以原態凝膠電泳系統分析鑑定亞硫酸化蛋白質

Abstract

人類的P-selectin glycoprotein ligand-1 (PSGL-1) 是一種常見於白血球和內皮細胞上約43 kDa醣蛋白，此醣蛋白需透過兩種重要的轉譯後修飾作用，再與細胞黏附分子Selectins產生交互作用。此兩種轉譯後修飾作用，其一為酪胺酸亞硫酸化作用，以酪胺酸亞硫酸基轉移酶 (Tyrosylprotein sulfotransferase, EC 2.8.2.20) 將亞硫酸基轉移至PSGL-1上N端的Tyr-46、Tyr-48、Tyr-51等三個潛在位置；其二為醣化作用，將Sialyl Lewis x tetrasaccharide (sLex)於Thr-57位置上形成O-Linked glycans。根據先前的研究，這類型的亞硫酸化蛋白質不僅參與調節發炎和凝血反應的生理過程，並扮演著調控與腸病毒71型 (EV71) 外殼蛋白VP1間的蛋白交互作用，影響病毒入侵的關鍵指標。然而，目前對於人體中存在亞硫酸化蛋白質的亞硫酸化程度和其穩定性功能仍然有許多疑問，肇因於亞硫酸化蛋白質和非亞硫酸化蛋白質的來源取得和純化分離困難。應用本實驗室已開發的PAPSS-TPST和PST-TPST亞硫酸化酵素連鎖反應平台系統，此論文將從傳統生化方式的角度，以原態凝膠電泳系統分析鑑定亞硫酸化蛋白質GST PSGL-1，並分別由GST PSGL-1 Y51K 和GST PSGL-1 Y51E單一胺基酸正負電荷的差異做為對照組，以辨識亞硫酸化和非亞硫酸化的GST PSGL-1蛋白質。綜合而言，本論文所描述的純化方法、亞硫酸化酵素連鎖反應法和凝膠電泳系統檢測方式提供探究亞硫酸化轉譯後修飾研究的基礎。

Human P-selectin glycoprotein ligand-1 (PSGL-1) is a 43 kDa glycoprotein and common found on leukocytes and endothelial cells. PSGL-1 needs two important post-translational modifications to regulate the binding affinity with cell adhesion molecules - Selectins. One is Tyrosine O-sulfation through tyrosylprotein sulfotransferase (TPST, EC 2.8.2.20) transferred sulfuryl groups to Tyr-46、Tyr-48、Tyr-51 three potential positions of PSGL-1 N-terminal; the other one is Glycosylation that added Sialyl Lewis x tetrasaccharide (sLex) to O-linked glycans of PSGL-1 Thr-57 position. According to previous studies, such tyrosine-sulfated proteins participate in many physiological processes of inflammatory and coagulation response and has been identified as a key marker to mediate protein-protein interaction with Enterovirus (EV71) capsid protein VP1 invasion. However, there are still many questions about the stability and sulfation degree of sulfated proteins, due to the difficulty of sulfated proteins purification. Combination of PAPSS-TPST and PST-TPST sulfation coupled enzyme assay that has been developed in our laboratory by traditional biochemical methods, i.e. The native gel electrophoresis system could quickly be to analyze and identify the sulfated GST PSGL-1, and the GST PSGL-1 Y51K and GST PSGL-1 Y51E with different Tyr-51 site residue’s charges as a control. Overall, the purification methods, to develope sulfation coupled enzyme assay and gel electrophoresis system, the post-translational modification research were described in detail in this thesis.