Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis

Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h, 12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab, Eriocheir sinensis

Apoptosis is a central regulatory feature of the immune system, and the most common form of death among immunological cells. However, the function of apoptosis, within the innate immune system of invertebrates, remains largely unknown. For this reason, we investigated the immune functionality of two apoptosis genes, caspase and nm23, in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The entire length caspase and nm23 cDNA genes were cloned using PCR, based on an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The caspase cDNA contained an 1119 bp open reading frame that encoded a putative 372 amino acid protein, while nm23 cDNA contained a 456 bp open reading frame that encoded a putative 151 amino acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of conserved enzyme active sites that were common among caspase and nm23 superfamilies. In brief, caspase and nm23 mRNA expression in E. sinensis were (a) both detected in all tissues, including the hemocytes, heart, hepatopancreas, gill, stomach, muscle, intestine, brain and eyestalk, and (b) responsive in hemocytes, gill and hepatopancreas to a Vibrio anguillarum immuno-challenge all appeared sharp increase. Collectively, the data presented here demonstrate the successful isolation of caspase and nm23 apoptosis genes from the Chinese mitten crab, and their role in the innate immune system of an invertebrate.     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

低氧胁迫和螺原体感染对中华绒螯蟹存活和细胞凋亡的影响

为了探究低氧胁迫对螺原体(Spiroplasma eriocheiris)感染中华绒螯蟹(Eriocheir sinensis)的影响,以中华绒螯蟹为研究对象,在低氧胁迫后,取中华绒螯蟹肝胰腺和鳃组织加入组织固定液,进行HE染色。在低氧条件下使螺原体感染中华绒螯蟹,计算螺原体拷贝数,并对血淋巴细胞进行细胞凋亡、细胞坏死和线粒体膜电位检测。结果显示,与对照组相比,处于长时间低氧胁迫状态下的中华绒螯蟹肝胰腺组织疏松,出现大量小空泡,鳃轴结构弥散,组织结构被破坏。此外,低氧组的中华绒螯蟹感染螺原体后的死亡速度相对于常氧组明显加快,血细胞内的螺原体数量、线粒体膜电位、血细胞凋亡率和坏死率相较于常氧组均显著升高。以上研究说明低氧胁迫可以加速螺原体的感染,使河蟹死亡速度变快,使血淋巴细胞凋亡和坏死更显著,不利于河蟹的生理生化。     

Molecular Cloning and Gene Expression Analysis of the Leptin Receptor in the Chinese Mitten Crab Eriocheir sinensis

Background

Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals.

Methodology/Principal Findings

In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5′-untranslated region (5′ UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3′ UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs.

Conclusions/Significance

We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.     

AiCTL-6, a novel C-type lectin from bay scallop Argopecten irradians with a long C-type lectin-like domain

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel C-type lectin gene from scallop Argopecten irradians (designated as AiCTL-6) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of AiCTL-6 was 1080 bp. The open reading frame encoded a polypeptide of 307 amino acids, including a signal sequence and a C-type lectin-like domain (CTLD) of 150 amino acid residues longer than any usual CTLD. It contained six conserved cysteine residues involved in the formation of three internal disulfide bridges and an EPD (Glu₂₆₉-Pro₂₇₀-Asp₂₇₁) motif at the Ca²⁺-binding site 2. The deduced amino acid sequence of AiCTL-6 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, AiCTL-6 mRNA was found mainly in hepatopancreas and gill, and marginally expressed in other tissues. After the scallops were challenged by Listonella anguillarum for 6 h, the mRNA expression of AiCTL-6 was up-regulated significantly to 7.2-fold compared to the blank group. While at 9 h post Micrococcus luteus challenge, its expression level was 60.1 times higher than that of the blank group. The functional activity of AiCTL-6 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiCTL-6 could agglutinate Gram-negative bacteria E. coli TOP10F′, Gram-positive bacteria M. luteus and Staphylococcus aureus. These results collectively suggested that AiCTL-6, as a novel member of C-type lectin family, contributed to the host defense mechanisms against invading microorganism in A. irradians.     

The C-type lectin-like receptors of Dectin-1 cluster in natural killer gene complex

Natural killer gene complex (NKC) encodes a group of proteins with a single C-type lectin-like domain, (CTLD) which can be subdivided several subfamilies according to their structures and expression patterns. The receptors containing the conserved calcium binding sites in the CTLD fold belong to group II of C-type lectin superfamily and are expressed on myeloid cells and non- myeloid cells. The receptors lacking conserved calcium binding sites in the CTLD fold have evolved to bind ligands other than carbohydrates independently on calcium and thereby are named as C-type lectin-like receptors. The C-type lectin-like receptors are previously thought to be exclusively expressed on natural killer (NK) cells and enable NK cells to discriminate self, missing self or altered self. However, some C-type lectin-like receptors are identified in myeloid cells and are intensely investigated, recently. These myeloid C-type lectin-like receptors, especially Dectin-1 cluster, have a wide variety of ligands, including those of exogenous origin, and play important roles in the physiological functions and pathological processes including immune homeostasis, immune defenses, and immune surveillance. In this review, we summarize each member of the Dectin-1 cluster, including their structural profiles, expression patterns, signaling properties as well as known physiological functions.     

ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures

Abstract C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca²⁺-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca²⁺-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis , which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors.
ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.     

Reproductive function of Selenoprotein M in Chinese mitten crabs (Eriocheir sinesis)

Selenoproteins are present in all major forms of life, including eukaryotes, bacteria and archaea. In eukaryotic animals, selenoproteins often function as antioxidants, but rare or absent in other phyla, such as plants and fungi (except for the green alga Chlamydomonas). Selenoprotein M (SelM) is a selenocysteine containing protein with redox activity, which is involved in the antioxidant response. However, information remains limited about SelM physiology and function in marine invertebrates, particularly in crustaceans. Hence, we investigated the reproductive functionality of SelM in the Chinese mitten crab (Eriocheir sinensis), which is a commercially important yet disease vulnerable aquaculture species. The full-length SelM cDNA (928bp) strand was cloned by using PCR, based on an initial expressed sequence tag (EST) that was isolated from a hepatopancreatic cDNA library. The SelM cDNA contained a 390bp open reading frame (ORF) that encoded a putative 129 amino acid (aa) protein. SelM mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in the hepatopancreas, testis, ovaries and intestines. Based on this information, we then detected the different stages of tissue expression for SelM in the testis, ovary, and male crab hepatopancreas and hemolymph, and the enzyme activity of SelM in the testis. Overall, SelM was isolated successfully from the Chinese mitten crab, and its involvement in the regulation of reproduction during the period of rapid development in E. sinensis was confirmed.     

镉胁迫对河南华溪蟹两种C型凝集素免疫应答的影响

C型凝集素是一类可以和糖类结合的蛋白质, 是先天性免疫系统中重要的模式识别受体。其中, 经典C型凝集素依赖Ca2+对糖类进行识别。Ca2+可作为细胞内第二信使, 参与多种信息传递。而重金属镉可导致细胞钙稳态失调, 干扰细胞内与Ca2+相关的信息传递。研究旨在探明镉胁迫对河南华溪蟹(Sinopotamon henanense) ShLec21和ShLec23两种C型凝集素免疫应答的影响。利用RACE方法, 克隆了ShLec21和ShLec23, 并进行了系统进化分析; 利用实时荧光定量PCR的方法, 研究了ShLec21和ShLec23的组织表达模式和镉联合嗜水气单胞菌(Aeromonas hydrophila)胁迫后肝胰腺和血淋巴中ShLec21和ShLec23表达模式。结果显示: ShLec21 cDNA全长863 bp, 编码152个氨基酸残基; ShLec23 cDNA全长681 bp, 编码164个氨基酸残基。ShLec21和ShLec23分别聚类为无脊椎动物的两个分支。ShLec21和ShLec23在血淋巴、鳃、肝胰腺、肠道、肌肉、卵巢和精巢中表达广泛, 但二者均主要在肝胰腺中表达。在胁迫条件下, 单独镉胁迫对肝胰腺和血淋巴中ShLec21和ShLec23表达量无显著影响; 在单独嗜水气单胞菌感染后, 肝胰腺中ShLec21和ShLec23表达量分别显著(P<0.05)与极显著(P<0.01)下调, 血淋巴中ShLec23表达量显著(P<0.05)下调; 而在镉胁迫后嗜水气单胞菌感染过程中,ShLec21和ShLec23表达量在肝胰腺和血淋巴中显著(P<0.05)或极显著(P<0.01)上调。研究结果表明, 河南华溪蟹ShLec21和ShLec23在响应嗜水气单胞菌感染过程中的表达, 在一定程度上能够被镉胁迫所上调。     

Identification and characteristics of a novel gene, <Emphasis Type="Italic">EJO1</Emphasis>, in the Chinese mitten crab (<Emphasis Type="Italic">Eriocheir japonica sinensis</Emphasis>) ovary

EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa. Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the ovary development.     

Effects of a novel peptide derived from human thrombomodulin on endotoxin-induced uveitis in vitro and in vivo

Thrombomodulin (TM) is a single-transmembrane glycoprotein receptor for thrombin, which is best known as a cofactor for thrombin-mediated activation of anticoagulant protein C. C-type lectin-like domain (CTLD) of TM has distinct coagulation/fibrinolysis-independent anti-inflammatory properties. Here we found anti-inflammatory effects of a novel peptide (GC31) from CTLD of TM in endotoxin-induced uveitis, which was characterized by a reduction of leukocyte counts, protein concentration, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 levels in aqueous humor. Through in vitro experiments, we further found that GC31 suppressed TNF-α and interleukin (IL)-6 expressions in lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and interrupted LPS-induced nuclear factor-κB (NF-κB) activation. These data indicate a beneficial role of peptide GC31 in preventing intraocular inflammatory response, especially uveitis.     

中华绒螯蟹MSTN基因SNPs多态性及与生长性状的关联分析

为研究中华绒螯蟹(Eriocheir sinensis)肌肉生长抑制素基因(myostatin, MSTN)的多态性及其与生长性状的相关性, 对中华绒螯蟹3个群体(育种群体、大赛群体、野生群体)共321个个体MSTN基因的多态性进行筛选, 发现该基因的第1外显子存在3个多态性SNP位点(S1: C714T; S2:G729A; S3:G753T), 均为处于Hardy-Weinberg平衡(P>0.05)的中、高度多态性位点。利用一般线性模型分析3个位点及其基因型组合与生长性状的相关性, 发现S1位点对中华绒螯蟹的体重和壳长等生长性状有显著影响(P≤0.05), 而其余2个位点与生长性状无显著关联性。结果表明S1位点的TT基因型对中华绒螯蟹的生长最为有利, 可作为分子标记辅助育种的候选标记。