Novel microbial fermentation for the preparation of iron-chelating scallop skirts peptides-its profile, identification, and possible binding mode.

Iron chelating peptides have been widely utilized as iron supplements due to their excellent absorption capacity, However, the high cost and cumbersome manufacturing process of these peptides significantly limit their industrial application. In this study, fermentation was used for the first time to prepare iron chelating peptides. Bacillus altitudinis 3*1-3 was selected as the most suitable strain from 50 strains. The hydrolysates of fermented scallop skirts showed excellent iron-chelating capacity (9.39 mg/g). Aspartic acid, glutamic acid, and histidine are crucial for the binding of peptides to ferrous ions. The heptapeptide (FEDPEFE) forms six binding bonds with ferrous irons. Compared with ferrous sulfate, peptide-ferrous chelate showed more stability in salt solution and simulated gastrointestinal juice (p < 0.05). Furthermore, the fermentation method could save >50% of the cost compared with the enzymatic method. The results can provide a theoretical basis for the preparation of ferrous-chelated peptides using the fermentation method.

Identification and Antihypertension Study of Novel Angiotensin I-Converting Enzyme Inhibitory Peptides from the Skirt of Chlamys farreri Fermented with Bacillus natto.

The aim of this study was to isolate the angiotensin I-converting enzyme (ACE) inhibitory peptides from the skirt of Chlamys farreri fermented with Bacillus natto and to explore the antihypertension effect through in vivo studies. ACE inhibitory peptides were purified from the fermentation mixture by ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography sequentially. The amino acids' sequence of the five novel ACE inhibitory peptides were identified by liquid chromatography-tandem mass spectrometry. Animal experiments demonstrated that the novel ACE inhibitory peptides significantly reduced the blood pressure in spontaneously hypertensive rats after a single or long-time treatment. Potential mechanisms were explored, and the results indicated that the novel peptides could regulate the renal renin-angiotensin system, improve vascular remodeling, inhibit myocardial fibrosis, and rebalance the gut microbial dysbiosis. Our results suggest that the fermentation products of the Chlamys farreri skirt by B. natto are potential sources of active peptides processing antihypertension activities.

Homeopathy outperforms antibiotics treatment in juvenile scallop Argopecten ventricosus: effects on growth, survival, and immune response.

BACKGROUND: Mortality from vibriosis in mollusk production is attributed to pathogenic bacteria, particularly Vibrio alginolyticus. Use of increasingly potent antibiotics has led to bacterial resistance and increased pathogenicity. Alternatives in sanitation, safety, and environmental sustainability are currently under analysis. To-date, homeopathy has been investigated in aquaculture of freshwater fish, but not in marine mollusks. The effect of the homeopathic complexes in the growth, survival, and immune response of the Catarina scallop Argopecten ventricosus were assessed. METHODS: A bioassay to assess the potential of homeopathy in improving cultivation of juvenile A. ventricosus was conducted for 21 days, with a final challenge of 120 h with V. alginolyticus. The experimental design included two homeopathic formulas The homeopathic complex Passival, consisting of Passiflora incarnata 30 CH, Valeriana officinalis 30 CH, Ignatia amara 30 CH and Zincum valerianicum 30 CH plus Phosphoricum acid 30 CH (treatment TH1) or Silicea terra 30 CH (TH2), two antibiotics (ampicillin = AMP, oxytetracycline = OXY), and two reference treatments (without homeopathic or antibiotic treatment = CTRL, ethanol 30° GL = ETH). Additionally, a negative control CTRL- (untreated/uninfected) is included in the challenge test. Juvenile scallops (4.14 ± 0.06 mm, 13.33 mg wet weight) were cultivated in 4 L tanks provided with aerated, filtered (1 µm), and UV-sterilized seawater that was changed every third day. They were fed a blend of the microalgae Isochrysis galbana and Chaetoceros calcitrans (150,000 cells mL-1 twice a day). All treatments were directly added to the tank water and then 500 mL challenge units were inoculated with 1 × 107 CFU/mL (LD50) of V. alginolyticus. RESULTS: Juveniles grew significantly larger and faster in height and weight with TH2 compared to the ETH and CTRL (P < 0.05, ANOVA). Higher concentrations of proteins occurred in scallops exposed to TH2 (160.57 ± 7.79 mg g-1), compared to other treatments and reference treatments. Higher survival rate during the challenge bioassay occurred with TH1 (85%), compared to AMP (53%), OXY (30%), and CTRL (0%), and superoxide dismutase (P < 0.05) was significantly higher in scallops treated with TH1, compared to other treatments and reference treatments. CONCLUSIONS: Homeopathic treatments improved growth and survival and enhanced survival against V. alginolyticus in juvenile A. ventricosus. This suggests that homeopathy is a viable treatment for this mollusk to reduce use of antibiotics in scallops and its progressive increase in pathogenicity in mollusk hatcheries.

Characterization of the TRAF3IP1 gene in Yesso scallop (Patinopecten yessoensis) and its expression in response to bacterial challenge.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an important adaptor that transmits upstream activation signals to induce innate immune responses. TRAF3 interacting protein 1 (TRAF3IP1) interacts specifically with TRAF3, but its function in innate immunity remains unclear, especially in marine invertebrates. In this study, to better understand the functions of TRAFs in innate immune responses, we identified and characterized the first bivalve TRAF3IP1 gene, PyTRAF3IP1, from Yesso scallop (Patinopecten yessoensis), one of the most important mollusk species for aquaculture. The PyTRAF3IP1 cDNA is 2,367 bp, with an open reading frame of 1,629 bp encoding 542 amino acids. Phylogenetic and protein structural analysis confirmed the gene's identity and revealed that PyTRAF3IP1 was more similar to vertebrate TRAF3IP1s than to those of invertebrates. PyTRAF3IP1 was expressed in all the adult tissues and developmental stages sampled, implying that it plays versatile roles in many biological processes. Furthermore, PyTRAF3IP1 expression was dramatically induced in the acute phase (3-6 h) after infection with both Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacteria, even stronger induction being observed after V. anguillarum challenge. This is the first report of the characterization and immune response involvement of TRAF3IP1 in marine invertebrates, and suggests that TRAF3IP1 contributes to innate immunity in bivalves.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C. farreri (denoted as CfFER). The full length of CfFER is 848 bp and contains a 5'-UTR of 113 bp, a 3'-UTR of 219 bp, and a complete open reading frame (ORF) of 516 bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95 kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12 h post-infection (hpi) and then normalized at 48 hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C. farreri and protection against pathogen challenge.

Genome-wide identification, characterization and expression analyses of two TNFRs in Yesso scallop (Patinopecten yessoensis) provide insight into the disparity of responses to bacterial infections and heat stress in bivalves.

Tumor necrosis factors receptors (TNFRs) comprise a superfamily of proteins characterized by a unique cysteine-rich domain (CRD) and play important roles in diverse physiological and pathological processes in the innate immune system, including inflammation, apoptosis, autoimmunity and organogenesis. Although significant effects of TNFRs on immunity have been reported in most vertebrates as well as some invertebrates, the complete TNFR superfamily has not been systematically characterized in scallops. In this study, two different types of TNFR-like genes, including PyTNFR1 and PyTNFR2 genes were identified from Yesso scallop (Patinopecten yessoensis, Jay, 1857) through whole-genome scanning. Phylogenetic and protein structural analyses were carried out to determine the identities and evolutionary relationships of the two genes. The expression profiling of PyTNFRs was performed at different development stages, in healthy adult tissues and in hemocytes after bacterial infection and heat stress. Expression analysis revealed that both PyTNFRs were significantly induced during the acute phase (3 h) after infection with Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacteria, though much more dramatic chronic-phase (24 h) changes were observed after V. anguillarum challenge. For heat stress, only PyTNFR2 displayed significant elevation at 12 h and 24 h, which suggests a functional difference in the two PyTNFRs. Collectively, this study provides novel insight into the PyTNFRs and the specific role and response of TNFR-involved pathways in host immune responses against different bacterial pathogens and heat stress in bivalves.

Algicidal effect of hybrid peptides as potential inhibitors of harmful algal blooms.

OBJECTIVES: To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems. RESULTS: We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms. CONCLUSIONS: These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Genome-wide identification and characterization of TRAF genes in the Yesso scallop (Patinopecten yessoensis) and their distinct expression patterns in response to bacterial challenge.

The tumor necrosis factor (TNF) receptor associated factors (TRAFs) are the major signal transducers for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily, which regulate a variety of cellular activities and innate immune responses. TRAF genes have been extensively studied in various species, including vertebrates and invertebrates. However, as one of the key component of NF-κB pathway, TRAF genes have not been systematically characterized in marine invertebrates. In this study, we identified and characterized five TRAF genes, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. In comparison with the TRAF genes from vertebrate species, the structural features were all relatively conserved in the PyTRAF genes. To gain insights into the roles of TRAF genes during scallop innate immune responses, quantitative real-time PCR was used to investigate the expression profiles in the different stages of scallop development, in the healthy adult tissues, and in the hemocytes after bacterial infection with Micrococcus luteus and Vibrio anguillarum. Based on the qRT-PCR analysis, the expression of most of the PyTRAFs was significantly induced in the acute phases (3-6 h) after infection with Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria, and many more dramatic changes in PyTRAFs expression were observed after V. anguillarum challenge. Notably, the strong response in the up-regulation of PyTRAF6 post-bacterial challenge was distinct from that previously reported in scallops and crabs but was similar to that of other shellfish, Echinodermata and even teleost fish. The high level expressions of PyTRAFs in the hemocytes and the gill, and their specific expression patterns after challenges provide insights into the versatile roles and responses of TRAFs in the innate immune system against Gram-negative bacterial pathogens in bivalves.

The immunomodulation of nicotinic acetylcholine receptor subunits in Zhikong scallop Chlamys farreri.

Nicotinic acetylcholine receptor (nAChR), the best-studied ionotropic neuron receptor protein, is a key player in neuronal communication, and it has been reported to play an important role in immunomodulation of vertebrates. Although nAChRs have also been identified in most invertebrates, the knowledge about their immunomodulation is still limited. In the present study, two scallop nAChR genes were identified from Chlamys farreri (designed as CfnAChR1 and CfnAChR2), which encoded 384 and 443 amino acids, respectively. The conserved disulfide-linked cystines, ion selectivity residues and the hydrophobic gating residues (L251, V255 and V259) were identified in CfnAChR1 and CfnAChR2. The immunoreactivities of CfnAChR1 and CfnAChR2 were observed in all the tested scallop tissues, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. After LPS (0.5 mg mL(-1)) stimulation, the expression of CfnAChR1 mRNA in haemocytes increased significantly by 9.83-fold (P < 0.05) and 12.93-fold (P < 0.05) at 3 h and 24 h, respectively. While the expression level of CfnAChR2 mRNA increased 43.94% at 12 h after LPS stimulation (P < 0.05). After TNF-α (50 ng mL(-1)) stimulation, the expression levels of CfnAChR1 and CfnAChR2 both increased significantly at 1 h, which were 21.33-fold (P < 0.05) and 2.44-fold (P < 0.05) of that in the PBS group, respectively. The results collectively indicated that the cholinergic nervous system in scallops could be activated by immune stimulations through CfnAChR1 and CfnAChR2, which function as the links between the cholinergic nervous system and immune system.

Molecular characterization and immune response expression of the QM gene from the scallop Chlamys farreri.

The scallop Chlamys farreri is an important aquaculture species in northern China. However, the sustainable development of the scallop industry is currently threatened by several pathogens that cause mass mortality of this mollusk. Therefore, a complete understanding of the immune response mechanisms involved in host-virus interactions is necessary. This study reports a novel QM gene from C. farreri. This gene was first identified as a putative tumor suppressor gene from human and then confirmed to participate in several functions, including immune response. The QM gene from C. farreri (CfQM) was identified by suppression subtractive hybridization, and its full-length (763 bp) cDNA was obtained through rapid amplification of cDNA ends. The cDNA of CfQM contained a short 5'-UTR of 22 bp and a 3'-UTR of 84 bp. Its ORF comprised 657 nucleotides that encode 218 amino acids with a molecular weight of approximately 28.3 kDa and an isoelectric point of 10.06. The deduced amino acid sequence of CfQM contained a series of conserved functional motifs that belong to the QM family. Phylogenetic analysis revealed that CfQM was closely related to other mollusk QM proteins, and altogether they form a mollusk QM protein subfamily that displays evolutionary conservation from yeast to human. The tissue-specific expression and transcriptional regulation of CfQM were investigated by quantitative real-time PCR in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges. The transcript level of CfQM was high in all of the examined tissues in a constitutive manner. The highest and lowest expression levels of CfQM were measured in the hepatopancreas and hemocyte, respectively. Upon bacterial and viral challenges, the relative mRNA expression of CfQM sharply increased at 6 h post-infection (hpi) and then normalized at 48 hpi. These findings suggest that CfQM can respond to and protect against pathogen challenge. To the best of our knowledge, this study is the first report of the QM gene from scallop. The results presented herein provided new insights into the molecular basis of host-pathogen interactions in C. farreri.

Characterizations and expression analyses of NF-κB and Rel genes in the Yesso scallop (Patinopecten yessoensis) suggest specific response patterns against Gram-negative infection in bivalves.

Rel/NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) genes are evolutionarily conserved and play a pivotal role in several physiological events. They have been extensively studied from various species, including both vertebrates and invertebrates. However, the Rel/NF-κB genes have not been systematically characterized in bivalves. In this study, we identified and characterized PyNF-κB and PyRel in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine the identities and evolutionary relationships of Rel/NF-κB genes in Yesso scallop. Compared with the Rel/NF-κB genes from vertebrate species, the PyNF-κB and PyRel are relatively conserved in their structural features, but there were no paralogs found in P. yessoensis or other invertebrates. To gain insights into the roles of Rel/NF-κB genes during the innate immune response in scallop, quantitative real-time PCR was used to investigate the expression profiles of these genes at different developmental stages, in healthy adult tissues and in the hemolymph after bacterial infection with Micrococcus luteus and Vibrio anguillarum. The real-time PCR results indicated the abundance of PyNF-κB in the first four embryonic stages, including oocytes, fertilized eggs, morulae and blastulae. By contrast, PyRel was abundantly expressed in blastulae, trochophores and D-shaped larvae. In adult scallops, PyNF-κB and PyRel were ubiquitously expressed in most healthy tissues and highly expressed in most of the immune related tissues. Both genes were significantly up-regulated during the acute phase (3 h) after infection with Gram-positive (M. luteus) and negative (V. anguillarum) bacteria, while the much higher expression level of PyNF-κB suggested the involvement of the extra immune deficiency (IMD)-like pathway against the Gram-negative bacterial infection. The complex pattern of Rel/NF-κB induced expression suggested that PyNF-κB and PyRel both have specific and cooperative roles in the acute immune responses to bacterial infection.

A low-density lipoprotein receptor-related protein (LRP)-like molecule identified from Chlamys farreri participated in immune response against bacterial infection.

Low-density lipoprotein receptor-related protein (LRP) is a group of important endocytic receptors contributing to binding ligands and maintaining internal environment. In the present study, an LRP-like molecule was identified from Zhikong scallop Chlamys farreri (CfLPR), and its mRNA expression profiles, tissue location, and immunology activities were analyzed to explore its possible function in the innate immune system. The ORF of CfLRP was of 1971 bp encoding a polypeptide of 656 amino acids with ten low-density lipoprotein-receptor YWTD (LY) domains and one scavenger receptor cysteine-rich (SRCR) domain. It shared similar structure with out-membrane domains of LRP family members in mammalian. The mRNA transcripts of CfLRP were dominantly expressed in hepatopancreas and mantle (P < 0.01), and its mRNA level in hemocytes was up-regulated (P < 0.01) significantly after the stimulations of lipopolysaccharides (LPS), peptidoglycan (PGN) and ß-glucan. Western blotting assay using polyclonal antibody specific for CfLRP revealed that CfLRP was localized in the plasma. The recombinant protein of CfLRP (rCfLRP) could bind acetylated low density lipoprotein (Ac-LDL), metalloprotease SPF1 of Vibrio splendidus and mannan, but could not bind other typical PAMPs such as LPS, PGN, ß-glucan and zymosan. Meanwhile, rCfLRP also exhibited strong bacteriostatic activity to Gram-negative bacteria Vibrio anguillarum and V. splendidus. These results indicated that CfLRP could serve as a receptor to recognize and eliminate the invading pathogens, which provided a new implication in the function of LRP-like molecules in invertebrate immunity.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri.

Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.

A novel C1qDC protein acting as pattern recognition receptor in scallop Argopecten irradians.

The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded ß-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, ß-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F' as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system.

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.

A multi-CRD C-type lectin with broad recognition spectrum and cellular adhesion from Argopectenirradians.

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins which play significant roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, a novel C-type lectin with four dissimilar carbohydrate-recognition domains (CRDs) was identified from Argopectenirradians (designated as AiCTL-9). The full-length cDNA of AiCTL-9 was of 2291 bp with an open reading frame of 1827 bp encoding a polypeptide of 608 amino acids with a signal sequence and four CRDs. The motifs determining carbohydrate binding specificity in each CRD of AiCTL-9 were different, and they were YPT in CRD1, EPD in CRD2, EPN in CRD3 and QPN in CRD4, respectively. All the four CRDs shared the similar potential tertiary structure of a typical double-loop structure with Ca(2+)-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The mRNA transcripts of AiCTL-9 were mainly detected in hepatopancreas as well as gonad, and also marginally detectable in mantle, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after the challenges of fungi PichiapastorisGS115 (P<0.05), Gram-positive bacteria Micrococcusluteus (P<0.05) and Gram-negative bacteria Vibrioanguillarum (P<0.01). The recombinant AiCTL-9 (rAiCTL-9) could bind various PAMPs, including LPS, PGN, mannan and glucan, and also displayed agglutinating activity to fungi P. pastorisGS115, Gram-positive bacteria Bacillussubtilis and Gram-negative bacteria EscherichiacoliTOP10F' as well as V. anguillarum in a Ca(2+) dependent manner. Moreover, rAiCTL-9 could initiate the cellular adhesion of hemocytes and enhance their encapsulation invitro. All these results implied that AiCTL-9 was a novel PRR involved in immune response of scallop against a large number of pathogens by recognizing different PAMPs and enhancing scallop hemocytes encapsulation.

Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-α.

Phenylalanine hydroxylase (PAH) is an important metabolic enzyme of aromatic amino acids, which is responsible for the irreversible oxidation of phenylalanine to tyrosine. In the present study, the full-length cDNA encoding PAH from Chlamys farreri (designated CfPAH) was cloned by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfPAH encoded a polypeptide of 460 amino acids, and its sequence shared 64.4-74.2% similarity with those of PAHs from other animals. There were an N-terminal regulatory ACT domain and a C-terminal catalytic Biopterin_H domain in the deduced CfPAH protein. The mRNA transcripts of CfPAH could be detected in all the tested tissues, including adductor muscle, mantle, gill, gonad, haemocytes and hepatopancreas. And its expression level in haemocytes was increased significantly during 3-48 h after bacteria Vibrio anguillarum challenge with the highest level (9.1-fold, P < 0.05) at 24 h. Furthermore, the mRNA expression of CfPAH in haemocytes also increased significantly to 2.6-fold (P < 0.05) at 4 h and 3.3-fold (P < 0.05) at 6 h after the stimulation of 50.0 ng mL(-1) human TNF-α. The cDNA fragment encoding the mature peptide of CfPAH was recombined and expressed in the prokaryotic expression system, and 1 mg recombinant CfPAH protein (rCfPAH) could catalyze the conversion of 192.23 ± 32.35 nmol phenylalanine to tyrosine within 1 min (nmol min(-1) mg(-1) protein) in vitro. These results indicated collectively that CfPAH, as a homologue of phenylalanine hydroxylase in scallop C. farreri, could be induced by cytokine and involved in the immunomodulation of scallops by supplying the starting material tyrosine for the synthesis of melanin and catecholamines.

A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response.

Galectins are a family of ß-galactoside-binding lectins that specifically bind to ß-galactoside residues and play crucial roles in innate immune responses of invertebrates and vertebrates. The cDNA of bay scallop Argopecten irradians galectin (designated as AiGal2) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiGal2 was of 2137 bp. The open reading frame encoded a polypeptide of 555 amino acids containing four carbohydrate-recognition domains. The deduced amino acid sequence and multi-domain organization of AiGal2 were highly similar to those of mollusk galectins. A typical galectin fold in ß-sandwich arrangement was identified in the potential tertiary structure of all the four CRDs in AiGal2. The mRNA transcripts of AiGal2 were found to be constitutively expressed in a wide range of tissues and mainly in hepatopancreas, adductor muscle and kidney. After scallops were challenged by Vibrio anguillarum or Micrococcus luteus, the mRNA expression level of AiGal2 was up-regulated significantly, while it did not changed remarkably after Pichia pastoris challenge. The recombined AiGal2 (rAiGal2) exhibited strong activity to agglutinate E. coli, V. anguillarum, Vibrio fluvialis, Edwardsiella tarda and M. luteus, and the agglutinating activities could be inhibited by both d-galactose and lactose. The in vitro encapsulation assay revealed that rAiGal2 could bind to hemocytes and enhanced its encapsulation of agarose beads. These results collectively suggested that AiGal2 functioned as a pattern recognition receptor in immune defense and contributed to the non-self recognition and elimination in cellular immune response of bay scallop.

cDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri.

The glutathione peroxidases are essential enzymes of the cellular antioxidant defence system. In the present study, the full-length cDNA sequence encoding an extracellular glutathione peroxidase (designated CfGPx3) was isolated from Zhikong scallop Chlamys farreri. The complete cDNA was of 1194 bp, containing a 5' untranslated region (UTR) of 50 bp, a 3' UTR of 490 bp and an open reading frame (ORF) of 654 bp encoding a polypeptide of 217 amino acids. CfGPx3 possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase, such as the selenocysteine encoded by stop codon UGA, the GPx signature motif (96LGVPCNQF103) and the active site motif (179WNFEKF184). The high similarity of CfGPx3 with GPx from other organisms indicated that CfGPx3 should be a new member of the glutathione peroxidase family. By fluorescent quantitative real-time PCR, the CfGPx3 mRNA was universally detected in the tissues of haemocytes, gill, gonad, muscle and hepatopancreas with the highest expression in hepatopancreas. After scallops were challenged by Listonella anguillarum, the expression level of CfGPx3 transcript in haemocytes was significantly up-regulated (P<0.05) at 8h post challenge. These results suggested that CfGPx3 was potentially involved in the immune response of scallops and perhaps contributed to the protective effects against oxidative stress.

An immune responsive multidomain galectin from bay scallop Argopectens irradians.

Galectins are a family of beta-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from bay scallop Argopectens irradians (designated AiGal1) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of AiGal1 was of 2235 nucleotides, encoding a polypeptide of 549 amino acids. SMART program analysis revealed that AiGal1 contained four galectin CRDs, and all the CRDs contained the two consensus motifs essential for ligand-binding. Quantitative real-time PCR was employed to investigate the tissue distribution of AiGal1 mRNA and temporal expression in haemocytes of scallops challenged with Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The AiGal1 mRNA could be detected in all tested tissues with the highest expression level in hepatopancreas. After challenged by V. anguillarum and M. luteus, the expression level of AiGal1 mRNA was both up-regulated and reached the maximum level at 9 h (1.52 fold, P < 0.05) and 18 h (2.89 fold, P < 0.01) post challenge, respectively. However, there was no significant difference in the mRNA expression of AiGal1 in haemocytes after P. pastoris challenge (P > 0.05). These results collectively indicated that AiGal1 was a new member of the galectin family and involved in the immune responses against bacterial infection.