- 學位論文
人類嗜酸性白血球陽離子蛋白進入細胞的分子機制
Molecular Mechanisms of Cellular Binding and Endocytosis of Human Eosinophil Cationic Protein
摘要
氣喘是一種與氣管發炎相關的複雜疾病，已知人類嗜酸性白血球陽離子蛋白(ECP)在氣喘發病原理中扮演很重要的角色。嗜酸性白血球顆粒中的嗜酸性白血球陽離子蛋白是一種能與肝素(Heparin)結合的核醣核苷酸水解酶(Ribonuclease)，會伴隨嗜酸性白血球的活化釋放到細胞外，進而對支氣管上皮細胞造成極強的細胞毒性，肝素則能抑制嗜酸性白血球陽離子蛋白的細胞毒性。嗜酸性白血球陽離子蛋白之細胞毒性與其進入細胞的內噬作用有關，但至目前為止嗜酸性白血球陽離子蛋白進入細胞路徑的詳細機制並不清楚。本篇論文中，我們證明細胞表面的肝黏醣硫酸鹽蛋白醣 (Heparan sulfate proteoglycan/HSPG)為嗜酸性白血球陽離子蛋白與細胞結合的主要受體。利用酵素切除細胞表面的肝黏醣硫酸鹽、或降低細胞表面的醣硫酸化程度皆能有效地抑制嗜酸性白血球陽離子蛋白與支氣管上皮細胞(Beas-2B)的結合。本研究並發現醣氨多醣(Glycosaminglycan)缺失的細胞能吸收嗜酸性白血球陽離子蛋白的量大幅降低，並能抵抗該蛋白的細胞毒性。利用各種藥物與小型干擾RNA(siRNA)阻斷不同的內噬作用，發現嗜酸性白血球陽離子蛋白係利用與網格蛋白(clathrn)或內陷素(caveolin)都無關的新穎細胞內噬作用進入細胞。此作用受到小G蛋白Rac1及Arf6調控，並與細胞中的膽固醇濃度、肌動蛋白的排列及PI3K的活性相關，這些特性十分符合透過細胞膜上脂肪筏(lipid Raft)區域進行的巨胞飲作用。 利用合成的高純度肝素與嗜酸性白血球陽離子蛋白結合的實驗，我們發現五單醣長度的合成肝素即能有效抑制蛋白與細胞的結合。利用點突變方法及合成的小胜肽分子研究則顯示位於脉2螺旋與刍1褶版間的L3環區域中的R34、W35、R36、K38等胺基酸參與嗜酸性白血球陽離子蛋白與肝素分子及細胞表面的結合。本研究合成的L3環區域胜肽具有與5單元長度的合成肝素之高度親合性，並能阻斷嗜酸性白血球陽離子蛋白與細胞的結合，R34A/W35A/R36A/K38A變異蛋白與細胞結合能力及細胞毒性均幅降低。本研究對於嗜酸性白血球陽離子蛋白及肝黏醣硫酸鹽之專一性分子識別的結構及功能提出創新的見解，本研究結果將有助於開發抑制嗜酸性白血球陽離子蛋白與細胞結合以治療氣喘的新穎方法。
關鍵字
嗜酸性白血球陽離子蛋白並列摘要
Eosinophil cationic protein (ECP) is currently used as a biomarker of airway inflammation. It is a heparin-binding ribonuclease (RNase) released by activated eosinophils and is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In first part of the study, we show that cell surface-bound heparan sulfate proteoglycans (HSPGs) serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells as compared to their wild-type counterparts. Furthermore, pharmacological treatment combined with si-RNA knock-down identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and Arf6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and PI3K activities, and is compatible with the characteristics of raft-dependent macropinocytosis. In terms of specific polysaccharide binding activity, ssynthetic heparin with 5 or higher monosaccharide units showed strong inhibition of ECP binding to cell surface. The heparin binding site in ECP is determined by site-directed mutagenesis and synthetic peptide segment derived from ECP. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that the charged and aromatic residues were involved in ECP binding to heparin and cell surface. Such binding motif is located in the loop L3 region between helix 脉2 and strand 刍1, outside the RNA binding domain. The peptide derived from the loop L3 region displayed strong pentasaccharide binding affinities and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin act as the primary step for protein endocyosis. These results provide new insights into the structure and function of ECP regarding specific molecular interaction with heparan sulfate. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.
並列關鍵字
ECP參考文獻
延伸閱讀
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