蛋白質酪氨酸亞硫酸化誘導PSGL-1與腸病毒71型VP1外鞘蛋白的結合作用

Abstract

酪氨酸亞硫酸化為蛋白質中常見的一種後轉譯修飾，並且在蛋白質的交互作用中扮演著關鍵性的角色，例如硫基化後的人類P-selectin glycoprotein ligand-1(PSGL-1)與enterovirus 71 (EV71)的外鞘蛋白VP1結合後會促進EV71的病毒進入細胞中。PSGL-1為免疫細胞上一個關鍵性的分子，它通常參與著初期的發炎反應並藉由後轉譯修飾的酪氨酸亞硫酸化來調節PSGL-1與P-selectin的交互作用。PSGL-1在N端位置的酪氨酸亞硫酸化也與神經細胞中腸病毒71型(EV71)的感染有關。我們實驗室的研究興趣為探討蛋白質中酪氨酸亞硫酸化如何引起蛋白質與蛋白質之間的交互作用進而促使病毒入侵。在本篇論文中，我們利用PSGL-1作為酪氨酸亞硫酸化時的受質蛋白，而EV71的外殼蛋白VP1則作為與亞硫酸化蛋白交互作用時的蛋白來驗證亞硫酸化對於蛋白質的交互作用的影響。利用我們實驗室之前所建立的酪氨酸亞硫酸化系統，PSGL-1的酪氨酸亞硫酸化可利用anti-sulfotyrosine抗體藉由酵素連結免疫吸附分析法(ELISA)及表面電漿共振儀(SPR)兩種方法證實外，我們實驗室更進一步利用ELISA設計一系列實驗，anti-GST抗體對於在相同GST及GST-fusion protein固定量的情況下的表現是一致的，並且證實VP1不會與其他蛋白，例如GST或GST-PSGL-1產生非特異性的鍵結。最後根據實驗的成果，我們證實亞硫酸化後的GST-PSGL-1比未亞硫酸化的GST-PSGL-1與VP1的交互作用高出10倍之多。我們的結果提供了在未來研究PSGL-1與VP1交互作用的重要的線索，也許最終能引導我們真正了解腸病毒感染的機制。

Tyrosine O-sulfation is a common post-translational modification of proteins and plays a crucial role in regulating the protein-protein interactions such as human P-selectin glycoprotein ligand-1(PSGL-1) and VP1 of enterovirus 71 (EV71) that enable viral infection. PSGL-1 on the immune cells is a key molecule which involves in early inflammatory events and its interactions with P-selectin are regulated through post-translational sulfation. Tyrosine sulfation at the N-terminal region of PSGL-1 is found to be critical for Enterovirus 71 (EV71) infection into neural cells. We are interested in how protein tyrosine sulfation induces protein-protein interactions that facilitates viral invasion. In this study, we use PSGL-1 as target for protein sulfation, and EV71 capsid protein VP1 as the binding protein to examine effect of sulfation on protein-protein interactions. Using sulfation system previously constructed in our lab, the sulfation of PSGL-1 was confirmed using anti-sulfotyrosine antibody by ELISA and surface plasmon resonance (SPR). We also designed a series experiment of ELISA to prove that anti-GST antibody appeared consistent to GST and GST-fusion protein when coating in the same amount. We further showed that there was no non-specificity binding between VP1 and other proteins, ex. GST or GST-PSGL-1. In conclusion, we have demonstrated that protein-protein interaction between VP1 and GST-PSGL-1-SO4 was ten-fold stronger than that between VP1 and GST-PSGL-1. Our results provide important clues for the further study on the interaction between PSGL-1 and VP1, which may eventually lead to understanding of the infection mechanism of enterovirus.