人類嗜酸性白血球陽離子蛋白肝素結合區域之功能鑑定與分子機制

Functional Characterization and Molecular Mechanisms of Heparin-binding Motifs of Eosinophil Cationic Protein

林台文(Tai-Wen Lin)

指導教授 ： 張大慈

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2009年)

摘要

人類嗜酸性血球在發炎反應發生時釋放之嗜酸性血球陽離子蛋白(ECP)可對抗致病源的入侵，其在人類先天性免疫中扮演重要的角色。此外，嗜酸性血球陽離子蛋白的呈現與呼吸道過敏性疾病高度相關，因此成為臨床檢測氣喘的指標分子。近年的研究發現嗜酸性血球陽離子蛋白與細胞表面的硫酸乙醯肝素結合進入支氣管表皮細胞內，進而造成細胞毒殺作用，並鑑定L3區域為主要肝素結合位。然而蛋白質與肝素結合的強度至今還未明確測定。本研究利用等熱滴定熱卡計測得重組嗜酸性血球陽離子蛋白與肝素間交互作用的解離常數為1.3 x 10-6 M，並發現突變的重組嗜酸性血球陽離子蛋白失去L3的肝素結合區後，仍然保存部份的肝素結合能力，顯示其它肝素結合區域存在的可能。經由等熱滴定熱卡計及肝素親合性管柱層析分析點突變的重組嗜酸性血球陽離子蛋白，證實在一級序列中兩個鹼性胺基酸群集區域都具有肝素結合之活性。另外，利用熱滴定熱卡計測得嗜酸性血球陽離子蛋白與五種肝素衍生物反應熱量變化，以及此五種衍生物對於嗜酸性血球陽離子蛋白與肝素膠質的競爭能力，證實嗜酸性血球陽離子蛋白與肝素之間的結合之高度的選擇性與N基硫酸化修飾關係密切。

關鍵字

人類嗜酸性白血球陽離子蛋白 ； 肝素

並列摘要

Eosinophil cationic protein (ECP) is secreted from activated eosinophil at inflammatory site, and it functions in immune system to against pathogens such as bacteria and virus. ECP shows highly correlation with airway allergic diseases and has been used as biomarker for asthma in clinical. Recent study revealed that the cytotoxic activity of ECP toward Beas-2B human bronchial epithelial cell line depended on cell surface heparan sulfate proteoglycans (HSPGs) and the heparin binding motif of ECP with a basic amino acid cluster located at loop 3 (L3) is identified. However, the binding affinity between ECP and heparin is not fully elucidated. In this study, the binding constant between ECP and heparin was quantified by isothermal titration calorimeter (ITC), and the dissociation constant (Kd) was 1.3 x 10-6 M. A mutated MBP-ECP lacking of heparin binding L3 remained a weaker binding ability with a Kd value of 7.4 x 10-6 M, indicating the existence of additional heparin binding region in ECP. MBP-ECP single point mutants were used for identification of key heparin binding residues by ITC and affinity column chromatography. Two basic amino acid clusters were verified to be a novel heparin binding path on ECP. In addition, five heparin derivatives with difference modifications were used for determining the specificity of heparin binding activity of MBP-ECP. The results obtained from ITC and competitive heparin affinity chromatography indicated that the N-sufation on heparin predominantly contributed in ECP binding.

並列關鍵字

eosinophil cationic protein ； heparin

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