The Studies On Molecular Biology And Function Of Innate Immune-related Proteins Of Oyster, Crassostrea Ariakensis

The studies on molecular biology and function of innate immune-related proteins in inverteberate species including oysters are currently a key topic in the frontier of pathogen and molecular immunology in the world. Rickettsia-like organisms (RLOs) are obligate intracellular Gram-negative bacteria which caused mass mortality of oysters. However, little is known about the mechanisms of anti-RLO infection in oyster. The studies on molecular biology and function of innate immune-related proteins were carried out in this scientific dissertation which included the immune function and mechanism of genes or proteins including sTRAIL,Tolloid-like,MAK3 and p38, and signal pathways of sTRAIL involved in the host defense under the induction of RLO. On the other hand, studies on the cytotoxicity and its mechanims and signal pathway of CasTRAIL on oyster hemocytes and HL-60 tumor cells were also carried out in this dissertation1.Study on the funtion of anti-tumor and anti-RLO immune response of CasTRAIL in oyster Crassostrea ariakensisTotal RNAwas extracted from hemocytes of RLO-challenged and unchallenged oysters using TRIzol method and was reverse transcribed into cDNA. Degenerate oligonucleotide primers were designed according to the sequences of TRAIL genes publisded in NCBI. Here, we first report that a homologue of human sTRAIL, CasTRAIL, was cloned and identified by RT-PCR in bivalves. The sequence of TRAIL cDNA includes an open reading frame of 501 nucleotide encoding a hypothetical protein of 167 amino acids with a molecular mass about 18.4kDa. Amino acid sequence analysis revealed that CasTRAIL shares conserved signature motifs with other TNF proteins and is highly homologous to sTRAIL of human (98%), it has a 99% similarity over the cDNA sequence and 98% over the amino acid sequence between them. On the contrary, it was only 42% of similarity compared to grass Carp (Ctenopharyngodon idella).The expression level of CasTRAIL in various tissues was investigated by RT-PCR and in situ hybridization(ISH).The results showed that CasTRAIL was ubiquitously expressed in all examined tissues, and expression levels in mantle, hemocytes and gills were higher than the others, while the lowlest level was detected in gonad, suggesting that CasTRAIL may be involved in immune responses. To investigate the biological functions of CasTRAIL, the recombinant plasmids (pET-CasTRAIL) were constructed and transformed into competent cells for expression. The recombinant fusion proteins were purified by affinity chromatography, then the antiserum against New Zealand white rabbits was prepared and used to study the subcellular location of oyster TRAIL protein. The results showed that: recombinant protein CasTRAIL was successfully expressed in E. coli, the valence of antibody raised with recombinant CasTRAIL was more than 1:4000.Oyster TRAIL protein was primarily located in the cell membrane, and it is a transmember protein. The results of SDS-PAGE, Flow cytometric analysis, DNA fragmentation ,real time RT-PCR and Western blotting showed that: CasTRAIL could induce apoptosis in HL-60 tumor cells, by binding to death receptors (DR4 and DR5) which were up-regulated by CasTRAIL induction. However, it showed no cytotoxicity to normal hemocytes of oyster,although the rapid increase in the phospho-ERK and phospho-p38 levels were observed, indicated that the MAPK pathway may be involved in CasTRAIL-mediated signaling.Then the results of ISH and real time RT-PCR showed that: obviously increased expression level were detected in mantle, hemolymph and muscle, while decreased in gill, and no obvious changes were found in gonad, suggesting that CasTRAIL is involved in anti-RLO defense mechanisms.2.Molecular cloning and identification of Tolloid-like,MAK3 and p38MAPK genes in oyster Crassostrea ariakensis after RLO chanllengeBased on our previous studies, other host genes, including Tolloid-like,MAK3 and p38 of oyster Crassostrea ariakensis were cloned, and their functions in the immune response against RLO were also investigated.â‘ The full-length cDNA of CaTLL spans 3492 nucleotides including an open reading frame of 2811 nucleotides which encodes a hypothetical protein of 936 amino acids, with a molecular mass of approximately 103 kDa. The CaTLL molecule possessed structural features of several motifs of astacin family including an N-terminal signal peptide sequence, a prodomain with a RTRR motif, an astacin-like domain that contains a conserved zinc-binding motif HELGHVIGFWHEH, five CUBs and two EGF domains with the arrangement CUB-CUB-EGF-CUB-EGF-CUB-CUB. The proteolytic domain of CaTLL shares more than 30% identity with other astacins of various animals from squail to mammals, indicating its conserved catalytic ability. RT-PCR and qantitative real-time PCR analyses revealed that CaTLL showed the lowest expression level in hemocytes of normal groups, but was affected significantly by RLO-challenge, suggesting that CaTLL is involved in the molluscan immune response.â‘¡The ORF of CaMAK3 consists 510bp encoding a protein of 169 amino acids, with a molecular mass of approximately 18.6kDa. The Acetyltransf₁ domain of CaMAK3 shares about 30% identity with yeast Saccharomyces cerevisiae, and 53% to 100% identity with animals selected, indicating that it is a very conserved domain. The results of RT-PCR showed that CaMAK3 was expressed in all examined tissues, and expression levels in gills and hemocytes were higher than the others. Meanwhile, obvious changes in the expression level of CaMAK3 were found after RLO challenge by RT-PCR, suggesting that it is involved in immune responses.â‘¢The full-length cDNA of Cap38 spans 1244bp,which contains an ORF of 1065 bp encoding a protein of 169 amino acids about 39kDa. Structure analysis founded that Cap38 shares conserved signature motifs--S_TKc with other p38 proteins, which shares 56% to 70% identity with other animals, indicating that it is a very conserved kinase. As RT-PCR indicated, Cap38 was ubiquitously expressed in all examined tissues, and expression levels in gills,mantle and hemocytes were higher than that in others.And obvious changes in the expression level of Cap38 were found in hemocytew after RLO challenge for 15min, suggesting that it play certain roles in immune responses.All together, we could draw the following conclusions:The expression level of CasTRAIL could be significantly up-regulated by RLO stimulation, indicating that CasTRAIL was involved in anti-RLO defense mechanisms. Moreover, CasTRAIL could up- regulate the expression levels p38 both from mRNA level and protein level, which suggesting that p38 may play a role in CasTRAIL-mediated anti-RLO signaling pathway, In addition, Tolloid-like,MAK3 and p38 are all involved in anti-RLO immune responses. These results support the hypothesis that the Pathogen/TRAIL/MAPKs/NFκB pathway may exist in the immune system of C. ariakensis and play an immportant role in the immune response against RLO infection, and Tolloid-like and MAK3 might also play some roles in regulation and acetylation modification of molecules involved in this pathway.In addition, CasTRAIL also showed an ability to induce apoptosis in HL-60 tumor cells through death receptors (DR4 and DR5) pathway but has no cytotoxicity to normal hemocytes of oyster.