Identification And Functional Characterization Of A Novel Tetraspanin Of The Oyster,Crassostrea Ariakensis

The molluscan immune system involves both humoral responses and various immunocyte reactions, including reactive oxygen species production, antimicrobial peptide secretion, encapsulation and phagocytosis. For molluscs, hemocytes play key role in host defense. Hemocyte adherence and mobility are essential for phagocytosis against foreign particles and migration toward inflammatory sites. Additionally, hemocyte adherence to different surfaces can result in important cellular behaviors; e.g. parasitic encapsulation and hemocyte-mediated clotting responses. These responses might be induced by specific membrane receptors of the hemocytes for receiving and transducing signals. Consequently, the elucidation of molluscan vector immunity is expected to help identify factors that influence the vectorial capacity of molluscs and to provide new avenues to understand parasite transmission by these organisms.Using SMARTTM technology, we construstructed an full-length cDNA library of oyster hemocytes which were challenged by LPS.120positive clones were selected for sequencing after screening and confirmation of PCR. We have got100ESTs which included seven novel genes:tetraspanin, FJ475123; FosB, FJ594438; ferritin, FJ529405; NADH, FJ495091; QM-like, FJ467930; SAHH, FJ495090:u-box, FJ495092. T identified by NCBI Blast and analysed by bioinformation software, hese genes were annotated to be involved in different biological processes including substance metabolism, respiratory metabolism, transcription regulation, protein degradation and native immune etc.Tetraspanins (also referred to as tetraspans or TM4SF proteins) are a family of four-transmembrane-domain proteins, found in mammals, insects, worms and fungi. Tetraspanin proteins regulate cell morphology, motility, invasion, fusion and signaling as organizers of multi molecular membrane complexes. The first tetraspanin protein, ME491/CD63, was characterized in1988and the hallmark family sequence motifs were reported in1990. In addition to four transmembrane domains, these proteins feature several conserved amino acid residues including a highly conserved CCG motif, where two cysteine residues are required for formation of essential disulphide bonds of the second extracellular loop. The mammalian immune cells,(T and B lymphocytes, granulocytes, monocytes, macrophages, dendritic cells and natural killer cells) all have at least eight tetraspanins. These tetraspanins associate directly or indirectly with numerous molecules—for example, T-cell receptors, B-cell receptors, major histocompatibility complex(MHC) class I, MHC class II, CD2, CD4. CD8, CD19, EWI-2,α4β1and α6β1integrins—that have crucial roles in the functions of immune cells.In this research, we identified a tetraspanin family member gene, named Ca-TSP, in the oyster Crassostrea ariakensis and found that the transcription profiles of Ca-TSP were variable in the oyster hemocytes. Three distinct patterns of variation of Ca-TSP were observed. Using immunofluorescence and immunoelectron microscopy, we show that Ca-TSP was present in granules and in vesicular structures of the oyster hemocyte. Sequence analysis, structural features and immunogold electron microscopy showed that Ca-TSP is an integral membrane glycoprotein of granules of hemocyte and may be a novel CD63-like gene of the tetraspanin family of molluscs. The gene expression analysis of Ca-TSP using isolated oyster hemocytes, was done following challenge of the oysters with LPS and PolyhC. The Ca-TSP mRNA levels increased in hemocytes in the first12hours after LPS and Poly Ⅰ:C stimulation, and decreased after the addition of H2O2. Western blot analysis using anti-Ca-TSP antibody indicated that gene expression and protein levels were similar. The recombinant Ca-TSP was found to significantly inhibit hemocytes aggregation. Our results suggested that Ca-TSP participates in the innate immunity of the oyster. In seeking an explanation for the functional properties of tetrasapnins, we focused on the interactions of acting proteins and signal transduction pathways. Immunofluorescence assay showed that Ca-TSP colocalizes with hemocyanin in oyster hemocytes in vivo. The analyses of protein interactions showed that Ca-TSP, hemocyanin and a lectin-like protein formed a complex. To our knowledge, there are no reports on the interaction between tetraspanin and hemocyanin. Although the HA of the unknown isolated proteins was detected, it is supposed that the unknown isolated proteins should be a complex. Therefore the immune response of Ca-TSP may be supported by compound action of oyster hemocyanin and the lectin-like protein. We used MEK inhibitors to survey the signal pathway which was involved in. However preincubation with MEK inhibitor U0126and PD98059. resulted in a reduction of p-MAPK after1h, but12h later level of p-MAPK increased a little. And treatment of hemocytes in vitro resulted in down-regulated level of Ca-TSP after12h. It was showned that there were obvious inhibition effects when both of inhibitors were used. It is suggested that the important kinase pathway might be involved in the regulation of Ca-TSP.