人類嗜酸性白血球陽離子蛋白之肝素結合區位的功能特性探討

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研究生：	吳偉碩 Wu, Wei-Shuo
論文名稱：	人類嗜酸性白血球陽離子蛋白之肝素結合區位的功能特性探討 Functional Characterization of Heparin Binding Regions in Human Eosinophil Cationic Protein
指導教授：	黎耀基 Lai, Yiu-Kay 張大慈 Chang, Margaret Dah-Tsyr
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	74
中文關鍵詞：	嗜酸性白血球陽離子蛋白、肝素結合區位
相關次數：	點閱：2 下載：0
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中文摘要
嗜酸性白血球陽離子蛋白（eosinophil cationic protein; ECP）是由活化的人類嗜酸性白血球分泌的蛋白質，屬於人類核醣核酸水解酶A家族，亦稱為核醣核酸水解酶三。ECP能與細胞表面的氨基葡聚醣，特別是硫酸乙醯肝素醣蛋白結合，並透過胞飲作用進入支氣管上皮細胞造成細胞死亡，最後引起呼吸道發炎而導致氣喘等疾病的發生。哺乳動物細胞表面的肝素/硫酸乙醯肝素會和肝素結合蛋白序列中帶正電的胺基酸結合，在先前的研究中，本實驗室利用生物資訊及點突變法找出在ECP序列中可能與肝素結合有關的胺基酸序列。為了更進一步探討預測之ECP肝素結合區位（heparin binding region, HBR）的功能，本研究建立包含三個可能肝素結合區域的七種ECP突變株。利用螢光標定醣電泳 (FACE) 及細胞表面酵素連結免疫吸附分析 (ELISA) 分別測定ECP與其突變株對肝素及支氣管上皮細胞的結合能力。本研究發現HBR2（75RSRFR79）與HBR3（101RPGRR105）是影響ECP與人類支氣管上皮細胞 (Beas-2B) 結合的主要二個區位，而HBR1（34RWRCK38）則在ECP進入細胞的過程中扮演重要的角色。此外在ECP突變株對Beas-2B之毒性試驗中，發現HBR3（101RPGRR105）是ECP造成細胞死亡的關鍵區域。本論文之具體貢獻為發現並報導ECP序列中肝素結合區位的功能特徵之嶄新視野。

Abstract
Eosinophil cationic protein (ECP) is secreted by activated granular eosinophilic leukocytes. It belongs to human ribonuclease (RNase) A superfamily and is also named as RNase 3. Endocytosis of ECP into Beas-2B, human bronchial epithelial cells, depends on the interaction between protein and cell surface glycosaminoglycan (GAGs), especially heparin sulfate proteoglycans (HSPGs), and this process results in cell death which in turn leads to airway inflammatory disease such as asthma. In general, heparin/ heparan sulfate on the surface of mammalian cells has been reported to interact with positively charged residues in heparin binding proteins. In previous studies, three putative heparin binding regions (HBRs) (34RWRCK38, 75RSRFR79, and 101RPGRR105) of ECP were identified by bioinformatics methods and point mutation analysis. To further examine functional characteristics of these HBRs on ECP, seven mutant ECPs with different HBRs were constructed in this study. The binding ability of each mutant ECP to LMWH and Beas-2B cell surface was evaluated by fluorescence-assisted carbohydrate electrophoresis (FACE) and cell based enzyme-link immunosorbent assay (ELISA), respectively. The results show that HBR2 (75RSRFR79) and HBR3 (101RPGRR105) are involved in cellular binding ability of ECP, whereas HBR1 (34RWRCK38) plays a crucial role on cell penetration ability of ECP based on ECP uptake assay. Furthermore, cell viability assay indicates that HBR3 (101RPGRR105) acts as the key component contributing the cytotoxicity of ECP towards Beas-2B cells. The present study provides a novel insight in functional characteristics of three HBRs of ECP.

Table of Contents
中文摘要    I
Abstract    II
Table of Contents    IV
List of Table    VII
List of Figures    VIII
List of Appendix    IX
Abbreviations    X
Chapter 1  Introduction    1
1-1  Eosinophil and asthma    1
1-2  ECP and EDN    2
1-3  ECP and Glycosaminoglycans    2
1-4  Cytotoxicity of ECP    4
1-5  RNase characteristics of ECP    5
1-6  Purpose of this study    6
Chapter 2  Materials and Methods    8
2-1  Microbial strain and plasmid    8
2-2  Cell line strain and cell culture    9
2-3  Construction of mutant ECP    9
2-4  in situ PCR    10
2-5  Competent cell preparation and transformation of E. coli    11
2-6  Mini-preparation of plasmid    12
2-7  Site-directed mutagenesis and DNA sequencing    13
2-8  Culture condition of E. coli    15
2-9  Expression and purification of recombinant ECP and mtECP    15
2-10  Determination of protein concentration    17
2-11  Fluorescence-assisted carbohydrate electrophoresis (FACE)    17
2-12  Cell enzyme-link immunosorbent assay (ELISA)    18
2-13  ECP uptake assays and Western blotting    20
2-14  Cell viability assay    20
2-15  RNase activity assay    21
Chapter 3  Results    22
3-1  Expression and purification of wild type and mutant ECP    22
3-2  Binding ability of wild type and mutant ECP to LMWH    23
3-3  Binding ability of wild type and mutant ECP to Beas-2B cells    25
3-4  Penetration ability of wild type and mutant ECP to Beas-2B cells    26
3-5  Cytotoxicity of wild type and mutant ECP to Beas-2B cells    27
3-6  RNase activity of wild type and mutant ECP    28
Chapter 4  Discussion    29
4-1  Three putative heparin binding regions were important for heparin binding    29
4-2  HBR2 and HBR3 played a critical role in ECP binding to Beas-2B cells    30
4-3  HBR1 of ECP played a critical role in cell penetration to Beas-2B cells    31
4-4  HBR3 contributed to the cytotoxicity of ECP    32
4-5  HBR1-mtECP lost RNase activity    33
4-6  Comparison of HBR functions in ECP and EDN    34
4-7  Comparison of ECP-6H and MBP-ECP    35
4-8  Conclusion    35
References    37

                                

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