黑腹果蠅蛋白質酪氨酸亞硫酸化酵素之鑑定與分析

Abstract

蛋白質酪氨酸的亞硫酸化為許多特定分泌性蛋白質或膜蛋白質很重要的後修飾。酪氨酸亞硫酸基轉移酶(tyrosylprotein sulfotransferase)負責的酪氨酸亞硫酸化，在細胞間的蛋白質與蛋白質交互作用，和許多重要的生物功能反應擔任關鍵的調節作用，其中包含人類免疫缺陷病毒(HIV)感染入侵、發炎反應、凝血機制、不孕...等等。然而這些生理與病理的機制都還是不清楚的。利用已經被解碼的染色體序列和生物資訊系統分析的協助，搜尋表達序列標記(EST)的資料庫，發現有一段基因可能負責黑腹果蠅體內酪氨酸亞硫酸化的反應。本研究將具有酵素活性的黑腹果蠅酪氨酸亞硫酸基轉移酶(DmTPST)表達在大腸桿菌BL21(DE3)pLysS寄主细胞，純化出高產率均質的酵素，並且探討其特性；利用polyEAY當作受質，定義出黑腹果蠅酪氨酸亞硫酸基轉移酶理想的最佳反應狀況。最後，藉由基因重組的酪氨酸亞硫酸基轉移酶催化了drosulfokinin的亞硫酸化，證明能夠進一步的解釋黑腹果蠅蛋白質酪氨酸的亞硫酸化未知的機制與功能。

Protein tyrosine sulfation, catalyzed by tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications towards secretory and transmembrane proteins. Protein tyrosine sulfation is a key modulator of extracellular protein-protein interactions and responsible for various important biological functions including HIV entry, inflammation, coagulation, and sterility. These physiological and pathological mechanisms, however, are not clear. With the assistances of decoded genome sequences and bioinformatic analysis, a promising gene competent for catalysis of protein tyrosine sulfation in Drosophila melanogaster was discovered by searching the expressed sequence tag (EST) database. Enzymatically active Drosophila melanogaster TPST (DmTPST) was first cloned, expressed in Escherichia coli BL21(DE3)pLysS host cells and purified to homogeneity in high yield. The homogeneous DmTPST was characterized through radioactive assay with polyEAY as substrate and its optimal reaction conditions were determined. Finally, the drosulfokinin sulfation catalyzed by recombinant DmTPST was firstly demonstrated, which provided direct link to tyrosine sulfation in Drosophila melanogaster and further opportunity to decipher the obscure mechanisms and functions of protein sulfation.