人類嗜酸性白血球陽離子蛋白 (human eosinophil cationic protein, hECP) 是一種具有毒性的鹼性蛋白質，是由活化的嗜酸性白血球所分泌來的。ECP能與細胞表面上的硫酸肝素醣蛋白結合，在細胞膜的脂肪筏 (lipid raft) 區域利用胞飲機制進入到支氣管上皮細胞中。先前研究發現ECP中含有三段肝素結合區域(HBRs)，34RWRCK38 (HBR1)、75RSRFR79 (HBR2)、101RPGRR105 (HBR3)，其中ECP序列中的第一段肝素結合區 34RWRCK38 (HBR1)，與肝素醣蛋白結合並促進進入細胞之功能極為相關，同時其他文獻亦指出32NYRWRCKN39序列可與長鏈單尾磷脂微粒(dodecylphosphocholine micelle) 結合。本研究將利用核磁共振技術研究新穎免疫調控胜肽ECP32-41與其點突變胜肽 ECP32-41 W4R以及同源胜肽 EDN32-41 ，鑒定那些胺基酸會與肝素結合，比較這三條胜肽分別與肝素的作用模式。另外，研究ECP32-41序列中的色胺酸與長鏈單尾磷脂微粒結合的情形。

Human eosinophil cationic protein (hECP) is a heparin-binding ribonuclease secreted by activated eosinophils. ECP is a basic, granule-stored protein known to be cytotoxic toward pathogens and several cell lines involved in the immune innate system. The mechanism of ECP internalization is including HS binding ability and lipid raft-associated micropinocytosis. Previous studies have discovered that the sequence motif 32NYRWRCKNQN41 of ECP was defined to possess in heparin binding and cell penetrating activities, while motif 32NYRWRCKN39 have been proven to bind with dodecylphosphocholine micelle. The binding between ECP32-41 (NYRWRCKNQN) and heparin was previously characterized, but the detail of binding mechanism is still unclear. To achieve the goal, we have used nuclear magnetic resonance (NMR) to evaluate the binding of ECP32-41、ECP32-41 W4R and EDN32-41 and to heparin. Additionally, we also investigated the tryptophan of ECP32-41 leading the interaction with membrane.
 

中文摘要 I
Abstract II
謝誌 III
縮寫(Abbreviations) IV
目錄 VI
第一章 前言 1
第二章 材料與方法 12
2.1 胜肽來源 12
2.2 ECP32-41、ECP W4R32-41、EDN32-41胜肽序列 12
2.3 肝素衍生低聚醣製備 13
2.4 核磁共振(Nuclear magnetic resonance, NMR) 14
2.4.1 NMR樣品製備 14
2.4.2 胜肽分子骨架的循序判定 14
2.4.3 TOCSY (Total Correlation Spectropscopy) 15
2.4.4 ROESY (Rotating-Frame Overhauser Spectroscopy) 16
2.4.5 胜肽二級結構預測 17
2.4.6 胜肽化學位移擾動(chemical shift perturbation) 17
2.4.7 DOSY (Diffusion ordered spectroscopy) 17
2.5 利用程式擬合化學位移得到動力學常數KD 20
2.6 螢光光譜儀(Fluorescence Spectrometry) 21
2.7 圓二色光譜儀(Circular Dichroism Spectroscopy, CD) 22
第三章 結果 24
3.1 人類嗜酸性陽離子胜肽， ECP32-41與肝素結合分析 24
3.2 人類嗜酸性陽離子胜肽，ECP32-41 W4R與肝素結合分析 24
3.3 人類嗜酸性白血球神經毒蛋白，EDN32-41 與肝素結合分析 25
3.4 比較ECP32-41 、ECP W4R32-41、 EDN32-41胜肽與肝素結合的差別 26
3.5 1H-13C HSQC光譜比較ECP32-41 、ECP W4R32-41、 EDN32-41胜肽在二級結構上的差異 28
3.6 ECP32-41 、ECP32-41 W4R胜肽與肝素結合能力分析 28
3.7 ECP32-41 胜肽與dodecylphosphocholine micelle結合 29
3.8 ECP32-41 胜肽與dodecylphosphocholine micelle結合的螢光光譜 30
3.9 圓二色光譜(CD)比較ECP32-41 、ECP32-41 W4R、EDN32-41胜肽在二級結構上的差異 31
3.10 ECP32-41胜肽與DPC micelle作用之DOSY實驗 32
第四章 討論 59
參考文獻 61

1. Spry CJ: The pathogenesis of endomyocardial fibrosis: the role of the eosinophil. In: Springer seminars in immunopathology: 1989. Springer: 471-477.
2. Broide DH, Gleich GJ, Cuomo AJ, Coburn DA, Federman EC, Schwarte LB, Wasserman SI: Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. Journal of Allergy and Clinical Immunology 1991, 88(4):637-648.
3. Trivedi S, Lloyd C: Eosinophils in the pathogenesis of allergic airways disease. Cellular and molecular life sciences 2007, 64(10):1269-1289.
4. OLSSON I, VENGE P: Cationic Proteins of Human Granulocytes: I. Isolation of the Cationic Proteins from the Granules of Leukaemic Myeloid Cells. Scandinavian journal of haematology 1972, 9(1‐6):204-214.
5. Olsson I, Venge P: Cationic proteins of human granulocytes. II. Separation of the cationic proteins of the granules of leukemic myeloid cells. Blood 1974, 44(2):235-246.
6. Olsson I, Venge P, Spitznagel J, Lehrer R: Arginine-rich cationic proteins of human eosinophil granules: comparison of the constituents of eosinophilic and neutrophilic leukocytes. Laboratory investigation; a journal of technical methods and pathology 1977, 36(5):493-500.
7. Fredens K, Dybdahl H, Dahl R, Baandrup U: Extracellular deposit of the cationic proteins ECP and EPX in tissue infiltrations of eosinophils related to tissue damage. Apmis 1988, 96(7‐12):711-719.
8. Silberstein DS: Eosinophil function in health and disease. Critical reviews in oncology/hematology 1995, 19(1):47-77.
9. Guilpain P, Guillevin L, Mouthon L: [Eosinophil granule cationic proteins: eosinophil activation markers]. La Revue de medecine interne/fondee par la Societe nationale francaise de medecine interne 2006, 27(5):406-408.
10. Niccoli G, Ferrante G, Cosentino N, Conte M, Belloni F, Marino M, Bacà M, Montone RA, Sabato V, Schiavino D: Eosinophil cationic protein: A new biomarker of coronary atherosclerosis. Atherosclerosis 2010, 211(2):606-611.
11. Khakzad MR, Mirsadraee M, Sankian M, Varasteh A, Meshkat M: Is serum or sputum eosinophil cationic protein level adequate for diagnosis of mild asthma? Iranian Journal of Allergy, Asthma and Immunology 2009, 8(3):155-160.
12. Koh GC-H, Shek LP-C, Goh DY-T, Van Bever H, Koh DS-Q: Eosinophil cationic protein: is it useful in asthma? A systematic review. Respiratory medicine 2007, 101(4):696-705.
13. Gleich GJ: Mechanisms of eosinophil-associated inflammation. Journal of Allergy and Clinical Immunology 2000, 105(4):651-663.
14. Driss V, Legrand F, Hermann E, Loiseau S, Guerardel Y, Kremer L, Adam E, Woerly G, Dombrowicz D, Capron M: TLR2-dependent eosinophil interactions with mycobacteria: role of α-defensins. Blood 2009, 113(14):3235-3244.
15. Lehrer RI, Szklarek D, Barton A, Ganz T, Hamann K, Gleich G: Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein. The Journal of Immunology 1989, 142(12):4428-4434.
16. Torrent M, de la Torre B, Nogues V, Andreu D, Boix E: Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment. Biochem J 2009, 421:425-434.
17. Hamann K, Gleich G, Checkel J, Loegering D, McCall J, Barker R: In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins. The Journal of Immunology 1990, 144(8):3166-3173.
18. Badar A, Saeed W, Hussain MM, Aslam M: Correlation of eosinophil cationic protein with severity of asthma. Age (years) 2004, 34(12.17):36.27-39.78.
19. Choi J, Callaway Z, Kim HB, Fujisawa T, Kim CK: The role of TNF‐α in eosinophilic inflammation associated with RSV bronchiolitis. Pediatric Allergy and Immunology 2010, 21(3):474-479.
20. Domachowske JB, Dyer KD, Adams AG, Leto TL, Rosenberg HF: Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. Nucleic acids research 1998, 26(14):3358-3363.
21. Maeda T, Kitazoe M, Tada H, de Llorens R, Salomon DS, Ueda M, Yamada H, Seno M: Growth inhibition of mammalian cells by eosinophil cationic protein. European Journal of Biochemistry 2002, 269(1):307-316.
22. Motojima S, Frigas E, Loegering DA, Gleich GJ: Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. American Review of Respiratory Disease 1989, 139(3):801-805.
23. Carreras E, Boix E, Navarro S, Rosenberg HF, Cuchillo CM, Nogués MV: Surface-exposed amino acids of eosinophil cationic protein play a critical role in the inhibition of mammalian cell proliferation. Molecular and cellular biochemistry 2005, 272(1-2):1-7.
24. Goto T, Morioka J, Inamura H, Yano M, Kodaira K, Igarashi Y, Abe S, Kurosawa M: Urinary eosinophil-derived neurotoxin concentrations in patients with atopic dermatitis: a useful clinical marker for disease activity. Allergology International 2007, 56(4):433-438.
25. Durack DT, Ackerman SJ, Loegering DA, Gleich GJ: Purification of human eosinophil-derived neurotoxin. Proceedings of the National Academy of Sciences 1981, 78(8):5165-5169.
26. Rosenberg HF, Tenen DG, Ackerman SJ: Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family. Proceedings of the National Academy of Sciences 1989, 86(12):4460-4464.
27. Rosenberg HF, Dyer KD: Eosinophil cationic protein and eosinophil-derived neurotoxin Evolution of novel function in a primate ribonuclease gene family. Journal of Biological Chemistry 1995, 270(37):21539-21544.
28. Slifman N, Loegering D, McKean D, Gleich G: Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein. The Journal of Immunology 1986, 137(9):2913-2917.
29. Sasisekharan R, Shriver Z, Venkataraman G, Narayanasami U: Roles of heparan-sulphate glycosaminoglycans in cancer. Nature Reviews Cancer 2002, 2(7):521-528.
30. Barker RL, Loegering DA, Ten RM, Hamann KJ, Pease L, Gleich G: Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases. The Journal of Immunology 1989, 143(3):952-955.
31. Larkin MA, Blackshields G, Brown N, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R: Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23(21):2947-2948.
32. Bishop JR, Schuksz M, Esko JD: Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 2007, 446(7139):1030-1037.
33. Munoz EM, Linhardt RJ: Heparin-binding domains in vascular biology. Arteriosclerosis, thrombosis, and vascular biology 2004, 24(9):1549-1557.
34. Capila I, Linhardt RJ: Heparin–protein interactions. Angewandte Chemie International Edition 2002, 41(3):390-412.
35. Hung T-J, Tomiya N, Chang T-H, Cheng W-C, Kuo P-H, Ng S-K, Lien P-C, Lee Y-C, Chang MD-T: Functional characterization of ECP-heparin interaction: A novel molecular model. 2013.
36. Fan T-c, Fang S-l, Hwang C-s, Hsu C-y, Lu X-a, Hung S-c, Lin S-C, Chang MD-T: Characterization of molecular interactions between eosinophil cationic protein and heparin. Journal of Biological Chemistry 2008, 283(37):25468-25474.
37. García-Mayoral MF, Moussaoui M, Beatriz G, Andreu D, Boix E, Nogués MV, Rico M, Laurents DV, Bruix M: NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics. Biophysical journal 2010, 98(11):2702-2711.
38. García-Mayoral MF, Canales An, Díaz D, López-Prados J, Moussaoui M, de Paz JL, Angulo Js, Nieto PM, Jiménez-Barbero Js, Boix E: Insights into the glycosaminoglycan-mediated cytotoxic mechanism of eosinophil cationic protein revealed by NMR. ACS chemical biology 2012, 8(1):144-151.
39. Sl F, Tc F, Fu H, Chen C, Hwang C, Antopolsky M: A novel cell-penetrating peptide derived from human eosinophil cationic protein. PloS one 2013, 8(3):e57318.
40. 陳勝智, 連金城: 為碳序法之化學結構產生法尋求資料豐富的核磁共振光譜? 2004.
41. Jelinski LW: Modern NMR Spectroscopy. Chemical and Engineering News 1984, 62(45).
42. Otting G: NMR studies of water bound to biological molecules. Progress in Nuclear Magnetic Resonance Spectroscopy 1997, 31(2):259-285.
43. Spera S, Bax A: Empirical correlation between protein backbone conformation and C. alpha. and C. beta. 13C nuclear magnetic resonance chemical shifts. Journal of the American Chemical Society 1991, 113(14):5490-5492.
44. Schwarzinger S, Kroon GJ, Foss TR, Chung J, Wright PE, Dyson HJ: Sequence-dependent correction of random coil NMR chemical shifts. Journal of the American Chemical Society 2001, 123(13):2970-2978.
45. Morris KF, Johnson Jr CS: Diffusion-ordered two-dimensional nuclear magnetic resonance spectroscopy. Journal of the American Chemical Society 1992, 114(8):3139-3141.
46. Johnson Jr C: Prog. NMR Spectrosc. 1999, 34, 203.[CrossRef],[CAS] Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications Johnson, CS, Jr. Progress in Nuclear Magnetic Resonance Spectroscopy 1999, 34(3):4.
47. 陳一帆: 以磁場梯度核磁共振技術測定幾丁聚醣分子量. 2006.
48. Lakowicz JR: Principles of Fluorescence Spectroscopy.
49. Teale F: The ultraviolet fluorescence of proteins in neutral solution. Biochemical Journal 1960, 76(2):381.
50. Komath SS, Swamy MJ: Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin. Journal of Photochemistry and Photobiology B: Biology 1999, 50(2):108-118.
51. Hennessey Jr JP, Johnson Jr WC: Information content in the circular dichroism of proteins. Biochemistry 1981, 20(5):1085-1094.
52. 李黠汝: 肝癌衍生生長因子 PWWP 區域第一個胺基酸對蛋白質穩定性與受體結合能力的影響. 清華大學生物資訊與結構生物研究所學位論文 2013:1-63.
53. Torrent M, Cuyás E, Carreras E, Navarro S, López O, de la Maza A, Nogués MV, Reshetnyak YK, Boix E: Topography studies on the membrane interaction mechanism of the eosinophil cationic protein. Biochemistry 2007, 46(3):720-733.
54. 杜慶璋: 體制素分子內氫鍵之研究. 2002.