大蹼铃蟾（Bombina maxima）皮肤分泌物中非晶状体βγ-晶状体蛋白与三叶因子复合物的结构和功能研究
文献类型：学位论文
| 作者 | 刘树柏 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-07 |
| 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 | 非晶状体betagamma-晶状体蛋白与三叶因子蛋白复合物 非晶状体betagamma－晶状体 三叶因子 孔道形成蛋白 溶血 寡聚体 ATP/GTP酶 |
| 其他题名 | The structure and function research of non-lens betagamma-crystallin and trefoil factor complex from Bombina maxima skin secretion |
| 学位专业 | 动物学 |
| 中文摘要 | 在脊椎动物中，非晶状体betagamma晶状体蛋白广泛的在各种组织中表达，然而对于其功能和详细的分子作用机制知之甚少！关于三叶因子作为胃黏膜修复启动者，关于其在肿瘤发生，创伤修复的详细的分子机制目前还很不清楚。我们从大蹼铃蟾皮肤分泌中分离纯化到的一类全新的大分子蛋白质复合物，由非晶状体betagamma-晶状体蛋白和三叶因子通过非共价键连接而成，分子形式为ab2，命名为非晶状体betagamma晶状体蛋白与三叶因子蛋白复合物 (non-lens betagamma-crystallin and trefoil factor complex，betagamma-CAT)，通过生物化学和分子生物学研究手段，分别获得了两个亚基的cDNA全克隆，结合肽指纹图谱，两个亚基N端和内肽序列信息，确定了两个亚基的蛋白质序列。表明betagamma-CAT的alpha beta两个亚基分别属于非晶状体betagamma-晶状体蛋白家族和三叶因子家族，其中alpha亚基与人源的黑色素瘤缺失蛋白（AIM1）和日本蝾螈表皮分化蛋白EP37家族具有很高的序列相似性；beta亚基与人源的三叶因子2/3（hTFF2/TFF3）具有很高的相似度，beta亚基含有三个三叶因子结构域，根据文献检索和序列比对，在高等哺乳动物中，目前还没有含有三个三叶因子结构域的三叶因子的报道。因此，betagamma-CAT作为一类全新的非晶状体betagamma-晶状体蛋白和三叶因子以非共价键连接而成的天然形式存在的蛋白分子，根据现有文献资料，在世界还是首次！ 分别采用anti-betagamma-CAT-alpha和anti-betagamma-CAT-beta的兔源多克隆抗体来检测betagamma-CAT的组织表达分布, 在大蹼铃蟾的皮肤，小肠，胃和心检测到阳性信号，而阴性对照无明显荧光信号。说明在大蹼铃蟾的皮肤, 小肠，胃和心等部位存在betagamma-CAT-alpha和betagamma-CAT-beta或者是其类似物的表达。 betagamma-CAT 对哺乳动物红细胞表现出很高的溶血活性。我们选取人红细胞作为研究模型，对betagamma-CAT溶血作用机制进行了系统研究。结果表明，betagamma-CAT 能够在人红细胞膜上形成孔道直径为1.6nm的孔道，从而导致细胞内钾离子迅速外流而导致溶血。betagamma-CAT不与细胞膜上的脂类结合（如胆固醇，卵磷脂等）。通过Westernblot分析，用兔源anti-betagamma-CAT-alpha抗体在人红细胞膜上检测到分子量大于240 kDa的大分子寡聚体。 通过对betagamma-CAT的alpha亚基的序列分析，表明betagamma-CAT的alpha亚基包含有典型的GTP/ATP酶的保守模体Walker A (IILYDEPS, residues 6-13)和Walker B (GQSLSGKS, residues 96-103), 同时在体外实验中检测到较弱的GTPase/ATPase 酶活性和ATP/GTP类似物结合活性，提示betagamma-CAT的alpha亚基类似的非晶状体betagamma-晶状体蛋白家族可能属于一类新的GTPase/ATPase 酶。因此，对betagamma-CAT分子作用机制和功能的研究很有可能为理解三叶因子和非晶状体betagamma-晶状体蛋白两个家族的功能和作用机制提供了一个全新的视角。 |
| 英文摘要 | In vertebrates, non-lens betagamma-crystallins are widely expressed in various tissues, however, little is known about the functions and action mechanisms of these proteins. The first-hand actions and molecular mechanisms involved of trefoil factors, initiators of mucosal healing and being greatly involved in tumor genesis, have remained elusive. Here we purified and cloned a naturally existing highly active of non-lens betagamma-crystallin and trefoil factor protein complex in frog Bombina maxima skin secretions. The protein is composed of two types of subunits connected non-covalently with a molecular structure of ab2. Surprisingly, sequence determination of the protein established that its alpha-subunit is homologous with ep37 proteins and mammalian absent in melanoma 1 gene family products. The mature beta-subunit is highly homologous to human trefoil factors. For the first time, a naturally existing complex of non-lens betagamma-crystallin and trefoil factor was identified in the world. With rabbit anti-betagamma-CAT alpha and anti-betagamma-CAT beta subunits antibodies, distribution of betagamma-CAT in frog skin were observed around the epidermis and granular glands imbedded in the dermis. In addition, immuno-reactivity was also detected in the heart, stomach and intestine of the frog, indicating either betagamma-CAT alpha-subunit and beta-subunit and/or its homologues are expressed in these tissues. betagamma-CAT show high activity of hemolysis to mammalian erythrocytes. We choose human erythrocyte as a research model to investigate the betagamma-CAT hemolysis mechanism. betagamma-CAT can formed a pore with diameter of 1.6nm on the erythrocyte membrane and caused intracellular potassium fastly efflux and hemoysis, without binding to the known lipids (such as PE, PI, PA, Cer, PS, Sph, S1P, PC, Cereb, PC，cholesterol, etc al). High molecular weight oligomers (over 240 kDa) were detected with anti-betagamma-CAT-alpha on the erythrocyte membrane by Westernblot analysis. A detail analysis of the alpha-subunit sequence of the protein indicated that, in fact, within its two betagamma-crystallin domains, there are two conserved regions (IILYDEPS, residues 6-13) and (GQSLSGKS, residues 96-103) that correspond to the Walker B and Walker A motifs, which are responsible for the formation of nucleotide binding site and can be commonly found in a number of ATP- and GTP-binding and hydrolyzing proteins. A weak but significant GTP hydrolyzing activity was also determined in vitro. The results imply that non-lens betagamma-crystallins might be a novel class of NTP-binding and hydrolyzing enzymes. The related research of betagamma-CAT molecular action mechanism will provide a new view to understand the two superfamilies protein in mammalians. |
| 语种 | 中文 |
| 公开日期 | 2010-11-12 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6453]  |
| 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 | 刘树柏. 大蹼铃蟾（Bombina maxima）皮肤分泌物中非晶状体βγ-晶状体蛋白与三叶因子复合物的结构和功能研究[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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