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 ... -  《-》

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 ... -  《-》

An interleukin-2 enhancer binding factor 2 homolog involved in immune response from Chinese mitten crab Eriocheir sinensis.

As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5' untranslated region (UTR) of 90bp, a 3' UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6-64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P<0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P<0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris.

Yang J ，Wang L ，Huang M ，Wang L ，Gai Y ，Qiu L ，Zhang H ，Song L ... -  《-》

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 ... -  《-》

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Gai Y ，Zhao J ，Song L ，Li C ，Zheng P ，Qiu L ，Ni D ... -  《FISH &amp; SHELLFISH IMMUNOLOGY》