臺灣博碩士論文加值系統

人類嗜酸性白血球陽離子蛋白(human eosinophil cationic protein, hECP)是由活化的嗜酸性白血球分泌且具有毒性之鹼性蛋白質。目前已知ECP能夠與細胞表面的硫酸乙醯肝素(heparan sulfate)或肝素(heparin)結合，並透過細胞膜上的脂肪筏(lipid raft)區域進行巨胞(macropinocytosis)作用。此外，ECP 會結合聚集於細胞表面並改變膜的通透性與離子平衡，進而造成細胞死亡。先前本實驗室研究發現ECP中32NYRWRCKNQN41序列與其結合並進入細胞之功能極為相關，同時其他文獻亦指出32NYRWRCKN39序列可結合長鏈單尾磷脂質微粒(dodecylphosphocholine micelle)。本研究將ECP 蛋白中32NYRWRCKNQN41區位的氨基酸個別點突變成丙氨酸(Ala)或性質相似之氨基酸，運用細胞表面酵素連結免疫吸附(cell ELISA)與生物資訊模擬分析，發現Arg34、Lys38、Asn41為重要的肝素結合位。另一方面，32NYRWRCKNQN41序列可與特定的細胞膜脂質結合，例如：PI3P、PI4P、PI5P、PI (3,4)P2、PI (3,5) P2、PI (3,4,5) P3、sulfatide等特定脂質結合。本研究證實ECP之32NYRWRCKNQN41區位兼具肝素、硫酸乙醯肝素、細胞表面、細胞膜脂質及細胞穿透等多樣化功能，有助釐清ECP與細胞的交互作用以及其細胞毒性機制。
Human eosinophil cationic protein (hECP) is a basic and cytotoxic granular protein released from activated eosinophils. So far, heparan sulfate (HS)/ heparin binding ability and lipid raft-associated macropinocytosis are implicated in the mechanism of ECP internalization. ECP binding to and aggregation on the cellular membrane account for its cytotoxicity, which in turn leads to cellular membrane permeability alteration and ionic equilibrium modification. The sequence motif 32NYRWRCKNQN41 of ECP was previously characterized to possess in HS binding and cell penetrating activities, while motif 32NYRWRCKN39have been proven to bind with dodecylphosphocholine micelle. Employing cell ELISA and in silico molecular docking, Arg34, Lys38 and Asn41 were found to be crucial heparin binding sites on ECP. In addition, membrane strip assay showed that both ECP and 32NYRWRCKNQN41 peptide interacted with specific membrane lipids including PI3P, PI4P, PI5P, PI (3,4) P2, PI (3,5) P2, PI (3,4,5) P3 and sulfatide, suggesting that it also possessed plasma membrane binding activity. In summary, we have demonstrated the key residues in 32NYRWRCKNQN41 motif for cell binding, and discovered multiple functions of ECP, which facilitates understanding of molecular interaction between ECP and cell surface and cell membrane components, as well as cytotoxicity mechanisms of ECP.

中文摘要 I
Abstract II
Acknowledgement III
List of Contents IV
List of Figures VI
List of Tables VII
List of Appendix VIII
Abbreviations IX
Chapter 1 Introduction 1
Chapter 2 Materials and Methods 14
2. 1 Microbial strains, plasmids and culture conditions 14
2. 2 Construction of plasmids 15
2. 3 Competent cell preparation and transformation of E. coli 17
2. 4 In situ PCR 18
2. 5 Mini-preparation of plasmid 19
2. 6 Site-directed mutagenesis and DNA sequencing 20
2. 7 Large scale expression of recombinant protein 22
2. 8 Purification of wild type and mutant MBP-ECPs and determination of protein concentration 23
2. 9 Isolation and solubilization of ECP-6His inclusion bodies 24
2. 10 Purification of recombinant ECP-6His 25
2. 11 In vitro folding of ECP-6His 26
2. 12 Protein concentration and quantification 27
2. 13 Peptide synthesis and mass determination 28
2. 14 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 28
2. 15 Cell line strains 29
2. 16 Cell enzyme-link immunosorbent assay (ELISA) 30
2. 17 Fluorescence-assisted carbohydrate electrophoresis (FACE) 31
2. 18 Membrane strip assay 33
2. 19 MBP-ECP uptake assays and Western blotting 34
2. 20 RNase activity assay 35
2. 21 Mass spectrometry determination 36
2. 22 Statistical analysis 36
Chapter 3 Results 38
3. 1 Expression and purification of wild type and mutant MBP-ECPs 38
3. 2 Expression and purification of ECP-6His 39
3. 3 Importance of LMWH and CS in ECP binding to cell surface 42
3.4 Identification of specific lipids interacting with ECP and CPPecp 46
3. 5 Cell surface binding activities of MBP-ECP 48
3. 6 Uptake of MBP-ECP into Beas-2B cells 51
3. 7 RNase activity of ECP-6His and EDN-6His 52
Chapter 4 Discussion 54
Reference 65
Figures 76
Tables 98
Appendix 127

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