anguillarum and Pichia pastoris GS115, as well as injury treatment. The cDNA encoding the mature EsDWD protein was cloned and expressed in Escherichia coli BL21 (DE3) pLysS, and the purified recombinant EsDWD (rEsDWD) protein exhibited antimicrobial activities against Gram-negative bacteria V. anguillarum, yeast P. pastoris GS115 and Candida parapsilosis. The results collectively suggested that EsDWD was a novel member of double WAP domain (DWD)-containing proteins, and involved in the immune defense against microorganism and wound healing in E. sinensis.

Li F ，Wang L ，Qiu L ，Zhang H ，Gai Y ，Song L ... -  《-》