Chang H H, Tsai W J, Lo S J
Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, R.O.C.
Toxicon. 1997 Feb;35(2):195-204. doi: 10.1016/s0041-0101(96)00121-3.
Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.
红口蝮蛇毒中的罗多斯汀（RHO）由68个氨基酸组成，含有精氨酸 - 甘氨酸 - 天冬氨酸（RGD）序列和12个半胱氨酸残基，是一种有效的血小板聚集抑制剂。我们之前证明，涂有细菌产生的谷胱甘肽S - 转移酶 - RHO融合蛋白[GST - RHO(RGD)]的细胞培养板可在15分钟内通过整合素相互作用促进人肝癌细胞附着。在本研究中，我们通过创建另外两种相关融合蛋白GST - RHO(RGE)和GST - (PS)RHO，进一步表征了RHO融合蛋白对血小板细胞的影响。前者是单个氨基酸取代的突变体，其中RGD的天冬氨酸残基被谷氨酸取代，后者是插入突变体，其中蛋白激酶A磷酸化位点的五肽插入GST和RHO之间。这两种突变蛋白与野生型GST - RHO(RGD)和天然形式的RHO一起用于研究对ADP诱导的血小板聚集的抑制作用。结果表明，GST - RHO(RGD)抑制血小板聚集的能力与天然RHO一样强，而另外两种突变体则无活性。此外，当未活化的血小板细胞附着在涂有GST - RHO(RGD)的板上时，它们会变成扁平的煎饼状。从融合蛋白包被板上细胞附着促进的结果来看，我们得出以下结论：(1) GST - RHO(RGD)融合蛋白在抑制血小板聚集和促进细胞附着方面具有同等功能，这是通过RGD与细胞膜上整合素的相互作用实现的；(2) GST - RHO(RGE)突变蛋白无法与整合素结合，导致功能丧失；(3) GST - (PS)RHO的插入突变体可能破坏RHO的正确构象，也导致功能丧失；(4) 细菌产生的融合蛋白GST - RHO(RGD)可适当地用作抗血栓形成剂和细胞外基质。
