大蹼铃蟾(Bombina maxima)皮肤新型血小板活性蛋白及信号转导的研究
文献类型：学位论文
| 作者 | 张杰 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 三叶因子蛋白 血小板活化 G一蛋白偶联受体 酪氨酸磷酸化 整合素Ilbp3 信号传导 磷脂酶C 胞内钙离子释放 膜联蛋白II |
| 其他题名 | Studies on Platelet actication Related Proteins from Amphibian Bombina Maxima Skin |
| 中文摘要 | 通过对两栖类动物大蹼铃蟾（B朋b了na勿日对朋）皮肤分泌物及匀浆物的分离，纯化，从其碱性组分中得到了全新的三叶因子多肤（BIn－－TFFZ）和膜联蛋白H相关蛋白（BAIIRP），它们分别具有诱导或抑制人血小板活化的全新功能。以血小板膜糖蛋白GPVI受体激动剂stejnulxin为对照，研究了Bm一TFFZ活化人血小板的信号传导途径。哺乳动物的三叶因子（TFF）蛋白的主要功能在于通过修复粘膜损伤，维持粘膜层的完整。它们在肿瘤抑制及癌症侵润方面也有一定程度的作用。我们从两栖类动物大蹼铃蟾皮肤分泌物分离，纯化而得到了一个全新的三叶因子多肤（Bm一TFFZ）。它是单亚基蛋白，表观分子量为13000，并具有全新的诱导人血小板活化的功能，我们研究了其诱导人血小板活化的活性的量效关系。它对血小板的激活涉及血小板整合素a：Ibp3的活化，Bm一TFFZ可诱导经阿斯匹林及三磷酸腺昔"双磷酸酶（Apyrase）处理的血小板的聚集，表明B，TFFZ的活性不依赖于血小板的ADP及血栓嗯烷A2（TXAZ）的自泌正反馈。F工TC标记的B爪一TFFZ蛋白能与血小板膜结合。通过专一性药理学抑制剂的研究，我们初步排除了Bm一TFFZ蛋白作用于血小板膜已知的G一蛋白偶联受体（如ADP，TXAZ及血小板活化因子受体）的可能性。以血小板GPVI激动剂stejnulxin为对照，我们仔细研究了Bm一TFFZ蛋白刺激血小板后的信号传导分子事件，发现其酪氨酸磷酸化图谱明显不同于血小板GPVI激动剂5tejnu1X如，尤其是磷脂酶CYZ并不磷酸化。因此，我们认为Bm一TFFZ蛋白激活血小板的信号传导通路是活化磷脂酶C，并刺激胞内钙离子的释放，活化血小板伪：。p3，进而导致血小板的聚集。从Bm－－TFFZ蛋白的cDNA克隆序列推知它由104个氨基酸组成，含有两个三叶因子结构域，与人TFFZ和非洲爪蟾xPZ序列中相同的氨基酸分别占28％及34％。为了确定两栖类的三叶因子蛋白具有诱导人血小板活化的功能，我们仔细地研究了整个分离纯化过程中所有组份的血小板聚集活性并比较了它们的相对活性差异。Bm一TFFZ是第一个来自两栖类动物的血小板激动剂，也是我们第一个报道的具有诱导血小板聚集活性的三叶因子多肤。·我们还通过阴离子交换，凝胶过滤和阳离子交换层析，从大蹼铃蟾皮肤匀浆物中纯化了一个表观分子量为33kD。的单链蛋白。N一末端序列比较分析显示，该·蛋白与来自非洲爪蟾、红色原鸡和人膜联蛋白H的N一末端序列相同的氨基酸分别占70％、64％和56％。该蛋白具有以钙依赖的方式抑制专一性血小板膜糖蛋白VI（GPVI）受体激动剂-Stejnu1Xin诱导洗涤人血小板聚集的生物学功能，最大抑制率达48％。结合其N一末端序列搜索结果及其活性的钙依赖性，推测该蛋白是与膜联蛋白H相关的一类蛋白质。 |
| 英文摘要 | From the Bombina maxima skin we have purified and characterized a novel trefoil factor protein (Bm-TFF2) and an annexin II related protein (BAIIRP). Bm-TFF2 induces platelet activation while BAIIRP plays an inhibition role. We also studied the difference of signal transduction pathway between Bm-TFF2 and stejnulxin a platelet membrabe glycoprotein GPVI agonist from Trimeresurus stejnegeri venomIn mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel TFF protein, named bomaggin, has been identified and purified from the skin secretions of frog Bombina maxima. It is a single chain protein with an apparent molecular weight of 13,000. It activated human platelets in a dose-dependent manner and activation of integrin anbP3 was involved. Aspirin and apyrase did not largely reduced platelet response to bomaggin (a 30% inhibition), suggesting the aggregation is not substantially dependent on ADP and thrornboxane A2 (TXA2) autocrine feedback. FITC-labeled bomaggin bound to platelet membranes. Selective pharmacological antagonist studies revealed that bomaggin activates platelets not via well-known G-protein coupled receptors, like those of ADP, TXA2 and platelet-activating factor. A tyrosine phosphorylation profile induced by bomaggin in platelets was different from that produced by stejnulxin, a glycoprotein VI agonist, and particularly phospholipase Cy2 (PLCV2) was not phosphorylated. Activation of PLC, then an elevation of cytosolic free Ca2+ and activation of protein kinase C are major molecular events involved in bomaggin induced platelet activation. Containing two TFF domains, bomaggin is composed of 104 amino acids as deduced from its coding cDNA, and its sequence exhibits 28% and 34% sequence identity with human TFF2 and Africa claw toad xP2 respectively. Bm-TFF2 is the first platelet agonist containing two trefoil factor domains from Amphibian.A single chain protein with an apparent molecular mass of 33 kDa was purified from skin of Bombina maxima by a combination of ion exchange and gel filtration chromatography steps. N-terminal amino acid sequence determination indicated that it shares 70%, 64% and 56% identity with those of annexin II from Africa claw toad, red junglefowl and human, respectively. The purified protein from Bombina maxina inhibits stejnulxin (a specific platelet agonist via platelet memdrane glycoproteinVI (GPVI) induced platelet aggregation in a Ca2+ dependent manner. Maximal inhibition rate reaches 48%. Based on the N-terminal amino acid sequence BLAST search results and its Ca2+ dependent activity, the purified protein might be structurally and functionally related to the annexin protein family. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6179]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 张杰. 大蹼铃蟾(Bombina maxima)皮肤新型血小板活性蛋白及信号转导的研究[D]. 北京. 中国科学院研究生院. 2005. |
入库方式： OAI收割
来源：昆明动物研究所
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