Highest Expression Level In Hepatopancreas Research Articles

PyLKB1 regulates glucose transport via activating PyAMPKα in Yesso Scallop Patinopecten yessoensis under high temperature stress

Liver kinase B1 (LKB1) is a classical serine/threonine protein kinase and plays an important role in maintaining energy homeostasis through phosphorylate AMP-activated protein kinase α subunit (AMPKα). In this study, a homologous molecule of LKB1 with a typical serine/threonine kinase domain and two nuclear localization sequences (NLSs) was identified in Yesso Scallop Patinopecten yessoensis (PyLKB1). The mRNA transcripts of PyLKB1 were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in hepatopancreas. PyLKB1 was mainly located in cytoplasm and nucleus of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyLKB1, PyAMPKα, and PyGLUT1 in hepatopancreas, the phosphorylation level of PyAMPKα at Thr170 in hepatopancreas, the positive fluorescence signals of PyLKB1 in haemocytes, glucose analogue 2-NBDG content in haemocytes, and glucose content in hepatopancreas, haemocytes and serum all increased significantly (p < 0.05) compared to blank group (15 °C). However, there was no significant difference at the protein level of PyLKB1 and PyAMPKα. After PyLKB1 was knockdown by siRNA, the mRNA expression level of PyGLUT1, and the glucose content in hepatopancreas and serum were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. However, there were no significant difference in PyGLUT1 expression, glucose content and glucose analogue 2-NBDG content in haemocytes. These results collectively suggested that PyLKB1-PyAMPKα pathway was activated to promote glucose transport by regulating PyGLUT1 in response to high temperature stress. These results would be helpful for understanding the function of PyLKB1-PyAMPKα pathway in regulating glucose metabolism and maintaining energy homeostasis under high temperature stress in scallops.

GST-Mu of Cristaria plicata is regulated by Nrf2/Keap1 pathway in detoxification microcystin and has antioxidant function

Glutathione S-transferase is a crucial phase II metabolic enzyme involved in detoxification and metabolism in aquatic organisms. This study aimed to investigate the regulation of Nrf2/Keap1 pathway on microcystin-induced CpGST-Mu expression and CpGST-Mu resistance to hydrogen peroxide. A mu class GST from Cristaria plicata (CpGST-Mu) was identified. The full-length cDNA was 1026 bp, with an open reading frame of 558 bp. Subcellular localization revealed that CpGST-Mu was localized in cytoplasm. The optimum pH and temperature for the catalytic activity of CpGST-Mu protein was pH 6 and 40 °C, respectively. The results of Real-time quantitative PCR showed that CpGST-Mu mRNA was constitutively expressed in tissues, with the highest expression level in hepatopancreas and the lowest expression level in gill. The mRNA level of CpGST-Mu was significantly increased under the stress of microcystins and hydrogen peroxide. CpGST-Mu had an antagonistic effect on hydrogen peroxide. In the knockdown experiments, the mRNA levels of CpGST-Mu exhibited corresponding changes while Nrf2 and Keap1 genes were individually knocked down. These findings indicated that GST-Mu exhibited antioxidant properties and its expression was regulated by Nrf2/Keap1 signaling pathway. The study provided new information on the function of GST-Mu and could contribute to future studies on how to excrete microcystins in molluscs.

CgATP synthase β subunit involved in the regulation of haemocytes proliferation as a CgAstakine receptor in Crassostrea gigas

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase β subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt_ab_N domain and an AAA domain in the CgATP synthase β subunit protein. The mRNA transcripts of CgATP synthase β subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase β subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase β subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase β subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase β subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase β subunit. There results suggested that CgATP synthase β subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas.

The first identification of a C-type lectin gene (CqCTL) in Cherax quadricarinatus: sequence features and expression profiles

As pattern recognition receptors (PRRs), C-type lectins (CTLs) have important roles in the recognition and clearance of pathogens by the innate immune system. In the present study, the first Cherax quadricarinatus CTL gene (designated CqCTL) was cloned and characterized. The complete cDNA sequence of CqCTL contained an open reading frame (ORF) of 543 bp, which encoded a protein of 180 amino acids. A carbohydrate recognition domain (CRD) containing four conserved cysteines (Cys48, Cys59, Cys76, Cys177) and the EPD (Glu80-Pro81-Asn82) and QPD (Gln146-Pro147-Asn148) motifs were identified in the deduced amino acid sequence of CqCTL. The deduced tertiary structure of CqCTL revealed two α helices，five β sheets and two disulfide bonds. CqCTL exhibited high similarity with previously identified CTLs from other species. The mRNA transcripts of CqCTL were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. These results provide useful information on the potential role of CqCTL in the innate immune system of C. quadricarinatus, and lay the foundation for further studies on the CTLs of crustacean.

Characterization and the role of α2-macroglobulin from Macrobrachium nipponense in response to ammonia-N stress

Ammonia-N is a major physiological challenge for prawns in culture. The alpha-2-macroglobulin (α2-macroglobulin, α2M) gene is a multifunctional protein with a significant role in the innate immunity. In this study, the full-length cDNA of α2M (Mnα2M) from M. nipponense was amplified and the 4805 bp cDNA contains a 4422 bp open reading frame (ORF). This cDNA encodes a polypeptide comprising 1473 amino acids, with a predicted molecular weight of 163.45 kDa. The sequence of Mnα2M contains three putative conserved functional domains including a bait region, a thiol ester domain and a receptor binding domain which are present in other invertebrate and vertebrate α2Ms. Multiple alignment analysis showed that α2M in M. nipponense and other crustaceans shared high similarity. Severe histological alterations were observed in M. nipponense hepatopancreas. Quantitative real-time PCR (qRT-PCR) confirmed that Mnα2M was expressed in all examined tissues, with the high expression level in the hepatopancreas, hemocyte and gills. The expression of Mnα2M in hepatopancreas, hemocyte and gills was significant up-regulated at 12 h, 24 h and 48 h under ammonia-N stress. Western blot analysis further revealed that the Mnα2M protein was distributed in all tissues, with the high expression level in hepatopancreas, hemocyte and gills, which was consistent with the qRT-PCR results. These results suggest that high concentrations of ammonia-N has significant effects on the M. nipponense and Mnα2M might be involved in the immune response to ammonia-N stress.

Stomach metabolic alterations in response to AHPND infection in shrimp

Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), and play crucial roles in immune responses. In the present study, a novel tetraspanin gene (designated MmTSPAN) was cloned and characterized from the hard clam Meretrix meretrix. The complete cDNA sequence of MmTSPAN contained an open reading frame (ORF) of 816 bp, which encoded a protein of 271 amino acids. MmTSPAN exhibited highly similarity with previously identified tetraspanins from other species. It contained four transmembrane domains (12–35 aa, 69–92 aa, 99–123 aa and 238–261 aa), characteristic CCG motif and four conservative cysteine residues. The mRNA transcripts of MmTSPAN were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. Temporal transcriptional levels in the hepatopancreas revealed significant up-regulation of MmTSPAN by Vibrio splendidus stimulation, with a 3.14-fold increase at 6 h compared to the control, and reaching 32.98-fold at 24 h. These results provide useful information for further study of the function of tetraspanin in the innate immune system of M. meretrix, and may offer a new therapeutic target for diseases of M. meretrix.

A preliminary attempt to explore the potential functions of a tetraspanin gene (MmTSPAN) in the innate immunity of hard clam Meretrix meretrix: Sequence features and expression profiles

Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), and play crucial roles in immune responses. In the present study, a novel tetraspanin gene (designated MmTSPAN) was cloned and characterized from the hard clam Meretrix meretrix. The complete cDNA sequence of MmTSPAN contained an open reading frame (ORF) of 816 bp, which encoded a protein of 271 amino acids. MmTSPAN exhibited highly similarity with previously identified tetraspanins from other species. It contained four transmembrane domains (12–35 aa, 69–92 aa, 99–123 aa and 238–261 aa), characteristic CCG motif and four conservative cysteine residues. The mRNA transcripts of MmTSPAN were ubiquitously detectable in all the tested tissues, with the highest expression level in hepatopancreas. Temporal transcriptional levels in the hepatopancreas revealed significant up-regulation of MmTSPAN by Vibrio splendidus stimulation, with a 3.14-fold increase at 6 h compared to the control, and reaching 32.98-fold at 24 h. These results provide useful information for further study of the function of tetraspanin in the innate immune system of M. meretrix, and may offer a new therapeutic target for diseases of M. meretrix.

Molecular characterization and transcriptional analysis of a crustacean heat shock protein 10 gene in shrimp Litopenaeus vannamei

Heat shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins which conserved phylogenetically in all living organisms from archaebacteria to humans. Recent research achievements indicated that HSP10s might not only be involved in the responses to environmental stresses, but also play a pivotal role in the host defenses mechanism. In the present study, a cDNA of 715 bp for the Pacific white shrimp Litopenaeus vannamei HSP10 (designated as LvHSP10) was cloned via rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of LvHSP10 contained an open reading frame (ORF) of 309 bp, which encoded a protein of 102 amino acids. The protein sequence of LvHSP10 shared over 80 % similarity with previously identified HSP10s. There were a CPN10 domain and a chaperonins HSP10/CPN10 signature in the protein sequence of LvHSP10. The mRNA transcripts of LvHSP10 were constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest expression level in hepatopancreas. The mRNA expression profiles of LvHSP10 in hepatopancreas could be significantly induced by the stimulation of Vibrio parahaemolyticus, white spot syndrome virus (WSSV), and low and high pH challenge. These results provided useful information of the potential roles of LvHSP10 in the defense mechanism of shrimp against various biological stimulations and multiple environmental stresses.

Two novel LRR and Ig domain-containing proteins from oyster Crassostrea gigas function as pattern recognition receptors and induce expression of cytokines

Leucine-rich repeat (LRR) domain and immunoglobulin (Ig) domain are both competent immune recognition modules, and the immunological roles of LRR and Ig domain containing- proteins (LRRIGs) are speculated to be multifunctional and worth investigating. In the present study, two novel LRRIGs, CgLRRIG-1 and CgLRRIG-2, were identified and characterized from oyster Crassostrea gigas. Both of them contained an N-terminal LRR region, an Ig domain, a transmembrane region, and a C-terminal cytoplasmic tail. The mRNA transcripts of CgLRRIG-1 and CgLRRIG-2 were constitutively expressed in muscle, gill, hepatopancreas, mantle, gonad and hemocytes with the highest expression level in hepatopancreas. Their mRNA expression levels in hemocytes were significantly up-regulated after the stimulations with four PAMPs including peptidoglycan (PGN), lipopolysaccharide (LPS), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C) and one bacteria Vibrio anguillarum. The recombinant proteins, rCgLRRIG-1 and rCgLRRIG-2, could bind to PGN, LPS, GLU and poly I:C, and rCgLRRIG-2 exhibited higher binding affinity. Additionally, rCgLRRIG-1 and rCgLRRIG-2 could significantly induce the expression of CgTNF-1 and CgIL17-5 in cultured oyster hemocytes, and the activity of rCgLRRIG-2 was higher than that of rCgLRRIG-1. All these results indicated that CgLRRIG-1 and CgLRRIG-2 could function as immune effectors or pro-inflammatory factors as well as PRRs in oyster.

Molecular characterization, expression and functional analysis of two Kazal-type serine protease inhibitors from Venerupis philippinarum

Kazal-type serine protease inhibitors (KSPIs) act as negative regulators in immune signaling pathway by controlling the extent of serine protease (SP) activities. In this study, the full-length cDNA of two KSPIs (designed as VpKSPI-1 and VpKSPI-2) were identified from Venerupis philippinarum by rapid amplification of cDNA ends (RACE) approaches. The open reading frame (ORF) of VpKSPI-1 and VpKSPI-2 was of 552 bp and 402 bp, encoding a polypeptide of 183 and 133 amino acids, respectively. The transcripts of VpKSPI-1 and VpKSPI-2 were ubiquitously expressed in all tissues tested with the highest expression level in hepatopancreas. After Vibrio anguillarum challenge, the relative mRNA expression of VpKSPI-1 and VpKSPI-2 in hepatopancreas was both up-regulated within 96 h. The recombinant VpKSPI-1 (rVpKSPI-1) displayed weak activities towards chymotrypsin, moderate inhibitory activity to trypsin, while rVpKSPI-2 showed significant inhibitory activities against chymotrypsin and trypsin. When the molar ratio of rVpKSPI-2 to chymotrypsin and trypsin reached 1:4 and 1:2, the protease activities could be almost entirely inhibited. All these results suggested that both VpKSPI-1 and VpKSPI-2 perhaps play a vital role in the innate immunity of V. philippinarum.

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress

Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.

The immunomodulation of a maternal translationally controlled tumor protein (TCTP) in Zhikong scallop Chlamys farreri

Translationally controlled tumor protein (TCTP) is initially described as a highly conserved protein implicated in cell growth, and it is subsequently confirmed to play important roles in mediating the innate immune response, especially the inflammatory. In the present study, the full-length cDNA sequence of a TCTP from Zhikong scallop Chlamys farreri (designed as CfTCTP) was cloned by rapid amplification of cDNA ends (RACE) technique based on the expression sequence tag (EST) analysis. It was of 1230 bp with an open reading frame (ORF) of 543 bp encoding a polypeptide of 180 amino acids. The deduced amino acid sequence contained a conserved TCTP signature sequence (from I47 to E58) and it shared 26.1%–48.9% similarities with previously identified TCTPs. CfTCTP was clustered with the TCTP from Argopectehs irradias in the phylogenetic tree and was designated into a single branch of mollusk with TCTP from Ruditapes philippinarum. The mRNA transcripts of CfTCTP were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression level of CfTCTP in oocytes and fertilized eggs kept at a higher level, and was down-regulated from 2-cell embryos to the lowest level in gastrula. Then it was up-regulated in trochophore and dropped down in the late veliger larvae to the similar level as that in oocytes. After pathogen-associated molecular patterns (PAMPs) stimulation, the expression of CfTCTP mRNA in haemocytes was increased at 3 or 6 h, and fall down to the normal level at 24 h. The recombinant protein of CfTCTP could induce the release of histamine from BT-549 cells. All these results indicated that CfTCTP was a pro-inflammatory factor and it could be maternally transferred from female gonad to oocytes and offspring, and play pivotal role in the embryonic development and immune protection of scallops.

The receptor for activated C kinase 1 (RACK1) functions in hematopoiesis through JNK activation in Chinese mitten crab Eriocheir sinensis

Receptor for activated C kinase 1 (RACK1) is a WD-domain repeating protein which involves in the mediation of various biological processes, including innate immune response. In the present study, a RACK1 (designed as EsRACK1) gene from Chinese mitten crab E. sinensis was cloned by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA sequence of EsRACK1 was of 1117 bp with an open reading frame (ORF) of 957 bp encoding a polypeptide of 318 amino acids containing seven WD repeats. EsRACK1 shared 62%–99% similarities with previously identified RACK1s in amino acid sequence, and it was clustered with the RACK1 from Pacifastacus leniusculus in the phylogenetic tree. The mRNA transcripts of EsRACK1 were constitutively expressed in various tissues with the highest expression level in hepatopancreas. The expression of EsRACK1 mRNA in hemocytes were significantly up-regulated post the stimulations with Vibrio anguillarum and Pichia pastoris. After exposure to CdCl2 and pentachlorophenol, the transcripts of EsRACK1 in hemocytes were up-regulated at the late phase from 12 h. When EsRACK1 was knocked down by dsRNA based RNAi, the total hemocyte counts, new-born hemocytes and phosphorylation of JNK were all significantly decreased. In addition, EsRACK1 transcription and phosphorylation of JNK were both decreased in hematopoietic tissue post Aeromonas hydrophila challenge. All the results suggested that EsRACK1 was involved in the innate immune response of the crab and participated in the production of new-born hemocytes through activation of JNK.

An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri

Accumulating evidence has demonstrated that leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein–protein interactions and were involved in the immune response. In the present study, an LRR-only protein (designed as CfLRRop-1) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfLRRop-1 contained an open reading frame (ORF) of 1377 bp, which encoded a protein of 458 amino acids. An LRRNT motif, an LRR_7 motif and seven LRR motifs were found in the deduced amino acid sequence of CfLRRop-1. And these seven LRR motifs contained a conserved signature sequence LxxLxLxxNxL. The mRNA transcripts of CfLRRop-1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hepatopancreas. After the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), the mRNA transcripts of CfLRRop-1 in haemocytes all increased firstly within the first 6 h and secondly during 12–24 h post stimulation. The mRNA expression level of CfLRRop-1 was continuously up-regulated, after the expression of CfTLR (previously identified Toll-like receptor in C. farreri) was suppressed via RNA interference (RNAi). The recombinant CfLRRop-1 protein could directly bind LPS, PGN, GLU and poly I:C, and induce the release of TNF-α in mixed primary cultured scallop haemocytes. These results collectively indicated that CfLRRop-1 would function as a powerful pattern recognition receptor (PRR) and play a pivotal role in the immune response of scallops.

Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus.

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89%). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.

A high mobility group box 1 (HMGB1) gene from Chlamys farreri and the DNA-binding ability and pro-inflammatory activity of its recombinant protein

High-mobility group box 1 (HMGB1) protein, a highly conserved DNA binding protein, plays an important role in maintaining nucleosome structures, transcription, and inflammation. In the present research, a cDNA of 1268 bp for the Zhikong scallop Chlamys farreri HMGB1 (designed as CfHMGB1) was cloned via rapid amplification of cDNA ends (RACE) technique and expression sequence tag (EST) analysis. The complete cDNA sequence of CfHMGB1 contained an open reading frame (ORF) of 648 bp, which encoded a protein of 215 amino acids. The amino acid sequence of CfHMGB1 shared 53–57% similarity with other identified HMGB1s. There were two HMG domains, two low complexity regions and a conserved acidic tail in the amino acid sequence of CfHMGB1. The mRNA transcripts of CfHMGB1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas, kidney and gonad, with the highest expression level in hepatopancreas. The mRNA expression profiles of CfHMGB1 in haemocytes after the stimulation with different pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (Glu), were similar with an up-regulation in the early stage and then recovered to the original level. The recombinant CfHMGB1 protein could bind double-stranded DNA and induce the release of TNF-α activity in mixed primary culture of scallop haemocytes. These results collectively indicated that CfHMGB1, with DNA-binding ability and pro-inflammatory activity, could play an important role in the immune response of scallops.

CDNA cloning, characterization and expression analysis of peroxiredoxin 5 gene in the ridgetail white prawn Exopalaemon carinicauda

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species. In this study, a full-length of peroxiredoxin 5 (designated EcPrx5) cDNA was cloned from the ridgetail white prawn Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of the EcPrx5 was of 827 bp, containing a 5′ untranslated region (UTR) of 14 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame of 585 bp encoding a polypeptide of 194 amino acids with the predicted molecular weight of 20.83 kDa and estimated isoelectric point of 7.62. BLAST analysis revealed that amino acids of EcPrx5 shared 89, 68, 66, 65, 53 and 51 % identity with that of Macrobrachium rosenbergii, Megachile rotundata, Harpegnathos saltator, Acromyrmex echinatior, Danio rerio, and Homo sapiens counterparts, respectively. The conserved Prx domain and the signature of peroxiredoxin catalytic center identified in EcPrx5 suggested that EcPrx5 belonged to the atypical 2-Cys Prx subgroup. Real time quantitative RT-PCR analysis indicated that EcPrx5 could be detected in all the tested tissues with highest expression level in hepatopancreas. As time progressed, the expression level of EcPrx5 both in hemocytes and hepatopancreas increased in the first 6 h after Vibrio anguillarum and white spot syndrome virus challenge, and showed different expression profiles. The results indicated that EcPrx5 involved in immune response against bacterial and viral infection in E. carinicauda.

A C-type lectin (AiCTL-3) from bay scallop Argopecten irradians with mannose/galactose binding ability to bind various bacteria

C-type lectins are a family of Ca2+-dependent carbohydrate-binding proteins playing crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a C-type lectin with one carbohydrate-recognition domain (CRD) of 127 amino acids was cloned from bay scallop Argopecten irradians (designated AiCTL-3) by rapid amplification of cDNA end (RACE) techniques based on expressed sequence tag (EST) analysis. The mRNA transcripts of AiCTL-3 could be detected in all the tested tissues including hepatopancreas, gonad, adductor muscle, heart, hemocytes, mantle and gill, with the highest expression level in hepatopancreas. After the challenges with Vibrio anguillarum and Micrococcus luteus, the mRNA expression level of AiCTL-3 was obviously up-regulated and reached the maximum level at 9h (11.87fold, P<0.01, and 20.02-fold, P<0.05, respectively). The recombinant AiCTL-3 (designated as rAiCTL-3) could bind LPS, PGN, and glucan in vitro, but could not bind mannan. And it also bound Gram-positive bacteria Staphylococcus aureus as well as Gram-negative bacteria Escherichia coli and V. anguillarum. With a Ca2+ binding site 2 EPN (Glu–Pro–Asn) motif, rAiCTL-3 could bind both mannose and galactose which was quite different from those in vertebrate. Meanwhile, it could significantly enhance the phagocytosis of scallop hemocytes in vitro. The results clearly suggested that AiCTL-3 could serve not only as a PRR participated in the immune response against various PAMPs and bacteria in non-self recognition via mannose/galactose binding specificity but an opsonin playing an important part in clearance of invaders.

Non-monotonous dose–response curves observed for some endpoints, after pre- and postnatal exposure of rats to the fungicide epoxiconazole

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase β subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt_ab_N domain and an AAA domain in the CgATP synthase β subunit protein. The mRNA transcripts of CgATP synthase β subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase β subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase β subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase β subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase β subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase β subunit. There results suggested that CgATP synthase β subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas.

Molecular cloning and functional characterization of a short peptidoglycan recognition protein (HcPGRPS1) from the freshwater mussel, Hyriopsis cumingi

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In the present study, a short-form PGRP, designated as HcPGRPS1 was identified from freshwater mussel Hyriopsis cumingi. The deduced amino acid sequence of HcPGRPS1 is composed of 235 residues which contains a conserved PGRP domain at the C-terminus. Sequence analysis showed that HcPGRPS1 shared high identities with other known PGRPs. The mRNA of HcPGRPS1 is constitutively expressed in a wide range of all tested tissues, with highest expression level in hepatopancreas, and its expression in tissues (gonad, nephridium, gill and foot) was up-regulated significantly after LPS or PGN stimulation (P<0.05). The recombinant protein of HcPGRPS1 exhibited binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and DAP-PGN from Bacillus subtilis. Furthermore, recombinant HcPGRPS1 displayed strong antibacterial activity to both Gram-negative bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas sobria and Gram-positive bacteria S. aureus in the presence of Zn2+. These results suggested that HcPGRPS1 plays a multifunctional role in the defense and protection mechanisms of mussel innate immunity against infections.