天然抗菌胜肽 Cecropin B及其衍生物之功能與結構探討

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研究生：	吳俊明 Jiun-Ming Wu
論文名稱：	天然抗菌胜肽 Cecropin B及其衍生物之功能與結構探討 Functional and Structural Studies of Natural Antimicrobial Peptide Cecropin B and its Derivatives
指導教授：	程家維 Jya-Wei Cheng 陳惠民 Hueih Min Chen
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：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	91
中文關鍵詞：	抗菌胜肽、脂膜通透性、脂膜融合、核磁共振光譜學、圓二色光譜、抗癌胜肽、肝素結合位置、螺旋-鉸鏈-螺旋
外文關鍵詞：	antimicrobial peptide, membrane permeability, membrane fusion, NMR spectroscopy, circular dichroism, anticancer peptide, heparin-binding motif, helix-hinge-helix
相關次數：	點閱：2 下載：0
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Cecropin B (CB)是一個從天蠶(Hyalophora cecropia) 血淋巴液分離出來的天然抗菌胜肽，由35個氨基酸組成，CB對於細菌具有廣泛的毒殺能力，但其對於人體紅血球細胞並不具有強烈的細胞溶破能力，圓二色光譜（circular dichroism, CD）的實驗結果顯示，CB在水溶液中為無序纏捲（random coil）的狀態，但是在極性溶劑中會轉換成alpha-螺旋，利用核磁共振光譜學的技術，研究CB在20% HFIP溶液中的立體結構，其由兩段alpha-螺旋（殘基 4-21 和殘基25-34）所組成，中間由一個脯氨酸扭結(proline kink) (殘基22-24)連接。為了仔細分析研究CB，合成了截斷型的短胜肽CB-N27 (殘基 1-27) 和 CB-C17 (殘基 19-35)，並研究其脂膜的通透性、融合效應與抗菌活性，此兩種截斷型的胜肽都不具有抗菌作用，最小抑制濃度的抗菌測試實驗都顯示其損失了原有的抗菌能力，顯示適當的胜肽長度對於CB的抗菌能力是必須的。另一個CB衍生物CB1，是由CB將C端的部分，以N端序列取代所組成，CB1的多正電荷特性使其保留了抗菌能力，而CB 之N端序列的多正電荷(polycationic)與兩面性(amphipathic)的特性，有助於其抗菌活性，此外，CB-N27 和 CB-C17 的溶破脂膜特性、脂質混合能力、抗菌能力都下降，代表適當的長度的CB，不論是N端帶正電序列以及C端斥水性的部分，對於CB的功能來說都是重要的，而CB的螺旋-鉸鏈-螺旋(helix-hinge-helix)式的結構，讓CB兩段alpha-螺旋有可彎曲性，促進與脂膜的互動作用。

幾種天然的抗菌胜肽，包括 cecropins、magainins和melittins，都被發現具有殺死癌細胞的特性，然而這些胜肽的功效並不一定適合發展成對抗癌細胞的藥劑，我們的研究裡，利用天然抗菌胜肽CB當做模版，製造新穎的抗癌胜肽。cecropin 家族裡的一致序列模式為W-x(0,2)-[KDN]-x-{L}-K-[KRE]-[LI]-E-[RKN] (PROSITE資料庫編號：PS00268) ，而此特徵段序列就是對應在CB的N端，我們將此特徵序列重複三次，並在C端夾入一個鉸鏈(hinge)，成為一個新的胜肽命名為CB1a，CB1a 在水溶液中並無構形，但在類似脂膜的環境中可以形成alpha-螺旋，而CB1a 溶在有機極性溶劑的立體結構也以核磁共振光譜技術進行研究，CB1a在20% HFIP 溶液中，其結構為螺旋-鉸鏈-螺旋 (helix – hinge - helix)的形式，而兩段螺旋片端中間的夾角約為 60~110度，在CB1a第一段螺旋結構的中間部位，有一個肝素結合位置 (heparin- binding motif)，恆溫熱卡計(isothermal titration calorimetry) 的實驗結果顯示， CB1a 在生理鹽度和25°C的環境下，與低分子量肝素的結合常數為1.66×105 M-1 ，CB1a 與肝素結合之後，造成很大的構形轉變，CB1a變的傾向於有二級結構的狀態。CB1a 對於幾株癌細胞展露出其具有發展前景的抗癌活性，但對於非癌細胞(non-cancer cells)的毒性卻是相對輕微，CB1a 對白血病與胃癌細胞的抑制濃度 IC50，大概低於CB的抑制濃度 2到8倍，此外，CB1a對於人類紅血球的溶血效應相當低，這些特性使得CB1a 可以被當作一個好的抗癌候選藥物。

Cecropin B (CB) is a 35-residue natural antimicrobial peptide isolated from the immune hemolymph of Hyalophora cecropia. CB shows a broad spectrum of activity against bacteria but has little cytolytic effect. CD measurements revealed that CB adopts random structure in aqueous solution but form helical conformation in polar solvent. The solution structure of CB in 20% HFIP was studied by using NMR spectroscopy. It consists of two amphipathic alpha-helices (residues 4-21 and 25-34) connected by a proline kink region (residues 22-24). In order to undertake the structural dissection of CB, CB-N27 (residues 1-27) and CB-C17 (residues 19-35) were synthesized to investigate the membrane permeabilities, fusogenic effect, and antimicrobial activities. The MIC test the two truncated form of CB both loss its antibacterial activity, suggesting the adequate peptide length of the CB is required in its antimicrobial function. CB1 was constructed by replacing the C-terminal segment with N-terminal sequence of CB, the polycationic property of CB1 make it restored the antimicrobial activity. The N-terminal polycationic and amphipathic sequence may contribute to antimicrobial activity. Furthermore CB-N27 and CB-C17 both inhibited membrane lysis, lipid mixing, and antibacterial activity. Suggesting that the optimal length of N-terminal positively charged residues and C-terminal hydrophobic segment are critical factors for its functions. The helix-hinge-helix structure of CB provides the flexibility for its two □-helices and facilitates the interactions to lipid membrane.

Several natural antimicrobial peptides including cecropins, magainins and melittins have been found to kill cancer cells. However, their efficacy may not be adequate for their development as anticancer agents. In this study, we used a natural antimicrobial peptide, cecropin B (CB), as a template to generate a novel anticancer peptide. The consensus pattern of cecropins is W-x(0,2)-[KDN]-x-{L}-K-[KRE]-[LI]-E-[RKN] (PROSITE: PS00268), and this signature sequence is located at N-terminus of CB. CB1a was constructed by repeating the signature sequence of CB three times and including a hinge near C-terminus. The circular dichroism spectra show that CB1a is unstructured in aqueous solution, but adopt a helical conformation in membrane-like environment. The solution structure of CB1a in polar solvent was also studied by NMR. CB1a formed a helix-hinge-helix in 20 % HFIP solution, and the bent angle between two helical segments was ranging from 60° to 110°. A heparin-binding motif is located in the central part of helix 1. Isothermal titration calorimetry reveals the association constant of CB1a bound to low molecular weight heparin is 1.66×105 M-1 at physiological ionic strength at 25°C. Binding of CB1a to heparin produces a large conformational change toward a more structural state. CB1a demonstrated promising activity against several cancer cells with low toxicity to the non-cancer cells. The IC50 of CB1a on leukemia and stomach carcinoma cells were in the range of 2- to 8-fold lower than those of CB. Besides, CB1a displayed low hemolytic property on human red blood cell. These properties might make CB1a a good candidate for use as an anticancer agent.

目錄
Table of Contents
                                                               Page
Abstract (中文)…………………………………………………………………i
Abstract (English version) ……………………………………………………iii
List of Tables ………………………………………………………………viii
List of Figures…………………………………………………………………ix

Chapter 1.  Functional and Structural Characterizations of Cecropin B, a Natural Antimicrobial Peptide from Hyalophora cecropia …………………………………………………………1

          Key Words and Abbreviations  …………………………………1
          Abstract   ……………………………………………………1

          Introduction………………………………………………………3

          Material and Methods ……………………………….…………5
               Materials……………….……………………………………..5
                Preparation of peptides ………………………………………5
                Antibacterial activity assay…………………………………...6
                Preparation of calcein-filled unilamellar vesicles………….…7
                Dye leakage experiments……………………………………..8
                Fluorescence resonance energy transfer (FRET) assay
for membrane fusion…………………………………….....…8
                Circular dichroism (CD) measurements…………………...….9
                Nuclear magnetic resonance (NMR) spectroscopy……..….…9
                Structure calculation…………………………………….......10

Results ……………………………………….………….……...…12
                Antibacterial activity………………………………………...12
Peptide-induced permeabilization on lipids vesicles………...12
Fusion monitored by lipid mixing……………………………13
Circular dichroism (CD) measurements……………………...13
NMR analysis………………………………………….……..14
Structure calculation………………………………………….15

          Discussion …………………………………………...……………17
                Antibacterial activity………………………………….……..17
                Dye leakage and membrane fusion……………………….…17
CD spectra………………….……………………………..…18
Structure of CB………………………………………………19

Chapter 2.  Structure of a Novel Anticancer Peptide CB1a, Based on the N-terminal Peptide Sequence of Cecropin B…………..……31

       Key Words and Abbreviations …………..………………………31
          Abstract ……………………………………..……………………31

Introduction………………………………………………………33

Material and Methods…………………………………………...36
               Materials……………………………………………….……36
Preparation of peptides………………………………….…..36
Circular dichroism (CD) measurements………………...…..37
Nuclear magnetic resonance (NMR) spectroscopy……...….38
Structure calculation…………………………………..…….39
Isothermal titration calorimetry (ITC)……….………….…..39
Cell culture……………………………………………….....40
Cytotoxicity assays……………………………….….…..….41
Confocal laser scanning microscopy (CLSM)………..…….41
Fluorescence-activated cell sorter (FACS) analysis….……..42
Hemolysis assay………………………………………..…...42

         Results……………….……………………………………….……43
Circular dichroism (CD) measurements……………….…...44
NMR analysis…………………………………………..…..44
Structure calculation…………………………………….….46
Heparin-induced secondary structure change of CB1a….....47
Isothermal titration calorimetry (ITC) analysis………….…48
Anticancer activity assays……………………………....…..48
Confocal images and flow cytometry analysis of peptide uptake………………………………………….….………..49
Hemolytic activity assays…………………………....…….49

          Discussion………………………………………………….……50
Structural characterization of CB1a……………..…………51
Heparin binding property of CB1a………………………...52
Anticancer and hemolytic activity…………………………52

Chapter 3.  Conclusions and Prospects   ……………………………69

References List……………………………………………………………75

Supplementary Materials (Appendix) ……………………………….…84

List of Tables
                                                              Page
Table 1-1  Amino acid sequences of peptides in this study……………………20
Table 1-2  Antimicrobial activity of cecropin B and its derivatives ………..…21
Table 1-3  Summary of structural constraints and structure statistics…………22
Table 2-1  Amino acid sequences of peptides in this study ……………..….…55
Table 2-2  Summary of structural constraints and structure statistics…………56
Table 2-3  IC50 values of various peptides and doxorubicin on different
cell lines ……………………………………………………….……57

List of Figures
                                                               Page
Figure 1-1  Dye leakage experiments for CB in POPGand POPC at 25°C …..…23
Figure 1-2  Membrane fusion experiments on POPG LUVs and POPC LUVs…24
Figure 1-3  CD spectra of cecropin B and its derivatives……………..…………25
Figure 1-4  NOESY spectra of CB in 20% HFIP at 298K ………………….……27
Figure 1-5  Solution structure of CB in 20% HFIP………………………….……28
Figure 1-6  The lowest energy structure of CB………………………………..…29
Figure 1-7  Helical wheel diagram of N-terminal helix of CB……………..……30
Figure 2-1  CD spectra of the peptides………………..…………………………58
Figure 2-2  NOESY spectrum and NOE connectivities of CB1a…..……………60
Figure 2-3  Calculated structures of CB1a.………………………………………62
Figure 2-4  Heparin-induced conformational change of CB1a………….………63
Figure 2-5  ITC titration of CB1a and CB with heparin ……………………..…64
Figure 2-6  Confocal images of cancer cells treated by CB1a…………..………65
Figure 2-7  Cellular uptake analyzed by FACS…………………………………66
Figure 2-8  Hemolytic activity assays……………………………………...……67
Figure 2-9  Helical wheel diagrams of N-terminal helices of CB1a and CB……68
Figure S1  Structure of NBD-PE (donor) and Rh-PE (acceptor) used in lipids mixing experiments………………………………………...………84
Figure S2  Molecular structure of 1,1,1,3,3,3-hexafluoro-2- propanol(HFIP)…...85
Figure S3  CD spectra of CB in 1 mM POPG LUVs, 20 mM SDS micelles, 40 % TFE and 40% HFIP solution (at 25 °C)…………………………...…86
Figure S4  CD spectra of CB1a in 20 mM SDS and in 20 mM DPC mcelles……87
Figure S5  CD spectra of CB with and without heparin………….………………88
Figure S6  SEM and TEM micrographs of CB1a on Jurkat leukemia cells…..…89
Figure S7  CLSM images of eukaryotic cells treated with FITC-CB
(60 min at 37°C)…………….………………………………………90
Figure S8  Confocal images of lung cancer cell line NCI-H661 treat with FITC-CB1a for 30 min…………………………………………...…91

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