摘要
软体动物的免疫系统包括体液应答和各种免疫细胞的细胞反应,这些反应包括活性氧的产生,抗微生物肽的分泌,包囊作用以及吞噬作用,例如,血淋巴细胞形成了宿主的主要防御系统,血淋巴细胞的粘附和运动在吞噬外来异物和向炎症部位转移过程中发挥了重要作用。由于血淋巴细胞的粘附行为,造成诸如对寄生虫的包囊作用和血淋巴细胞介导的凝血反应等,这些反应可能是由于位于细胞表面的特异的受体分子诱导的,且这些受体接收和传递不同的生物反应信号。因此,鉴定影响软体动物免疫系统的因子如血淋巴细胞的表面受体分子将有助于我们搞清楚作为病原载体的软体动物的免疫系统,同时为理解寄生虫的传播开辟一条新途径。
本研究利用SMARTM技术构建了近江牡蛎(Crassostrea ariakensis)LPS刺激下血淋巴细胞的全长cDNA文库。经筛选和PCR鉴定后我们挑选了120个阳性克隆进行了测序分析,共获得了100个EST序列,其中包含了7个未报道的近江牡蛎血淋巴细胞的全长序列(四跨膜素tetraspanin, FJ475123;癌基因FosB, FJ594438;铁蛋白ferritin, FJ529405;烟酰胺腺嘌呤脱氢酶NADH, FJ495091;QM样蛋白QM-like, FJ467930;腺苷高半胱氨酸水解酶SAHH, FJ495090;泛素因子u-box, FJ495092)。通过NCBI匕对和生物信息学软件分析,这些基因分别与物质代谢、转录调控、呼吸代谢、蛋白降解以及天然免疫等作用相关。
四跨膜素(又称为四跨膜蛋白)是一种具有四段跨膜结构的大基因家族,该家族成员在哺乳动物、昆虫、蠕虫和真菌中都有发现。四跨膜素蛋白可以调控细胞的形状、运动、入侵、融合以及膜表面复合体的信号传导。第一种四跨膜素蛋白(ME491/CD63)在1988年进行了报道,而标志性的序列特征报道则出现在1990年。除了四段跨膜结构以外,四跨膜素还具有几个保守的氨基酸残基包括高度保守的CCG,而月.还有两个半胱氨酸残基,这对于形成四跨膜素的胞外环结构至关重要。在哺乳动物的免疫细胞(如T和B淋巴细胞,粒细胞,单核细胞,巨噬细胞,树突细胞和自然杀伤细胞)至少具有八种四跨膜素。这些四跨膜素直接间接通过大量的免疫分子发生作用,例如T细胞受体,B细胞受体,主要组织相容性复合体1类分子等等。
我们在牡蛎血淋巴细胞cDNA文库中鉴定了一种四跨膜素分子命名为Ca-TSP,并且发现在血淋巴细胞的转录表达中具有变异,发现三种不同的Ca-TSP分子。利用免疫荧光和免疫电镜检测我们发现Ca-TSP位于牡蛎血淋巴细胞的颗粒及囊泡状结构中。序列分析、结构预测以及免疫电镜检测显示Ca-TSP是一种位于颗粒上的整合的糖蛋白,可能是一种和CD63类似的蛋白。通过对分离的牡蛎的血淋巴细胞的基因表达分析,在LPS和PolyⅠ:C刺激的前12小时中Ca-TSP的表达显示上调,而H2O2刺激下Ca-TSP的表达显示降低。Western blot得出了类似的结果。实验同时发现：重组表达的Ca-TSP可以显著抑制血淋巴细胞的聚集,结果显示Ca-TSP可能参与牡蛎血淋巴细胞的天然免疫过程。为了寻求牡蛎四跨膜素的功能性解释,我们进行了蛋白质相互作用和信号传导途径方面的检测。免疫荧光实验结果显示Ca-TSP与血蓝蛋白共同位于牡蛎的血淋巴细胞中,蛋白质相互作用检测发现Ca-TSP和血蓝蛋白以及一种类凝集素蛋白形成复合体。未知蛋白检测出血细胞凝集特性,但它仍然可能是一种复合物,因此牡蛎的Ca-TSP的免疫应答可能有血蓝蛋白和这种类凝集素的参与。我们利用MEK的抑制剂来检测可能涉及的信号传导通路,当血细胞同U0126和PD98059两种抑制剂混合后检测,发现MAPK的磷酸化水平在1小时后是下降的,在12小时后升高了一点,而Ca-TSP的表达量是下调的,因此我们推测Ca-TSP可能是受MAPK诱导的,并且其中可能有其它的信号途径参与。
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.
引文
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