人類嗜伊紅血球陽離子蛋白進入細胞及毒性機制

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研究生：	吳嘉茂 Chia-Mao Wu
論文名稱：	人類嗜伊紅血球陽離子蛋白進入細胞及毒性機制 Mechanism of cell entry ability and signal peptide toxicity of human eosinophil cationic protein
指導教授：	張大慈 Margaret Dah-Tsyr Chang
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生命科學系 Department of Life Sciences
論文出版年：	2004
畢業學年度：	92
語文別：	英文
論文頁數：	101
中文關鍵詞：	嗜伊紅血球、嗜伊紅血球陽離子蛋白、生長抑制、細胞毒性、訊號序列、蛋白質水解脢、訊號序列水解酵素
外文關鍵詞：	eosinophil, eosinophil cationic protein, growth inhibition, cytotoxicity, signal peptide, carboxypeptidase E, signal peptide peptidase
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人類嗜伊紅血球陽離子蛋白是嗜伊紅血球顆粒中蛋白之主要組成之一，它通常被當作氣喘或過敏性疾病之臨床生物診斷指標。文獻指出嗜伊紅血球陽離子蛋白是由活化的嗜伊紅血球中所分泌的一個毒性蛋白。嗜伊紅血球陽離子蛋白已經被證實可以損害許多不同組織的細胞膜，但目前造成這種損害的機制並不清楚。在此論文中我們利用蛋白質重組技術融合嗜伊紅血球陽離子蛋白、綠色螢光蛋白及組胺酸標示，並成功利用大腸桿菌表現此一融合蛋白(mECP-eGFP-6H)。經由組胺酸標示親和性管柱層析純化過後之融合蛋白加入大白鼠神經內分泌細胞株(GH3)中，並藉此觀察嗜伊紅血球陽離子蛋白進入神經內分泌細胞的能力。我們發現嗜伊紅血球陽離子蛋白融合蛋白不僅可進入神經內分泌細胞中還可以抑制細胞的生長，其IC50為0.8微莫爾濃度。此外，我們利用酵母菌雙雜和及免疫沈澱的實驗方法篩選出一個蛋白質C端水解脢(CPE)，並證實它和嗜伊紅血球陽離子蛋白間有直接的蛋白質-蛋白質交互作用。我們更進一步證實在CPE胺基酸序列中第318到387的胺基酸序列片段在此蛋白質-蛋白質作用中扮演不可或缺的角色，此一胺基酸序列片段在CPE中的功能目前並不明。除此之外，我們利用大量表現CPE突變蛋白(preproHA-CPES471A,E472A)的方法阻止其由細胞膜上回收的步驟，藉此可抑制嗜伊紅血球陽離子蛋白融合蛋白進入細胞的能力。由此可證，嗜伊紅血球陽離子蛋白進入細胞的機制與CPE在細胞中的回收過程有關。另一方面，過去已經有許多文獻針對成熟嗜伊紅血球陽離子蛋白特性的研究被發表，然而目前並沒有對嗜伊紅血球陽離子蛋白的前導序列(signal peptide)的研究。我們利用在大腸桿菌、嗜甲醇酵母菌及人類上皮腫瘤細胞中表現數個在胺基端含有嗜伊紅血球陽離子蛋白前導序列的融合蛋白來研究嗜伊紅血球陽離子蛋白的前導序列的功能。我們發現到表現數嗜伊紅血球陽離子蛋白前導序列融合蛋白會造成大腸桿菌及嗜甲醇酵母菌的生長抑制，然而對人類上皮腫瘤細胞卻沒有此一影響。藉由分析嗜伊紅血球陽離子蛋白前導序列的氨基酸序列及利用試管內轉錄/轉譯的實驗方式，我們也發現到嗜伊紅血球陽離子蛋白前導序列可能是人類蛋白質前導序列水解脢(human signal peptide peptidase)的受質，它是一個位於內質網膜上的蛋白質水解脢。此外，以小型干擾核醣核酸(siRNA)的方式壓制人類蛋白質前導序列水解脢的表現後，表現嗜伊紅血球陽離子蛋白前導序列融合蛋白的人類上皮腫瘤細胞也出現生長抑制的情形。另一方面，在表現嗜伊紅血球陽離子蛋白前導序列融合蛋白的嗜甲醇酵母菌中同時表現人類蛋白質前導序列水解脢可以讓酵母菌的生長恢復。總而言之，此論文中我們發現了CPE協助嗜伊紅血球陽離子蛋白進入神經內分泌細胞的新功能，經由此胞飲作用進入細胞中的嗜伊紅血球陽離子蛋白因此可抑制細胞的生長。此外，我們亦發現嗜伊紅血球陽離子蛋白前導序列是一個毒性氨基酸序列，而細胞中表現的蛋白質前導序列水解脢可以保護細胞不被嗜伊紅血球陽離子蛋白前導序列的毒性影響。

Eosinophil cationic protein (ECP) is a major component of eosinophil granule proteins and is used as a clinical bio-marker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In this study, mECP-eGFP-6H, a recombinant fusion protein containing mature ECP (mECP), green fluorescence protein (eGFP) and Histag, has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP-eGFP-6H entered into GH3 neuroendocrine cells and inhibited the growth of the cells with the IC50 of 0.8 □M. By yeast two-hybrid screening and immunoprecipitation, we have identified a specific protein-protein interaction between mature mECP and carboxypeptidase E (CPE), a well characterized metalloprotease. Further in vivo yeast two-hybrid screening has also revealed that residues 318 to 387 located in a region of unknown function in mature CPE are indispensable for association with mECP. In addition, the uptake of mECP-eGFP-6H is suppressed by dominant-negative expression of the recycling defect mutant preproHA-CPES471A,E472A in GH3 cells, suggesting that the entrance of mECP-eGFP-6H is associated with the recycling of CPE in GH3 cells. On the other hand, the properties of mature ECP have been well studied but that of the signal peptide of ECP (ECPsp) are not clear. In this thesis, several chimeric proteins containing N-terminal fusion of ECPsp were generated, and introduced into Escherichia coli, Pichia pastoris and human epidermoid carcinoma cell line A431 to study the function of ECPsp. We found that expression of ECPsp chimeric proteins inhibited the growth of E. coli and P. pastoris but not A431 cells. Primary sequence analysis and in vitro transcription/translation of ECPsp have revealed that it is a potential substrate for human signal peptide peptidase (hSPP), an intramembrane protease located in endoplasmic reticulum. In addition, knockdown of the hSPP mRNA expression in ECPsp-eGFP/A431 cells caused the growth inhibitory effect, whereas complementary expression of hSPP in Pichia pastoris system rescued the cell growth. Taken together, we have demonstrated that CPE possesses a novel function to facilitate the entry of ECP to neuroendocrine cell, and such endocytosis process allows the cytotoxic ECP to inhibit growth of the target cells. In addition, we also demonstrated that ECPsp is a toxic signal peptide, and expression of hSPP protects the cells from growth inhibition.

中文摘要………………………………………………………………………………………1
Abstract ……………………………………………………………………………..............3
Abbreviations……………………………………………………………………………….5
Chapter 1  Background…………………………………………………………………….7
1-1. Human ribonuclease A superfamily…………………………………………………7
1-2. Eosinophils………………………………….……………………………………….7
1-2-1. Biological function of eosinophil…………………………………………………8
1-2-2. Component of eosinophils granule protein………………………………….…….8
1-2-3. Eosinophil granule ribonucleases………………………………………………....9
Chapter 2 The Cell entry of Eosinophil Cationic Protein………………..…………...…..11
2-1. Introduction………………………………………………………………………...11
2-2. Materials and Methods…………………………………………………….………14
2-2-1. Cell culture and transfection…………………………………………………….14
2-2-2. Preparation of recombinant mECP-eGFP-6H and eGFP-6H fusion proteins…..14
2-2-3. MTT assay for GH3 cell growth………………………………………………..15
2-2-4. Uptake of mECP-eGFP6H into GH3 cells……………………………………...16
2-2-5. Yeast two-hybrid assay………………………………………………………….16
2-2-6. Constructions of CPE deletion mutants……………………………………….....18
2-2-7. Immunoprecipitation and Western blot analysis………………………………...18
2-2-8. Construction of preproHA-CPES471A,E472A mutation……………………………..19
2-3. Results……………………………………………………………………………...20
2-3-1. Preparation of Recombinant mECP-eGFP-6H and eGFP-6H…………………...20
2-3-2. mECP-eGFP-6H inhibits the growth of neuroendocrine cells…………………..20
2-3-3. Secretagogues and lysosomotrophic agents affect mECP-eGFP-6H uptake into cells……………………………………………………………………………..21
2-3-4. CPE interacts with mature ECP in yeast cells…………………………………...22
2-3-5. mECP is associated with CPE in vitro…………………………………………..22
2-3-6. The region from 318 to 387 of mature CPE is indispensable for association with mECP in yeast…………………………………………………………………..23
2-3-7. The uptake of mECP-eGFP-6H fusion protein is blocked by dominant-negative expression of the preproHA-CPES471A,E472A in GH3 cells………………………24
2-4. Discussion……………………………………………………………………….…26
Chapter 3  The Toxicity of Eosinophil Cationic Protein Signal Peptide……………….41
3-1. Introduction…………………………………………………………………….….41
3-2. Materials and Methods……………………………………………………………44
3-2-1. Cell culture………………………………………………………………………44
3-2-2. RNA isolation, RT-PCR and Northern blotting…………………………………44
3-2-3. Plasmids preparation for E. coli, P. pastori and mammalian expression systems………………………………………………………………………….46
3-2-4. Construction of recombinant pSilencer expressing siRNA205 specific to hSPP......................................................................................................................47
3-2-5. Transfection……………………………………………………………………...48
3-2-6. In vitro transcription and translation…………………………………………….48
3-2-7. Monitoring the cell proliferation by MTT assay…………………………..…….49
3-2-8. eGFP detection by fluorescence microscopy and Western blotting……………..50
3-2-9. De novo protein synthesis in P. pastoris……………………………………..….50
3-3. Result………………………………………………………………………………52
3-3-1. Expression of ECPsp chimeric proteins causes the growth inhibitory effect on E. coli and P. pastoris cells………………………………………………………….52
3-3-2. Expression of ECPsp chimeric proteins in mammalian cell line A431 do not cause the growth inhibitory effect………………………………………………….….53
3-3-3. ECPsp is a potential substrate for signal peptide peptidase in vitro……………..54
3-3-4. Human SPP mRNA was expressed in ECPsp-eGFP/A431 stable clone and HL60 clone- 15………………………………………………………………………..55
3-3-5. Knockdown of the hSPP mRNA level in ECPsp-eGFP/A431 stable clone decreased the proliferation rate………………………………………………....55
3-3-6. Complementary expression of human SPP restores the growth of ECPsp-eGFP/GS115…………………………………………………………….56
3-4. Discussion………………………………………………………………………….57
Chapter 4 Conclusion……………………………………………………………………….72
References……………………………………………………………………………...……75
Appendix…………………………………………………………………………………….85
Supplement figures………………………………………………………………………..89

                                

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