【作者】 高茜；

【导师】 张云；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 βγ-CAT是一个从中国特有的两栖动物大蹼铃蟾（Bombina maxima）皮肤分泌物中分离得到的,由非晶状体βγ-晶状体蛋白（α-亚基, CAT-α）和三叶因子蛋白（β-亚基, CAT-β）天然结合在一起形成的复合物,α-亚基与β-亚基通过非共价的方式交联形成αβ₂,分子量为72-kDa。CAT-α的N-末端部分（CAT-αN, 1-170氨基酸残基）是两个βγ-晶状体结构域,C-末端部分（CAT-αC）与产气荚膜梭菌ε毒素的膜插入结构域有序列同源性。为了分析βγ-CAT的βγ-晶状体蛋白结构域的生物化学特性,我们在大肠杆菌中分别表达了CAT-αN, CAT-αC和CAT-β。对天然纯化的βγ-CAT的免疫共沉淀实验表明CAT-α与CAT-β形成了一个紧密的复合物。Pull-down实验进一步证明重组表达的CAT-β能结合在重组表达的CAT-αN上。此外,我们还发现CAT-αΝ具有钙离子结合模体,存在于其β-折叠构象上。重组的CAT-αΝ可以结合钙离子探针铽离子,而且CAT-αΝ在结合钙离子后构象没有发生明显的改变。近年来,研究发现血小板也会发生凋亡,并且这个凋亡过程可能在血小板清除中起作用。βγ-CAT在哺乳动物中体内会引起很多毒理学作用,通过血液学分析发现βγ-CAT会引起兔子体内血小板数量发生显著的下降。因此,为了研究βγ-CAT对血小板的作用,我们将洗涤后的人血小板与不同浓度的βγ-CAT孵育30分钟,我们发现βγ-CAT使人血小板表现出了多种凋亡特征,如caspase-3的激活,磷脂酰丝氨酸外翻,线粒体膜电位去极化,细胞色素c的释放以及促凋亡蛋白Bax和Bak的表达上调。同时,通过血小板激活检测实验,如P-选择素在人血小板表面的表达,GPIIb/Ⅲa的激活和血小板聚集等,发现βγ-CAT在引起人血小板凋亡的同时并不引起血小板的激活。前期的研究表明,βγ-CAT可以在人红细胞上形成寡聚体并具有很强的溶血活性。我们通过实验进一步发现βγ-CAT （1 nM）可以引起红细胞内钙离子浓度急剧升高从而引起溶血。然而,在没有细胞外钙离子存在的情况下（反应体系中加入20 mM EGTA）,虽然βγ-CAT引起的溶血得到显著抑制,但是βγ-CAT仍然可以结合在红细胞膜上并寡聚化。另外,我们发现βγ-CAT在人血小板上引起的磷脂酰丝氨酸外翻和线粒体膜电位的下降也是钙离子依赖的。综上所述,我们的数据表明CAT-β（三叶因子蛋白）和CAT-αN（βγ-晶状体结构域）在体外可以相互结合,这为βγ-晶状体蛋白与三叶因子的蛋白在体外形成复合物形成了结构基础。此外,CAT-αN具有钙离子结合活性,而且βγ-CAT引起的溶血和血小板凋亡都是钙离子依赖的,这为进一步研究βγ-CAT生物学活性的作用机制提供了一个新的线索。更多还原

【Abstract】 βγ-CAT is a naturally existing 72-kDa complex of non-lensβγ-crystallin （α-subunit, CAT-α） and trefoil factor （β-subunit, CAT-β）, with a non-covalently linked form ofαβ₂, identified from frog Bombina maxima skin secretions. The N-terminal part （CAT-αN, 1-170 residues） of CAT-αare twoβγ-crystallin domains, while the rest C-terminal part （CAT-αC） shows sequence homology to membrane insertion domain of Clostridium perfringens epsilon toxin. To examine the biochemical characteristics of theβγ-crystallin domains ofβγ-CAT, CAT-αN, CAT-αC and CAT-βwere expressed in Escherichia coli, respectively. Co-immunoprecipitation of naturally purifiedβγ-CAT comfirmed the constant existence of CAT-αand CAT-βcomplex. Pull-down assays showed that recombinant CAT-βcould bind to recombinant CAT-αN that is composed of twoβγ-crystallin domains. Ca²⁺ -binding motifs were found in CAT-αΝthat folds mainly inβ-sheet conformation. Recombinant CAT-αΝwas able to bind the calcium probe terbium, and the conformation of the protein is not significantly altered upon binding Ca²⁺ .In recent years, it has been reported that apoptosis may occur in platelets and play a role in the clearance of effete platelets.βγ-CAT caused several in vivo toxic effects on mammals. Through determined hematological parameters of rabbits, it has been found thatβγ-CAT significantly reduced the number of platelets in a time-dependent manner. Here, in order to explore the effect ofβγ-CAT on platelet, washed platelets were incubated with various concentrations ofβγ-CAT for 30 min. We found thatβγ-CAT induced several apoptosis events in human platelets, including caspase-3 activation, phosphatidylserine （PS） exposure, depolarization of mitochondrial inner transmembrane potential （ΔΨm）, cytochrome c release and strong expression of pro-apoptotic Bax and Bak proteins. However,βγ-CAT did not significantly induce platelet activation as detected by P-selectin surface expression, GPIIb/IIIa activation and platelet aggregation.βγ-CAT protein possesses strong hemolytic activity on human erythrocytes. Treatment of the erythrocytes withβγ-CAT （1 nM） resulted in rapid Ca²⁺ influx in the cells and eventually led to hemolysis. However, in the absence of extracellular Ca²⁺ （in the presence of 20 mM EGTA）, though the binding and oligomerization ofβγ-CAT in erythrocyte membranes were observed, no significant hemolysis could be detected. In addition, we observed thatβγ-CAT-induced PS exposure andΔΨm depolarization in platelets are Ca²⁺ -dependent. Taken together, our data reveal the binding capacity of CAT-β（a trefoil factor） to CAT-αN （βγ-crystallin domains）, providing a basis for the formation ofβγ-crystallin and trefoil factor complex in vivo. Furthermore, theβγ-crystallin domains ofβγ-CAT are able to bind Ca²⁺ , andβγ-CAT-induced hemolysis and platelet apoptosis are Ca²⁺ -dependent.更多还原