Development of GST fusion system for the identification of protein sulfation site
|關鍵字:||亞硫酸化;亞硫酸化催化位;谷胱甘肽S-轉移酶融合系統;Sulfation;sulfation site;GST fusion system;PSGL-1;Tyrosylprotein Sulfotransferase|
|摘要:||蛋白質酪氨酸亞硫酸化是由酪胺酸亞硫酸基轉移酶 (tyrosylprotein sulfotransferase)所催化的一種常見蛋白質轉譯後修飾作用。酪氨酸亞硫酸基轉移酶 (tyrosylprotein sulfotransferase)負責將腺苷3’-磷酸-5'-磷醯硫酸 (PAPS)所提供的亞硫酸基轉移到許多分泌性蛋白或高基氏體的膜蛋白質酪氨酸。蛋白質酪氨酸亞硫酸化的蛋白質參與了蛋白質交互作用及生理過程，其中包含凝血反應、白血球貼附血管細胞、病毒入侵、以及趨化素的訊息傳遞。目前對於酪氨酸亞硫酸基轉移酶調控生物功能性的機制仍未知。標的蛋白基質上的酪胺酸殘基數目不一，並且亞硫酸化酪胺酸殘基的位置及數目會影響蛋白質間的交互作用。而酪氨酸亞硫酸基轉移酶的催化速率及特異性可能會受到標的蛋白基質上的酪胺酸間的影響。本研究的目標是發展谷胱甘肽S-轉移酶融合胜肽及蛋白質系統辨別蛋白質亞硫酸化催化位去探討酪胺酸對於酵素的重要性。利用先前所開發的酚醛亞硫酸基轉移酶-酪氨酸亞硫酸基轉移酶耦合酶分析及酶聯免疫吸附試驗去分析酪氨酸亞硫酸基轉移酶對於標的蛋白基質及抑制劑的特異性。利用定點突變標的蛋白基質的酪胺酸來探討酪胺酸對於酵素催化的重要性。根據酵素動力學，酪氨酸亞硫酸基轉移酶對於野生型GST-PSGL-1的Km值為52 M、Vmax值為0.016 mol/min/mg。酪氨酸亞硫酸基轉移酶對於野生型與突變型GST-PSGL1的Km值有差異，但Vmax值沒差異。發現當野生型GST-PSGL-1上的第三位酪胺酸被突變，並且在腺苷3',5'-二磷酸 (PAP)存在下會誘導酪氨酸亞硫酸基轉移酶產生沉澱。根據酵素動力學，將GST-PSGL-1 3F胜肽作為抑制劑，其Ki數值為0.171 M。其有助於未來PB型腸病毒71型抑制劑之研究。為了解酪胺酸亞硫酸基轉移酶與GST-PSGL-1之間的交互作用，其兩者的結構分析更顯得重要。隨著谷胱甘肽S-轉移酶融合系統發展，蛋白質亞硫酸化基質大量表達且快速純化，並且利用新穎的分析方式可快速探討胺酸亞硫酸基轉移酶與基質的關係。|
Protein tyrosine sulfation, catalyzed by tyrosylprotein Sulfotransferase (TPST), is one of the most common protein posttranslational modifications. TPST catalyzes the transfer of the sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residue of various secreted and membrane-bound proteins that transit to the Golgi. Protein tyrosine sulfation is a key modulator of extracellular protein-protein interactions, and mediates many physiological processes, including coagulation, leukocyte adhesion, virus infection, chemokine signaling. At present, it remains much unknown about the mechanisms of TPST regulated biological functions. It is shown that the majority of target proteins surround more than one tyrosine near sulfation site, an indication that position and number of sulfated tyrosine residues on target protein may affect protein-protein interaction. It is also possible that neighboring tyrosine may affect the rate or specificity of other tyrosine sulfation in a protein. The goal of this research is to develop a GST fusion system to study the significance of the multiple tyrosine residues for protein sulfation. Using PST-TPST coupled enzyme assay and enzyme-linked immunosorbent assay previously developed in our lab to analyze activity of TPST toward specifically designed substrates/inhibitors. The significance of tyrosine residues on protein sulfation substrates was investigated by site-specific mutagenesis for TPST catalysis. The Km and Vmax of TPST values were 52 M and 0.016 mol/min/mg, respectively. Significant variation in Km values were observed when tyrosines around sulfation site were mutated. However, similar Vmax values were obtained as long as tyrosine 3 of the sulfation site remained intact. Interestingly, the phenomenon of that precipitation of TPST was induced by GST-PSGL-1 was discovered when the tyrosine 3 residue was substituted by a variety of other amino acid and in the presence of PAP. The inhibition kinetics constants toward PSGL-1 3F peptide as inhibitor gave Ki value as 0.171 M. This result strongly indicated that an inhibitor for EV-71 PB entry can be developed in the future base on the similar strategy. Structural analysis using known TPST structure and PSGL-1 is needed to more clearly describe the interaction between TPST and PSGL-1. The interesting protein sulfation substrates would be overexpressed and purified quickly with development of GST system, and then the relationship between TPST and substrates using novel analyzed method could be explored rapidly.
|Appears in Collections:||Thesis|