Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor.

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.

Ge H ，Wang G ，Zhang L ，Zhang Z ，Wang S ，Zou Z ，Yan S ，Wang Y ... -  《-》

Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor.

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone.

Ge H ，Wang G ，Zhang L ，Wang S ，Zou Z ，Yan S ，Wang Y ，Zhang Z ... -  《-》

Insulin-like growth factor binding protein 7, a member of insulin-like growth factor signal pathway, involved in immune response of small abalone Haliotis diversicolor.

Insulin-like growth factor binding protein 7 (IGFBP7), the only member of the IGFBP superfamily that binds strongly to insulin, may have different functions from other IGFBPs. Unlike other IGFBPs, there is no knowledge available on aquatic invertebrate IGFBP7. In this study, a molluscan IGFBP7 gene, saIGFBP7, was cloned for the first time from the small abalone Haliotis diversicolor. Its full-length cDNA sequence is 1812 bp, with a 720 bp open reading frame encoding a protein of 239 aa. The molecular mass of the deduced protein is approximately 25.37 kDa with an estimated pI of 5.00, and it shares highest 41% identity to IGFBP7 of Amblyomma americanum. Analysis of conserved domains revealed the presence of an IGFBP N-terminal domain (IB), a kazal-type serine proteinase inhibitor domain (KI), and an immunoglobulin-like C2 domain (IgC2) in saIGFBP7. Furthermore, the 12 cysteine residues and the signature amino acid motif 'xCGCCxxC' which are characterized by the amino terminus region of the IGFBP superfamily are all presented in saIGFBP7. Quantitative real-time PCR and western blot were employed to investigate the tissue distribution of saIGFBP7, and its expression under bacterial challenge. The saIGFBP7 mRNA and protein could be detected in all examined tissues, with the highest expression level in hemocytes, higher expression level in gills, and was up-regulated in hemocytes and gills after bacterial injection. In addition, saIGFBP7 mRNA transcripts were observed in a subset of the branchial epithelium and the nucleus of hemocytes using the in situ hybridization method. Interestingly, saIGFBP7 was detected mainly in the goblet-like cell of the branchial epithelium by immunohistochemistry. These results suggested that saIGFBP7 was likely to be involved in a function associated with pathogenic infection and may play an important role in the adult abalone immune system.

Li N ，Zhang Z ，Zhang L ，Wang S ，Zou Z ，Wang G ，Wang Y ... -  《-》

Molecular cloning and expression analysis of GABA(A) receptor-associated protein (GABARAP) from small abalone, Haliotis diversicolor.

GABA(A) receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5'-UTR, and a 333 bp 3'-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae.

Bai R ，You W ，Chen J ，Huang H ，Ke C ... -  《-》

First molecular cloning of a molluscan caspase from variously colored abalone (Haliotis diversicolor) and gene expression analysis with bacterial challenge.

Mammal caspases have been demonstrated to possess important functions in apoptosis and immune signaling, but there is less knowledge available on abalone caspases. In the present study, a molluscan caspase gene, abCaspase, was cloned for the first time from the variously colored abalone (Haliotis diversicolor) and its full-length cDNA sequence was 2427 bp, with a 1008 bp of open reading frame encoding a protein of 336 aa. The molecular mass of the deduced protein was approximately 36.97 kDa with an estimated pI of 5.28. The predicted amino acid sequence of abCaspase contained two domains of p20 and p10 which were conserved in the caspase family, including the cysteine active site pentapeptide "QSCRG" and the histidine active site signature "HTVYDCVVVIFLTHG". Homology analysis showed that abCaspase shared high similarity with apoptotic caspases and it was grouped together with vertebrate caspase-8s and caspase-10s using phylogenetic analysis, suggesting that abCaspase belonged to a typical apoptotic caspase and might possess the characteristic of human caspase-8 and -10. The mRNA transcripts of abCaspase were widely distributed in various tissues of H. diversicolor. Expression of the abCaspase gene was significantly induced in the tissues tested, especially in the hemocytes, gill and mantle with bacterial challenge. This study suggested that abCaspase may be an initiator caspase associated with the induction of apoptosis which is potentially involved in the immune defense of H. diversicolor.

Huang WB ，Ren HL ，Gopalakrishnan S ，Xu DD ，Qiao K ，Wang KJ ... -  《-》