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Thioester-containing proteins (TEPs), characterized by a unique intrachain β-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in Cf TEP, a previously identified thioester-containing protein in scallop Chlamys farreri , were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of Cf TEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The Cf TEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the Cf TEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of Cf TEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or β-glucan. Two bands (100 and 55 kDa) were detected using anti- Cf TEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942–1472), and a single band (46 kDa) was detected by using anti- Cf TEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452–496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of Cf TEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum . All these results suggested that Cf TEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops. [ABSTRACT FROM AUTHOR]

Abstract: Tumor necrosis factor (TNF) signaling pathway plays crucial roles in the regulation of various immune responses. In the present study, a TNF signaling pathway related regulatory factor, TRAF and TNF receptor-associated protein (TTRAP), was firstly identified from the mollusk Zhikong scallop Chlamys farreri (designated as CfTTRAP). The full-length cDNA of CfTTRAP was of 2326bp, containing an open reading frame (ORF) of 1008bp encoding a polypeptide of 335 amino acids with the predicted molecular weight of 38.4kDa. There was an Exo_endo_phos domain in CfTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. The recombinant protein of CfTTRAP exhibited prominent endonuclease activity to digest the genome DNA from C. farreri in the presence of Mg2+, but it could not digest genome DNA of Escherichia coli and Bacillus subtilis, indicating CfTTRAP was a new member of Mg2+/Mn2+-dependent phosphodiesterase enzymes (MDP) superfamily. The mRNA transcripts of CfTTRAP were detected in all tested tissues of scallop, including muscle, mantle, gonad, gill, kidney and hemocytes. The expression level of CfTTRAP mRNA in hemocytes varied greatly after the stimulation of LPS, PGN or β-glucan. LPS induced significant down-regulation (P <0.05) of CfTTRAP mRNA expression, while PGN or β-glucan up-regulated the expression significantly (P <0.01), indicating that this regulatory factor was involved in modulating immune responses towards different stimulus. The present results provided new evidences for the potential roles of such molecule in C. farreri, and further confirmed the existence of TTRAP modulated TNF signaling pathway in mollusk. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which participate in nonself-recognition and clearance of invaders. In invertebrate, some multidomain C-type lectins have been identified, but their relative functions and binding mechanism are still meager. In the present study, A C-type lectin (CfLec-4) with four CRDs from Chlamys farreri was selected to investigate its possible function in innate immunity. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated (P <0.01) after the stimulations of β-glucan, LPS or PGN, and reached the highest expression level at 3, 6, 12h post-stimulation, which was 27.9-, 22.6- or 47.9-fold of that in blank group, respectively. Immunohistochemistry assay with polyclonal antibody specific for CfLec-4 revealed that the endogenous CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4 (rCflec-4) could bind LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broader bacteria binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as Gram-negative bacteria Escherichia coli, Vibrio anguillarum and fungi Pichia pastoris. Meanwhile, rCfLec-4 could significantly (P <0.01) enhance the phagocytosis of hemocytes in vitro. The results clearly suggested that four-CRD containing CfLec-4 not only served as PRR with wider recognition spectrum, but also functioned as an opsonin participating in the clearance of invaders in scallops. It could be inferred that the diversity and complexity of CRDs in C-type lectins endowed these receptors with comprehensive recognition spectrum and multiple immune functions against complex living environment. [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P <0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0h to 9.7U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55μmol/L), and reached the top concentration at 24h (42.27μmol/L), then recovered to the normal level after 48h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent Km was 0.82±0.11 and 1.24±0.13mM for l-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri. [Copyright &y& Elsevier]

Abstract: Monoamine oxidase (MAO) is an essential enzyme in the catabolism of monoamines, and implicated in the immune response of vertebrates. In the present study, the full-length cDNA encoding monoamine oxidase (designated CfMAO) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfMAO cDNA encoded 519 amino acids, which shared 73.9% similarity with that from oyster Crassostrea gigas, and 64.5–66.3% similarity with those from vertebrates. A conserved Amino_oxidase domain and a transmembrane domain were identified in the deduced CfMAO protein. The mRNA transcripts of CfMAO could be detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, mantle, gill and gonad. The mRNA expression of CfMAO was up-regulated significantly in haemocytes of scallops during 6–48h after bacteria Vibrio anguillarum challenge, and it reached the peak (25.9-fold, P <0.05) at 12h. The cDNA fragment encoding the mature peptide of CfMAO was expressed in the prokaryotic expression system, and 1mg of the recombinant protein (rCfMAO) could catalyze the deamination of 3665.59nmol serotonin, 2061.89nmol norepinephrine, 2104.85nmol epinephrine or 3040.34nmol dopamine within 1min (nmolmin−1 mg−1) in vitro. When the reaction mixture was coincubated with 0.1mmolL−1 MAO inhibitor clorgyline, its catalyzing activity to deaminize serotonin and dopamine was decreased significantly to 1603.69 and 955.39nmolmin−1 mg−1 (P <0.05) respectively. These results indicated that CfMAO, as the homologue of monoamine oxidase in scallop C. farreri, could modulate the immune response of scallops through the deamination of monoamines. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of Ca2+ dependent carbohydrate-recognition proteins which play significant diverse roles in nonself-recognition and clearance of invaders. In the present study, a C-type lectin (CfLec-2) from Zhikong scallop Chlamys farreri was selected to investigate its functions in innate immunity. The mRNA expression of CfLec-2 in hemocytes was significantly up-regulated (P <0.01) after scallops were stimulated by LPS, PGN or β-glucan, and reached the highest expression level at 12h post-stimulation, which was 72.5-, 23.6- or 43.8-fold compared with blank group, respectively. The recombinant CfLec-2 (designated as rCfLec-2) could bind LPS, PGN, mannan and zymosan in vitro, but it could not bind β-glucan. Immunofluorescence assay with polyclonal antibody specific for CfLec-2 revealed that CfLec-2 was mainly located in the mantle, kidney and gonad. Furthermore, rCfLec-2 could bind to the surface of scallop hemocytes, and then initiated cellular adhesion and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-2 serum. These results collectively suggested that CfLec-2 from the primitive deuterostome C. farreri could perform two distinct immune functions, pathogen recognition and cellular adhesion synchronously, while these functions were performed by collectins and selectins in vertebrates, respectively. The synchronous functions of pathogen recognition and cellular adhesion performed by CfLec-2 tempted us to suspect that CfLec-2 was an ancient form of C-type lectin, and apparently the differentiation of these two functions mediated by C-type lectins occurred after mollusk in phylogeny. [Copyright &y& Elsevier]