藉由P -選擇素糖蛋白配體亞硫酸化探討酪胺酸亞硫酸機轉移酶之受質選擇機制

Abstract

蛋白質酪胺酸亞硫酸化是屬於蛋白質後修飾的其中一種，主要發現在分泌型蛋白質或穿膜蛋白質，調控許多蛋白質與其他蛋白質的交互作用。蛋白質亞硫酸後修飾影響許多生理反應，例如凝血反應、白血球黏附、病毒入侵和分子傳遞路徑。最著名的例子是P -選擇素糖蛋白配體Ｎ端的三個酪胺酸，此位置之亞硫酸化能增加P -選擇素糖蛋白配體與P -選擇素或特定腸病毒的結合能力。本研究使用P -選擇素糖蛋白配體作受質，探討酵素酪胺酸亞硫酸基轉移酶如何辨認三個潛在酪胺酸。但是發現與先前文獻不同，惟有兩個酪胺酸會被催化，且亞硫酸化的程度也有差異。除此之外，我們也發現第一個酪胺酸的亞硫酸化影響第二個酪胺酸的催化。其中我們也探討周圍的酸性胺基酸影響酪胺酸的催化與否。雖然周圍之酸性胺基酸有助於酪胺酸亞硫酸化，緊靠的兩個穀胺酸則會抑制該酪胺酸的亞硫酸化。天門冬胺酸與穀胺酸兩者酸性胺基酸皆有助於酪胺酸亞硫酸化，然而卻分別帶有迥然不同的效果。我們也發現酪胺酸在短胜肽的所在位置影響著酪胺酸亞硫酸化，靠近胜肽C端的酪胺酸提高被催化之可能性。利用此研究所得到的資訊，我們能夠建立蛋白質酪胺酸亞硫酸化的催化模型，解釋酵素如何選擇受質上的酪胺酸，此外可以改進蛋白質後修飾資料庫，使日後對於蛋白質相關的研究更精準更有利。

Protein tyrosine sulfation (PTS) is a common post-translational modification frequently found in secreted and transmembrane proteins and a key modulator for extracellular protein-protein interactions. PTS mediates many physiological processes, including coagulation, leukocyte adhesion, virus infection, and chemokine signaling. One of the prominent examples is the sulfation of tyrosine residue on the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1), which enforces its interaction with P-selectin and specific enterovirus. Using PSGL-1 as a model substrate, we investigate how tyrosylprotein sulfotransferase (TPST) recognizes one of the three potential tyrosine sulfation sites in the protein. On the contrary to what generally proposed in the literature, we determined that there are one major and one minor sulfation sites among the three tyrosines. In addition, we found that the second tyrosine is sulfated only after the first PTS near the C-terminal was completed. The selection of the tyrosine for sulfation is variously regulated by the two surrounding acidic amino acids, aspartic and glutamic acids. While surrounding acidic amino acids are critical for the tyrosine sulfation, two near-by glutamic acids prohibit one of the three PSGL-1 tyrosine for sulfation. In addition, mutation at the tyrosine sulfation site produced strong TPST inhibitors. We proposed that the location of tyrosine on the protein/peptide substrate was also an important factor to determine the priority of which tyrosine to get sulfated. Our proposed model and molecular modeling explained why mutant of PSGL-1 peptide become strong TPST inhibitors with the change of specific active site tyrosine.