Identification, expression pattern and potential role of variable lymphocyte receptor Aj-VLRA from Apostichopus japonicus in response to bacterial challenge.

The variable lymphocyte receptors (VLRs) are found in jawless vertebrates (agnathans), and specifically recognize bacteria and viruses via their leucine-rich repeats (LRRs). VLRs are believed to be adaptive immune response molecules. Echinoderms do not have adaptive immune systems; however, in the present study, a VLR cDNA named Aj-VLRA was cloned and characterized from sea cucumber, Apostichopus japonicus. The complete cDNA of Aj-VLRA was 3072 bp, including a 1995 bp open reading frame encoding 664 amino acids comprising LRR domains, a predicted transmembrane helix and an N-terminal signal peptide. As determined by quantitative real-time reverse transcription polymerase chain reaction, Aj-VLRA transcripts are ubiquitously expressed in the body wall, longitudinal muscles, intestine and respiratory tree of A. japonicus. The expression level of Aj-VLRA was upregulated after challenge with four common marine bacteria. In situ hybridization showed that the expression of Aj-VLRA was widely distributed in the four tissues, particularly in the cytoplasm of epidermal cells. Recombinantly expressed Aj-VLRA (including the LRR domains) could bind to bacteria including Micrococcus lysodeikticus (Gram+) and Vibrio anguillarum (Gram-). Collectively, the results suggested that Aj-VLRA is related to an innate immune response of A. japonicus.

Identification, expression pattern and functional characterization of As-MyD88 in bacteria challenge and during different developmental stages of Artemia sinica.

Myeloid differentiation factor 88 (MYD88), a key adapter protein in Toll-like receptor signaling, affects the immune response and the formation of the dorsal-ventral axis. Here, the 1555bp full-length cDNA of MyD88 from Artemia sinica (As-MyD88) was obtained. Molecular characterization revealed that the sequence includes an 1182bp open reading frame encoding a predicted protein of 393 amino acids. The predicted protein contains a death domain in the N-terminus, and box1 and 2 motifs of the TIR domain in the C-terminus. Real-time quantitative PCR, Western blotting and immunohistochemistry were used to determine the expression level, protein production and location of As-MYD88 during embryonic development and bacterial challenge. The highest expression level during embryonic development was at the 0h and 5h stages of A. sinica. As-MYD88 was remarkably upregulated after bacterial challenge. Our results suggested that As-MYD88 plays a vital role in response to bacterial challenge, and during post-diapause embryonic development of A. sinica.

Identification and expression patterns of extracellular matrix-associated genes fibropellin-ia and tenascin involved in regeneration of sea cucumber Apostichopus japonicus.

Sea cucumbers have a strong regenerative capacity. Many important genes involved in the molecular mechanism of regeneration and associated with intercellular signaling pathways of regeneration have been identified. The product of the fibropellin-ia gene forms a layer known as the apical lamina that surrounds the sea cucumber embryo throughout development. Meanwhile, the tenascin gene displays highly restricted and dynamic patterns of expression in the embryo and is expressed in the adult during normal processes such as wound healing, nerve regeneration and tissue involution. In this study, we cloned for the first time full-length cDNAs of fibropellin-ia (1390 bp, encoding a 199 amino acid protein) and tenascin (1366 bp, encoding a 179 amino acid protein) from Apostichopus japonicus (designated Aj-fnia and Aj-tenascin, respectively) using rapid amplification of cDNA ends. The structures and characteristics of these two genes were analyzed bioinformatically, and their expression patterns associated with extracellular matrix remodeling in regeneration of A. japonicus were investigated by real-time PCR and in situ hybridization (ISH). Expression levels of Aj-fnia and Aj-tenascin in the regeneration tissues were higher than those in normal tissues. The highest expression levels of Aj-fnia and Aj-tenascin were shown in the intestine and respiratory tree on the 15th and 20th days after sea cucumbers were eviscerated. In the body wall, the highest expression levels of Aj-fnia and Aj-tenascin occurred at 35 and 45 min during early regeneration and then emerged between 5 and 7 days again during late regeneration after the body wall was injured. ISH analysis revealed expression of these genes in the body wall, longitudinal muscle, intestine and respiratory tree. These findings suggest that Aj-fnia and Aj-tenascin are crucial genes that play important roles in the regeneration of the sea cucumber.