Characterization of a novel molluscan MyD88 family protein from manila clam, Ruditapes philippinarum

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein which is required for signal transduction of TLR/IL-1R family. In this study, a novel molluscan MyD88 family member protein (named as RpMyD88) was identified from manila clam, Ruditapes philippinarum. It was identified using BLAST algorithm from GS-FLX? sequencing data. The cDNA of RpMyD88 consists of 1416 bp open reading frame (ORF) encoding 471 amino acid residues. The RpMyD88 contains death domain and Toll/interleukin-1 receptor (TIR) domain which are typical features of MyD88 family proteins. The predicted amino acid sequence of RpMyD88 shares 27% identity with scallop MyD88. The expression level of RpMyD88 mRNA was investigated in healthy and challenged clams by quantitative real-time RT-PCR. The RpMyD88 gene expression is ubiquitous in all selected tissues. The RpMyD88 mRNA was strongly expressed in hemocyte, gill and mantle. In contrast, it was weakly expressed in siphon, foot and adductor muscle. RpMyD88 was up-regulated in gill and hemocyte after immune challenge with both Vibrio tapetis and LPS challenge. All results considered, sequence characterization, comparison and gene expression data suggesting that MyD88-dependent signaling pathway is presence in manila clam and RpMyD88 plays an important role in innate immune response against bacteria.     

Cloning and characterization of two ferritin subunit genes from bay scallop, <Emphasis Type="Italic">Argopecten irradians</Emphasis> (Lamarck 1819)

We have cloned two full-length cDNAs from two ferritin genes (Aifer1 and Aifer2) of the bay scallop, Argopecten irradians (Lamarck 1819). The cDNAs are 1,019 and 827 bp in length and encode proteins of 171 and 173 amino acids, respectively. The 5′ UTR of each contains a conserved iron response element (IRE) motif. Sequence analyses reveal that both proteins belong to the H-ferritin family with seven conserved amino acids in the ferroxidase center. Highest expression of Aifer1 is found in the mantle and adductor muscle, while that of Aifer2 is only in the latter tissue. These Aifer genes are differentially expressed following bacterial challenge of the scallop. The expression level of Aifer1 was acutely up-regulated (over 10 fold) at 6 h post-bacteria injection, whereas Aifer2 expression was not significantly changed by bacterial challenge. Both genes were effectively expressed in E. coli BL21 (DE3), producing proteins of similar molecular weight, approximately 23 kDa. Purified Aifer1 and Aifer2 proteins exhibited iron-chelating activity of 33.1% and 30.4%, respectively, at a concentration of 5 mg/ml. Cations, Mg²⁺, Zn²⁺ and Ca²⁺, depressed iron-chelating activity of both proteins. Additionally, the E. coli cells expressing recombinant Aifer1 and Aifer2 showed tolerance to H₂O₂, providing a direct evidence of the antioxidation function of ferritin. The results presented in this study suggest important roles of Aifer1 and Aifer2 in the regulation of iron homeostasis, immune response, and antioxidative stress in A. irradians.     

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207bp with a 678bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3h post-injection with V. anguillarum and then slightly recovered from 6 to 48h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V. anguillarum infection.     

Molecular cloning and expression analysis of interferon-γ inducible lysosomal thiol reductase (GILT)-like cDNA from disk abalone (Haliotis discus discus)

Interferon Gamma (IFN-γ) Inducible Lysosomal Thiol reductase (GILT) has been described as a key enzyme in processing and presentation of major histocompatibility complex (MHC) class II restricted antigen (Ag) by catalyzing disulfide bond (S–S) reduction in mammals. Abalone GILT-like (AbGILT) full-length cDNA was isolated from the normalized disk abalone cDNA library. The 807-bp AbGILT cDNA consists of an open reading frame of 684-bp, encoding 228 amino acid residues. The predicted AbGILT protein has a molecular weight of 25 kDa and an isoelectric point of 7.8. The N-terminus of the AbGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19–20. AbGILT contains two active site C-XX-C motifs, (²³CLDC²⁶ and ⁴⁶CPYC⁴⁹) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence ⁹²CQHGX₂ECX₂NX₄C¹⁰⁷ and 12 cysteine residues representing 5% in the mature peptide. Phylogenetic analysis showed that AbGILT has been derived from a common ancestor with other GILT proteins. RT-PCR results showed that AbGILT expression was up-regulated in the gill, mantle and digestive tract 24 h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48 h. In contrast, AbGILT expression was not up-regulated by poly inosinic–cytidylic acid (poly I:C) during the 48 h induction. However, AbGILT was constitutively expressed in gill, mantle, and digestive tract tissues suggesting that it may maintain first line of innate immune defense at basal level in disk abalone.     

The involvement of HSP22 from bay scallop <Emphasis Type="Italic">Argopecten irradians</Emphasis> in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd²⁺, Pb²⁺ or Cu²⁺ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu²⁺, Pb²⁺ and Cd²⁺. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu²⁺, Pb²⁺ and Cd²⁺ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.