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由馬來亞蝮蛇 Calloselasma rhodostoma 毒腺中純化出之disintegrin蛇毒蛋白rhodostomin是一個非常有效的血小板凝集抑制劑，由68個胺基酸所組成，其中包含12個cysteine，而其第49至51的胺基酸序列為Arginine-Glycine-Aspartic acid (RGD)。基因工程生產之蛇毒蛋白GST-rhodostomin可經由與細胞表面分子結合作用，促進細胞的黏貼，而各種不同之GST-rhodostomin與細胞的黏貼效果也各有不同，說明了蛋白質三度空間的構形與其具有之生物活性息息相關。若將基因工程生產之蛇毒蛋白以生物素(Biotin)標定後，蛇毒蛋白依然保有其生物活性，因此可當作探針使用於細胞與介質間作用的研究上。人類血小板模式一直是出血性蛇毒研究中的好材料，本論文以此模式建立了一系列的實驗，在GST攫取實驗中，得到了兩個主要膜蛋白，分子量為130 kDa及110 kDa，還有另五個次要膜蛋白，其分子量介於50至80 kDa。進一步以抗αⅡb integrin和抗β3integrin抗體進行西方墨點實驗，及以抗β3 integrin和抗α6integrin抗體進行免疫沉澱實驗，證實110 kDa及130 kDa之血小板膜蛋白是β3及αⅡb integrin，即血小板上與GST-rhodostomin結合作用的部位是αｖⅡb β3 integrin，而非他種integrin。
B16-F1及B16-F10是由小鼠黑色素腫瘤所篩選出之兩細胞株，B16-F10細胞的轉移能力(Metastasis)較B16-F1強，而且吸附在覆有GST-rhodostomin之玻片上的效果也較B16-F1細胞好。利用GST攫取實驗比較這兩株細胞的表面蛋白，B16-F10細胞上有一130kDa的表面蛋白與rhodostomin是專一性的結合，而這是B16-F1細胞上所沒有的。至於此130 kDa之膜蛋白是integrin或是其它蛋白？與腫瘤細胞之轉移擴散能力是否有關？還需要更進一步的實驗證實。
Rhodostomin, a disintegrin from Calloselasma rhodostoma venom, is a potent inhibitor of platelet aggregation. It contains 68 amino acids which including 12 residues of cysteine and an RGD (Arginine-Glycine-Aspartic acid) sequence at positions of 49-51. The recombinant fusion proteins of GST-rhodostomin could facilitate cell attachment via integrins on cell membrane. The biotinylated recombinant rhodostomin maintained its biological functions and could be used as a probe for cell-matrix interaction studies. In order to characterize the cell surface proteins which can interact with recombinant rhodostomin, here we used GST pulldown assay, immunoprecipitation, and Western blotting to address this question by using a human platelet system. From the GST pulldown assay, two surface proteins, about 110 kDa and 130 kDa, on human plateletmembrane were specifically bound to the recombinant GST-rhodostomin, in addition to 5 proteins ranging 50-80 kDa.Immunoprecipitation and Western blotting confirmed that the 110 kDa and 130 kDa proteins were β3 and αⅡb integrin, respectively. Taken together, the results demonstrated that the rhodostomin-binding protein on human platelets was αⅡb β3 integrin, but not α6 β1 integrin.
B16-F1 and B16-F10 cell lines from mouse melanoma were selected by repeated colonization. B16-F10 melanoma cells have not only a higher metastatic rate than B16-F1, but also have a higher ability to adhere on the recombinant rhodostomin -coated plates. The GST-rhodostomin pulldown assay identified a 130 kDa surface protein on B16-F10, but not B16-F1, specifically interacted with rhodostomin. It might be an integrin subunit or integrin-associated protein on cell surface, the nature of which requires further investigation.
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