【作者】 徐存拴； 辛泽华； RAHMAN Salman；

【Author】 XU Cun-shuan, Xin Zhe-hua, RAHMAN Salman(College of Life Sciences, Henan Normal University, XinXiang 453002, China;Key Laboratory of Henan Province for Bioengineering, XinXiang 453002, China;Hemophilia Research Center, University of London, London SEl 7EH, United Kingdom)

【机构】 河南师范大学生命科学学院； 河南省生物工程重点实验室； 英国皇家医学院血友病研究中心；

【摘要】 解联蛋白（disintegrin）是一类存在于蛇毒中的血小板凝集抑制因子。研究表明,Arg-GLT-Asp（RGD）序列与解联蛋白的生物学活性有密切关系,紧靠RGD序列两翼的氨基酸残基有调节解联蛋白抑制ADP-诱导血小板凝集作用,RGD序列侧翼的第45位氨基酸残基位于该类蛋白内部形成的小槽内,这个小槽在RGD袢形成发夹结构方面起重要作用。在此基础上,本文进一步分析了RGD袢上游区域氨基酸残基在调节解联蛋白功能中的作用。具体方法是:用基因点突变技术将华丽蛇毒素的K⁴¹K⁴²K⁴³R⁴⁴T⁴⁵I⁴⁶突变成腹蛇毒素的S⁴¹K⁴²A⁴³G⁴⁴T⁴⁵I⁴⁶和S⁴¹K⁴²A⁴³G⁴⁴I⁴⁶,然后分析突变蛋白的结构、性质和功能变化。结果表明,前一种突变K⁴¹K⁴²K⁴³R⁴⁴T⁴⁵I⁴⁶→S⁴¹K⁴²A⁴³G⁴⁴T⁴⁵I⁴⁶显著地降低了华丽蛇毒素对血小板凝集的抑制活性,后一种突变S⁴¹K⁴²A⁴³G⁴⁴I⁴⁵T⁴⁶→S⁴¹K⁴²A⁴³G⁴⁴I⁴⁶有效地恢复了华丽蛇毒素抑制血小板凝集的作用。同时,这两种突变也改变了含点突变基因的pGEX-3X质粒在受体细胞（E.coli）内的表达,例如,重组于pGEX-3X质粒的野生型腹蛇毒基因和华丽蛇毒素基因以可溶性形式表达,而重组于相同载体的点突变基因表达的目的蛋白是不溶性的,为此,本文又通过构建表达难溶蛋白载体的方法获得了突变基因的可溶性表达产物。本文首次证实了RGD上游的39.45位氨基酸残基与解联蛋白的结构和功能有密切关系。更多还原

【Abstract】 Disintegrins are a family of platelet aggregation inhibitors present in the venoms of the viper idea. It is known that the disintegrins exert their biological activity by a common mechanism of action involving in the Arg-Gly-Asp (RGD) sequence. A molecular model for the disintegrin elegantin inferred that Thr45 amino acid residue flanking the RGD sequence in most disintegrins creates a cavity within the protein core, which maybe is important on regulating the hairpin RGD loop structure. In the previous reports was demonstrated that the amino acid residues close flanking the RGD sequence were important for action of disintegrins in regulating their binding with integrin α11bβ3. to inhibit the ADP-induced platelet aggregation. In the present work, we have analyzed the role of the amino acid residues in the domain preceding the RGD loop were important for this action of disintegrins. Our approach was to develop hybrids between the two disintegrins kistrin and elegantin targeting residues in this domain and within the RGD loop. The basic sequence within elegantin K41K42K43R44T45I46 was changed by mutagenesis to S41K42A43C44T45I46 and to S41K42A43G44I46, thereby resembling the corresponding S39RAGT43 sequence in kistrin. This change K41K42K43R44T45I46→S41K42A43G44T45I46 dramatically reduced the activity of elegatin as an inhibitor of platelet aggregation. In contrast, delation change of T45 (S41K42A43G44T45I46→S41K42A43G44I46) restored activity of elegantin as an inhibitor of platelet aggregation. Eeanwhile these mutations also altered their expression capability. For example, the GST-kistrin and GST-elegantin in pGEX-3X could be easy and soluble expressed, whereas their variants were difficult to express and insoluble. Therefore, new recombinant vectors, which can express the difficult expressed proteins, were reconstructed. These data highlight the importance of the domain encompassing residues 39-45 on disintegrin structure-function.更多还原