臺灣博碩士論文加值系統

草菇毒蛋白 A（Volvatoxin A, VVA）。已知VVA是由一種孔洞形成的心臟毒素草菇毒蛋白 A2（VVA2）及草菇毒蛋白 A1（VVA1）所組成。先前的研究發現VVA2具有溶血作用及細胞毒性，但是對VVA1的活性仍不清楚。
由胜肽圖譜分析，我們運用RACE方法選殖到全長為1179個核苷酸，並得知VVA1是一個具有393個胺基酸的蛋白不含半胱胺酸及訊號胜肽。有趣的是經由BLAST比對，我們發現VVA1的胺基酸序列與VVA2 tandem repeat 相似性很高。二級結構分析得知VVA1有兩對amphipathic alpha-helices，但是缺乏與肝磷脂結合(HBS)之結構。由溶血及細胞毒性的實驗，得知VVA1本身無溶血或是造成細胞死亡的能力，然而在VVA2比VVA1之分子數比為2或比值更小，VVA1能完全抑制VVA2的溶血及細胞毒性。此結果說明VVA1與VVA2可能具有交互作用的能力，但是VVA1無法與細胞膜作用。利用酵素免疫結合吸附法(ELISA) 和超高速離心，我們發現VVA1與liposomes作用很弱，但是，在VVA2比VVA1之分子數比為2時VVA1抑制VVA2與liposomes作用，同時抑制VVA2聚合體的產生。利用免疫螢光染色及共軛焦顯微鏡觀察，證實VVA1不結合在細胞膜上，在分子數比為2時，抑制VVA2對細胞膜的結合。以pull-down 的實驗，在可模擬磷脂質兩親性（amphipathic）的Triton-X-100緩衝液存在下，發現VVA1可與VVA2直接作用。進一步透過co-pull-down的實驗及利用胜肽片段競爭的實驗，顯示一個分子VVA1所攜帶的兩對amphipathic alpha-helices可能與兩個分子VVA2的amphipathic alpha-helix直接作用形成聚合物。
另一方面，在本研究也發現VVA1在分子數比大於2時已無法抑制VVA2的溶血及細胞毒性的能力，但在VVA2/VVA1比值為 8時毒性達到最強。此分子數比所影響的毒性進一步以動物實驗來佐證，VVA1和VVA2單獨時都不具明顯的毒性，若以不同分子數比混合VVA2與VVA1則發現毒性漸強，在VVA2比VVA1 為8的毒性提升至最強，死亡的老鼠經抽血檢測發現有溶血現象伴隨產生。探討為何VVA2於老鼠體內失去毒性功能，我們由血漿中分離及純化能降低VVA2活性的成分。血漿經通過分子篩、ProteomeLab PF 2D系統及質譜的分析得知，apoB100促使VVA2形成不可逆的大分子聚合體，因此降低了VVA2的毒性。
綜上所述，本研究中證實VVA1與VVA2利用彼此的amphipathic alpha-helix直接交互作用，透過不同分子數比VVA1在體內或是體外都可直接調節VVA2的活性，這樣的調節模式對於治療癌症可提供一個新的研究方向。

Volvatoxin A (VVA) is a cardiotoxic protein isolated from edible mushroom Volvariella volvacea. It was demonstrated that VVA consists of a pore-forming cardiotoxic protein volvatoxin A2 (VVA2) and volvatoxin A1 (VVA1). Previous studies suggest that only VVA2 is endowed with hemolytic and cytotoxic activity but the function of VVA1 is unclear.
The primary structure of VVA1, elucidated by peptide mapping and cDNA nucleotide sequencing was found to consist of 393 amino acid residues without cysteine residue and signal peptide. Interestingly, the amino acids sequence of VVA1 closely resembles a tandem-repeat of VVA2. VVA1 contains two pairs of amphipathic alpha-helices but it lacks a heparin binding site (HBS). VVA1 itself has no hemolytic and cytotoxic activities, however, it inhibits the toxicity of VVA2 at the molar ratio of 2 or less. This suggests that VVA1 may interact with VVA2 but not with the cell membrane. By ELISA assay and ultracentrifugation analysis, VVA1 did not interact with phospholipids as VVA2 did. In contrast, at the molar ratio of 2, VVA1 inhibited the VVA2 binding on the phospholipids and abolished the VVA2 oligomerization which is essential for pore formation. Furthermore, the study with confocal microscopy, demonstrated that VVA1 could not bind to the cell membrane, however, it inhibited VVA2 binding to the cell membrane at the molar ratio of 2. By pull-down experiment, VVA1 directly interacted with VVA2 in the amphipathic environment established by Triton-X-100, which was similar to the environment of cell membranes. By co-pull-down experiment and peptide competition assay we found that VVA1 and VVA2 could form complex by the two pairs of amphipathic alpha-helices. The results suggest that this occurs via the interaction of one molecule of VVA1 with two molecules of VVA2.
In the present study, we demonstrated that VVA1 could not inhibit hemolytic and cytotoxic activity of VVA2 at the VVA2/VVA1 molar ratios higher than 2, while the maximum toxicity was at the molar ratio of 8. The toxicity assay by experimental animals showed that the toxicity of either VVA1 or VVA2 only was low, however, the toxicity was enhanced at molar ratios higher than 4. The degree of hemolysis in the mice that were i.p. injected with a mixture of VVA2 and VVA1 was correlated with that of the lethality. In order to study why VVA2 lost toxicity in vivo, the putative inhibitor was purified from the human plasma by gel filtration chromatography, and ProteomeLab PF 2D system, and identified with a mass spectrum. The lipoprotein, apoB100, was found to interact with VVA2 and induced VVA2 oligomerization, therefore, inhibited the toxicity of VVA2.
Taken together, this study revealed a novel mechanism by which VVA1 and lipoporotein apoB100 regulates the cytotoxicity of VVA2 by direct interaction of amphipathic alpha-helix, which provides us the new mechanism of the potential clinical research of pore-forming toxin for cancer therapy.

ABBREVIATIONS 5
ABSTRACT 6
摘要 8
OVERVIEW and RATIONALE 10
INTRODUCTION 11
MATERIALS and METHODS 14
Materials 14
Purification of VVA1, and its primary structure 14
cDNA cloning of VVA1 and construction of expression vectors 15
Site-directed mutagenesis 17
Hemolysis Assay 18
Cytotoxicity assay 18
Animal toxicity assay 18
Preparation of liposomes 19
Oligomerization regulation of VVA1 to VVA2 by SDS-PAGE Analysis 19
Enzyme-linked immunosorbent assay (ELISA assay) 20
Binding Analysis by Surface Plasmon Resonance (SPR) 21
Preparation of reVVA1 and its mutants 21
Pull-down assay 22
Co-pull-down assay 23
Peptide competition assay 23
Confocal microscopy 24
DNA ladder analysis 25
TUNEL assay 25
Analysis of VVA2 binding protein(s) in human plasma 26

RESULTS 28
cDNA cloning and characteristics of VVA1 28
Effects of VVA1 on the hemolytic and cytotoxic activity of VVA2 29
Effects of VVA1 on VVA2 in the presence of liposomes and cell membrane 30
Interactions between VVA1 and VVA2 32
Number of VVA1 binding sites for VVA2 34
Interaction of VVA1 and VVA2 by amphipathic a-helix 35
VVA2 induced cell membrane damage and apoptosis 36
Protein internalization via the pore induced by VVA2 36
Animal toxicity 37
Inhibition of VVA2 toxicity by apoB100 protein 38

DISCUSSION 40
Two component toxins, VVA1 and VVA2 40
VVA1 is a novel regulator of VVA2 41
Clinical application of VVA in cancer therapy 42
A model for VVA1 regulate the activity of VVA2 43

FIGURES and LEGENDS 45
Fig. 1. The fruit bodies of Volvariella volvacea 45
Fig. 2. Amino Acid Sequence of VVA1 46
Fig. 3. cDNA cloning of VVA1 47
Fig. 4. Alignment of the deduced amino acid sequence of VVA1-NTD and VVA1-CTD with that of VVA2 50
Fig. 5. Hydrophobic moment of amphipathic a-helix-C and -D of VVA1-NTD, and a-helix-D’ and -E’ of VVA1-CTD 52
Fig. 6. Regulation of VVA2 activity by VVA1 53
Fig. 7. Interactions of phospholipids with VVA1 and VVA2 54
Fig. 8. Interaction of VVA1 and VVA2 analyzed by SPR Assay 55
Fig. 9. Inhibition of VVA2 binding on cell membrane by VVA1 56
Fig. 10. Effects of Triton-X-100, SDS or sodium deoxycholate and liposomes on the oligomerization of VVA2 57
Fig. 11. Pull-down experiments 58
Fig. 12. Co-pull-down experiments 59
Fig. 13. Peptide competition assay 60
Fig. 14. VVA2 induced HeLa cell apoptosis identified by DNA gel electrophoresis and TUNEL assay 61
Fig. 15. Co-localization of VVA1 and VVA2 63
Fig. 16. Hemolysis of the mice 64
Fig. 17. Human plasma protein inhibits the hemolytic activity of VVA2 65
Fig. 18. Separation human plasma by the Sephadex G100 column chromatography 66
Fig. 19. 1st dimension of ProteomLab PF 2D system fractionation of the first peak obtained by Sephadex G100 coulmn chromatography. 68
Fig. 20. Purification the inhibitor(s) of VVA2 in human plasma 69
Fig. 21. ApoB100 inhibited the activity of VVA2 by inducing the oligomerization of VVA2 70
Fig. 22. Schematic representation of a structural model of VVA1 71
Fig. 23. Model of VVA1 controls VVA2 activity 72

TABLES 73
Table I 73
Table II 74

REFERENCES 75

APPENDEX I (LC-MS) 80
APPENDEX II (CV) 83
APPENDEX III (publication) 86

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