系統性探討蛋白質酪氨酸硫化作用-從生化觀點至蛋白質交互作用之研究

Abstract

酪氨酸亞硫酸基轉移酶 (Tyrosylprotein sulfotransferase, TPST, EC 2.8.2.20) 所催化的蛋白質酪氨酸亞硫酸化是作用於分泌蛋白和膜蛋白的一種常見轉譯後修飾。蛋白質酪氨酸亞硫酸化作用也是細胞外蛋白質與蛋白質間產生交互作用的關鍵調節劑，目前認為多種重要的生物活性與它有密切的相關，調節主要的生物運作，如病毒入侵，發炎反應，凝血反應與不孕症。在本研究中，我們有系統地檢測蛋白質酪氨酸亞硫酸化反應，由生化階段的酵素活性研究到其所誘導的蛋白質與蛋白質間交互作用的關係。酪氨酸亞硫酸基轉移酶的製備和特性對於理解酪氨酸亞硫酸化蛋白質的合成與生物中蛋白質酪氨酸亞硫酸化作用的研究是非常重要的。因此，我們將使用酶偶聯的亞硫酸化系統來製備亞硫酸化蛋白質，並且使用原子力顯微鏡來測量蛋白質酪氨酸亞硫酸化所誘導的蛋白質與蛋白質間交互作用關係。我們的結果顯示，重組的酪氨酸亞硫酸基轉移酶、基質和交互作用物可以容易的使用原核表達系統大量製備。亞硫酸化蛋白質的製備也透過同位素測定法，螢光測定法和免疫化學測定法進行交叉確認。相對地，使用3’ 磷酸腺甘酸5’ 磷酸硫酸合成酶 (PAPSS) 與酪氨酸亞硫酸基轉移酶的偶聯系統與果蠅酪氨酸亞硫酸基轉移酶 (Drosophila melanogaster Tyrosylprotein sulfotransferase, DmTPST) 的酵素，製備亞硫酸化蛋白質特別有效。原子力顯微鏡將進一步用於測量亞硫酸化的P-選擇素糖蛋白配體-1 (PSGL-1) 和腸病毒71型 (EV71) 的VP1蛋白質之間的交互作用。我們發現蛋白質酪氨酸亞硫酸化作用處理後的P-選擇素糖蛋白配體-1與特異性的酪胺酸亞硫酸化抗體的結合強度增加6.7倍。若是將特異性的酪胺酸亞硫酸化抗體替換為VP1蛋白質時也獲得相似的結果。我們發現，GST融合的P-選擇素糖蛋白配體-1的酪氨酸-51的殘基，是主要負責蛋白質酪氨酸亞硫酸化所誘導的蛋白質與蛋白質間交互作用的位置。本研究提供許多的生化工具和方法，且發掘蛋白質酪氨酸亞硫酸化的重要性，這將有利於解釋其機制、功能與未來更多的應用。

Protein tyrosine sulfation, catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20), is one of the most common post-translational modifications of secretory and transmembrane proteins. Protein tyrosine sulfation, a key modulator of extracellular protein–protein interactions, accounts for various important biological activities and regulates principal biological processes, namely virus entry, inflammation, coagulation, and sterility. In this study, we systematically examined the protein tyrosine sulfation activity of TPST from its biochemical level to its induced protein–protein interaction study. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying protein tyrosine sulfation in biology. Therefore, the sulfated protein was prepared using a TPST-coupled protein sulfation system and the atomic force microscopy (AFM) was used to measure the protein tyrosine sulfation induced protein–protein interactions. Our results revealed that recombinant TPSTs, substrates, and interactors was particularly easy to prepare in large quantities by the prokaryote expression system. The preparation of sulfated proteins was cross-confirmed through radiometric, fluorimetric and immunochemical assays. Relatively, DmTPST, through the use of PAPS synthetase (PAPSS)- TPST coupled enzyme system, was particularly useful due to its catalytic efficiency for preparation of sulfated proteins. Atomic force microscopy was further used to measure the interaction between sulfated or mutated P-selectin glycoprotein ligand-1 (PSGL-1) and VP1 protein of enterovirus 71 (EV71). We found that the binding strength increased by 6.7-fold following protein tyrosine sulfation treatment on PSGL-1 with a specific anti-sulfotyrosine antibody. Similar results were obtained when the anti-sulfotyrosine antibody was replaced with the VP1 protein of EV71. We also found that protein tyrosine sulfation on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the protein tyrosine sulfation induced protein–protein interaction. The research provides feasible biochemical tools and pioneering approaches to uncover the significance of protein tyrosine sulfation that is conducive to deciphering the mechanisms, functions, and future applications.