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研究生:翁育萍
研究生(外文):Yui-Ping Weng
論文名稱:草菇毒蛋白VVA2與細胞膜作用機制及其功能性區域之探討
論文名稱(外文):Studies on the mechanism of VVA2 interacting with cell membrane and its functional domains analysis
指導教授:林榮耀林榮耀引用關係
指導教授(外文):Jung-Yaw Lin
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:114
中文關鍵詞:草菇毒蛋白A2孔洞形成毒素寡聚合體形成溶血作用機制孔洞形成前假說定點突變法功能性區域限制性蛋白質水解
外文關鍵詞:Volvatoxin A2pore-forming toxinoligomerizationmechanism of hemolysispre-pore modelsite-directed mutagenesisfunctional domainslimited proteolysis
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草菇毒蛋白A2 (Volvatoxin A2;VVA2)是由草菇子實體純化出來的一種溶血毒素 (hemolysin)又稱為心臟毒素 (cardiotoxin)。胺基酸定序結果得知VVA2共由199個胺基酸組成,但不具雙硫鍵。運用RACE方法,已選殖到全長為597個nucleotides的VVA2 cDNA序列,且其所轉譯 (encode)出的胺基酸序列與由蛋白質分析所獲得的一致。VVA2與已知的蘇力菌 (Bacillus thuringiensis subspecies Kynshuensis)分離出的孔洞形成毒素 (pore-forming toxin)-CytB有48.7 %相似性,故我們推測VVA2造成細胞溶解 (cytolysis) 的機制,可能是藉由孔洞形成方式造成的。由免疫螢光染色 (immunofluorescence)實驗,可觀察到VVA2與細胞作用後,會分佈並聚集在細胞膜上。利用穿透式電子顯微鏡 (transmission electron microscopy)觀察,則發現VVA2可在微脂體 (liposomes)表面產生約20-30 nm的孔洞。利用非洲爪蟾之培養肌細胞 (cultured Xenopus myocytes)進行patch-clamp電生理實驗,更顯示VVA2的作用機制是可穿透細胞膜,且其孔洞可讓分子量為70 KDa的dextran進出。使用蔗糖梯度超高速離心 (sucrose-density gradient ultracentrifugation)及gel filtration實驗則證明VVA2在溶液中是以單體 (monomer)型式存在,但其與細胞膜作用後會轉變成寡聚合體 (oligomer)。利用圓二色光譜 (circular dichroism)及螢光儀(fluorimeter)可分析出VVA2由單體型式轉變成寡聚合體的過程中,會產生二級及三級蛋白質結構變化。運用SPR、stop-flow fluorescence 及NuPAGE技術分析VVA2與細胞膜作用之動力學特徵,結果顯示VVA2與細胞膜結合後,大部分不解離,我們亦發現VVA2在寡聚合反應 (oligomerization)發生之時間與其蛋白質結構之改變 (conformational change)是相伴發生的。
此外,利用蛋白質限制水觧 (limited proteolysis)及重組蛋白實驗,我們亦鑑定出一段N端片段 (N-terminal fragment; NTF)(1-127殘基),並發現其與微脂體作用相似,具抑制VVA2溶血活性及促進其寡聚合物形成之效果,且其可在reNTF/VVA2分子數比例低於0.01時仍具抑制活性。利用BIAcore系統亦證實reNTF與VVA2兩者具有直接交互作用。此現象說明了reNTF是經由seeding effect造成VVA2之寡聚合反應。利用定點突變 (site-directed mutagenesis),我們發現VVA2之二級結構中的amphipathic alpha-helix B,對兩者之活性上扮演著必要的角色。此外,運用Western blot及BIAcore系統亦鑑定出重組C尾片端 (reC-terminal fragment; reCTF)(128-199殘基)是VVA2結合至細胞膜的功能區。經由酵素水觧VVA2與微脂體作用之複合物 (VVA2-liposomes complexes),則確認VVA2孔洞形成功能區位置乃包含C端beta-6, 7, 8 strands。
統整以上實驗結果,我們可以歸納VVA2包含二個功能性區域,NTF及CTF。NTF主要負責VVA2寡聚合之形成,而CTF則擔任其與細胞膜結合及嵌入細胞膜之角色。總之,我們的實驗結果支持寡聚合形成過程發生在細胞膜嵌入之前,即pre-pore模式之假說。

Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the edible mushroom Volvariella volvacea. The primary structure of VVA2, elucidated by protein sequencing and cDNA nucleotide sequencing was found to be comprised of 199 amino acid residues without cysteine residues. VVA2 had 48.7 % homology to delta-endotoxin CytB, an insecticidal and pore-forming protein isolated from Bacillus thuringiensis subspecies Kynshuensis. By immunofluorescence staining, VVA2 was found to predominately localize and aggregate on cell membrane. VVA2 forms pores in liposomes with diameters of 30-50 nm corresponding to oligomer formation analyzed by sucrose density-gradient ultracentrifugation. By patch clamp analysis, membrane permeabilization and passage of 70-kDa dextran rhodamine B in cultured Xenopus myocytes by VVA2 were demonstrated. Conformational change of VVA2 upon interacting with liposome was revealed by circular dichroism (CD) and tryptophan fluorescence emission spectra. The irreversible binding of VVA2 to the membrane was demonstrated by surface plasmon resonance (SPR) analysis. Furthermore, the molecular events of VVA2 interacting with phospholipids accompanied by the oligomerization and conformational change were shown by stopped-flow fluorescence and NuPAGE system.
In addition, we identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 obtained by tryptic digestion as a domain for oligomerization of VVA2. On pre-incubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic alpha-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the reC-terminal fragment (reCTF) (128-199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing beta-6, 7, and 8, which is protected from protease digestion due to its insertion of a membrane.
Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.

TABLE OF CONTENTS
page
I. Abbreviations used -------------- 2
II. Abstract (Chinese) ------------- 4
III. Abstract -------------- 6
IV. Introduction -------------- 8
V. Materials --------------- 13
VI. Methods ---------------- 17
VII. Results ---------------- 33
VIII. Discussion ---------------- 47
IX. Figures ---------------- 55
X. Tables ---------------- 101
XI. References ---------------- 105

REFERENCES
1. Lin, J.Y., Jeng, T.W., Chen, C.C., Shi, G.Y. and Tung, T.C. (1973) Isolation of a new cardiotoxic protein from the edible mushroom, Volvariella volvacea. Nature, 246, 524-5.
2. Fassold, E., Slade, A., Lin, J.Y. and Nayler, W. (1976) An effect of the cardiotoxic protein volvatoxin A on the function and structure of heart muscle cells. J. Mol. Cell Cardiol., 8, 501-19.
3. Rowe, G.E. and Welch, R.A. (1994) Assays of hemolytic toxins. Methods Enzymol., 235, 657-67.
4. Bhakdi, S., Valeva, A., Walev, I., Zitzer, A. and Palmer, M. (1998) Pore-forming bacterial cytolysins. Soc. Appl. Bacteriol. Symp. Ser., 27, 15S-25S.
5. Titball, R.W. (1999) Membrane-damaging and cytotoxic phospholipases. In Alouf, J.E., and Freer, J. H. (ed.), The Comprehensive Sourcebook of Bacteria Protein Toxins. Acedimic press, London, pp. 310-329.
6. Alouf, J.E., and Palmer, M. W. (1999) Streptolysin O. In Alouf, J.E., and Freer, J. H. (ed.), The Comprehensive Sourcebook of Bacteria Protein Toxins. Academic press, London, pp. 457-475.
7. Alouf, J.E. (1999) Introduction to the family of the structurally related cholesterol-binding cytolysins. In Alouf, J.E., and Freer, J. H. (ed.), The Comprehensive Sourcebook of Bacteria Protein Toxins. Academic press, London, pp. 443-456.
8. Menestrina, G., Serra, M.D. and Prevost, G. (2001) Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family. Toxicon, 39, 1661-72.
9. Prevost, G., Mourey, L., Colin, D.A. and Menestrina, G. (2001) Staphylococcal pore-forming toxins. Curr. Top. Microbiol. Immunol., 257, 53-83.
10. Tomita, T. and Kamio, Y. (1997) Molecular biology of the pore-forming cytolysins from Staphylococcus aureus, alpha- and gamma-hemolysins and leukocidin. Biosci. Biotechnol. Biochem., 61, 565-72.
11. Bhakdi, S., Bayley, H., Valeva, A., Walev, I., Walker, B., Kehoe, M. and Palmer, M. (1996) Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins. Arch. Microbiol., 165, 73-9.
12. Song, L., Hobaugh, M.R., Shustak, C., Cheley, S., Bayley, H. and Gouaux, J.E. (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science, 274, 1859-66.
13. Rossjohn, J., Feil, S.C., McKinstry, W.J., Tsernoglou, D., van der Goot, G., Buckley, J.T. and Parker, M.W. (1998) Aerolysin--a paradigm for membrane insertion of beta-sheet protein toxins? J. Struct. Biol., 121, 92-100.
14. Buckley, J.T. (1999) The channel-forming toxin aerolysin. In Alouf, J.E., and Freer, J. H. (ed.), The Comprehensive Sourcebook of Bacteria Protein Toxins. Academic press, London, pp. 362-372.
15. Abrami, L., Fivaz, M. and van der Goot, F.G. (2000) Adventures of a pore-forming toxin at the target cell surface. Trends. Microbiol., 8, 168-72.
16. Gilbert, R.J. (2002) Pore-forming toxins. Cell Mol. Life Sci., 59, 832-44.
17. Montoya, M. and Gouaux, E. (2003) Beta-barrel membrane protein folding and structure viewed through the lens of alpha-hemolysin. Biochim. Biophys. Acta, 1609, 19-27.
18. Shatursky, O., Heuck, A.P., Shepard, L.A., Rossjohn, J., Parker, M.W., Johnson, A.E. and Tweten, R.K. (1999) The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell, 99, 293-9.
19. Lally, E.T., Kieba, I.R., Sato, A., Green, C.L., Rosenbloom, J., Korostoff, J., Wang, J.F., Shenker, B.J., Ortlepp, S., Robinson, M.K. and Billings, P.C. (1997) RTX toxins recognize a beta2 integrin on the surface of human target cells. J. Biol. Chem., 272, 30463-9.
20. Diep, D.B., Nelson, K.L., Raja, S.M., Pleshak, E.N. and Buckley, J.T. (1998) Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin. J. Biol. Chem., 273, 2355-60.
21. Heuck, A.P., Tweten, R.K. and Johnson, A.E. (2001) Beta-barrel pore-forming toxins: intriguing dimorphic proteins. Biochemistry, 40, 9065-73.
22. Patel, H.V., Vyas, A.A., Vyas, K.A., Liu, Y.S., Chiang, C.M., Chi, L.M. and Wu, W.G. (1997) Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action. J. Biol. Chem., 272, 1484-92.
23. Vyas, K.A., Patel, H.V., Vyas, A.A. and Wu, W.G. (1998) Glycosaminoglycans bind to homologous cardiotoxins with different specificity. Biochemistry, 37, 4527-34.
24. Sue, S.C., Chien, K.Y., Huang, W.N., Abraham, J.K., Chen, K.M. and Wu, W.G. (2002) Heparin binding stabilizes the membrane-bound form of cobra cardiotoxin. J. Biol. Chem., 277, 2666-73.
25. Fukushima, K., Ikehara, Y., Kanai, M., Kochibe, N., Kuroki, M. and Yamashita, K. (2003) A beta -N-acetylglucosaminyl phosphate diester residue is attached to the glycosyl-phosphatidylinositol (GPI) anchor of human placental alkaline phosphatase: A target of the channel-forming toxin aerolysin. J. Biol. Chem., 278, 36296-303.
26. Bayley, H. (1997) Building doors into cells. Sci. Am., 277, 62-7.
27. Panchal, R.G. (1998) Novel therapeutic strategies to selectively kill cancer cells. Biochem. Pharmacol., 55, 247-52.
28. Provoda, C.J. and Lee, K.D. (2000) Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules. Adv. Drug Deliv. Rev., 41, 209-21.
29. Panchal, R.G., Smart, M.L., Bowser, D.N., Williams, D.A. and Petrou, S. (2002) Pore-forming proteins and their application in biotechnology. Curr. Pharm. Biotechnol., 3, 99-115.
30. Herlax, V. and Bakas, L.S. (2002) [Therapeutic applications of pore-forming lytic toxins: potential use of Escherichia coli alpha-hemolysin]. Medicina (B Aires), 62, 66-72.
31. Gentschev, I., Dietrich, G. and Goebel, W. (2002) The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol., 10, 39-45.
32. Palmer, M., Harris, R., Freytag, C., Kehoe, M., Tranum-Jensen, J. and Bhakdi, S. (1998) Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization. EMBO J., 17, 1598-605.
33. Hotze, E.M., Wilson-Kubalek, E.M., Rossjohn, J., Parker, M.W., Johnson, A.E. and Tweten, R.K. (2001) Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane beta-sheet from a prepore intermediate. J. Biol. Chem., 276, 8261-8.
34. Hotze, E.M., Heuck, A.P., Czajkowsky, D.M., Shao, Z., Johnson, A.E. and Tweten, R.K. (2002) Monomer-monomer interactions drive the prepore to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin. J. Biol. Chem., 277, 11597-605.
35. Kumar, T.K., Jayaraman, G., Lee, C.S., Arunkumar, A.I., Sivaraman, T., Samuel, D. and Yu, C. (1997) Snake venom cardiotoxins-structure, dynamics, function and folding. J. Biomol. Struct. Dyn., 15, 431-63.
36. Vyas, A.A., Pan, J.J., Patel, H.V., Vyas, K.A., Chiang, C.M., Sheu, Y.C., Hwang, J.K. and Wu, W.G. (1997) Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif. J. Biol. Chem., 272, 9661-70.
37. Ludwig, A., and Goebel, W. (1999) The family of the multigenic encoded RTX toxins. In Alouf, J.E., and Freer, J. H. (ed.), The Comprehensive Sourcebook of Bacteria Protein Toxins. Academic press, London, pp. 330-348.
38. Shimada, Y., Nakamura, M., Naito, Y., Nomura, K. and Ohno-Iwashita, Y. (1999) C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin. J. Biol. Chem., 274, 18536-42.
39. Palmer, M., Saweljew, P., Vulicevic, I., Valeva, A., Kehoe, M. and Bhakdi, S. (1996) Membrane-penetrating domain of streptolysin O identified by cysteine scanning mutagenesis. J. Biol. Chem., 271, 26664-7.
40. Heuck, A.P., Hotze, E.M., Tweten, R.K. and Johnson, A.E. (2000) Mechanism of membrane insertion of a multimeric beta-barrel protein: perfringolysin O creates a pore using ordered and coupled conformational changes. Mol Cell, 6, 1233-42.
41. Hsu, H.C., Hsu, C.I., Lin, R.H., Kao, C.L. and Lin, J.Y. (1997) Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem. J., 323, 557-65.
42. Liu, C., Yang, Z., Yang, J., Xia, Z. and Ao, S. (2000) Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation. J. Biol. Chem., 275, 31972-8.
43. Cordoba, O.L., Linskens, S.B., Dacci, E. and Santome, J.A. (1997) 'In gel' cleavage with cyanogen bromide for protein internal sequencing. J. Biochem. Biophys. Methods, 35, 1-10.
44. Takeuchi, K., Nakamura, K., Fujimoto, M., Kaino, S., Kondoh, S. and Okita, K. (2002) Heat stress-induced loss of eukaryotic initiation factor 5A (eIF-5A) in a human pancreatic cancer cell line, MIA PaCa-2, analyzed by two-dimensional gel electrophoresis. Electrophoresis, 23, 662-9.
45. Liu, C.L., Tsai, C.C., Lin, S.C., Wang, L.I., Hsu, C.I., Hwang, M.J. and Lin, J.Y. (2000) Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis. Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity. J. Biol. Chem., 275, 1897-901.
46. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 76-85.
47. Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K. (1999) In Short Protocols in Molecular Biology, pp. 14-22.
48. Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch., 391, 85-100.
49. Nieuwkoop, P.D., Faber, J. (1967) Normal table of Xenopus Laevis. In, Amsterdam, North-Holland.
50. Tabti, N., Poo, M. M. (1991) In Banker, G.a.G., K. (ed.), Culturing Spinal cord neurons and muscle cells from Xenopus embryos, Cambridge, MIT, MA.
51. Nakamura, M., Sekino, N., Iwamoto, M. and Ohno-Iwashita, Y. (1995) Interaction of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, with liposomal membranes: change in the aromatic side chains upon binding and insertion. Biochemistry, 34, 6513-20.
52. Wang, W., Smith, D.K., Moulding, K. and Chen, H.M. (1998) The dependence of membrane permeability by the antibacterial peptide cecropin B and its analogs, CB-1 and CB-3, on liposomes of different composition. J. Biol. Chem., 273, 27438-48.
53. Danelian, E., Karlen, A., Karlsson, R., Winiwarter, S., Hansson, A., Lofas, S., Lennernas, H. and Hamalainen, M.D. (2000) SPR biosensor studies of the direct interaction between 27 drugs and a liposome surface: correlation with fraction absorbed in humans. J. Med. Chem., 43, 2083-6.
54. Bartlett, G.R. (1959) Phosphorus Assay in Column Chromatography. J. Biol. Chem., 234, 466-468.
55. Galvan, D.L., Borrego-Diaz, E., Perez, P.J. and Mignery, G.A. (1999) Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor. J. Biol. Chem., 274, 29483-92.
56. Lee, S.F., Wang, C.T., Liang, J.Y., Hong, S.L., Huang, C.C. and Chen, S.S. (2000) Multimerization potential of the cytoplasmic domain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41. J. Biol. Chem., 275, 15809-19.
57. Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E. and Hashimoto, Y. (1998) Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell. Biochem. J., 333, 129-37.
58. Sugawara, N., Tomita, T. and Kamio, Y. (1997) Assembly of Staphylococcus aureus gamma-hemolysin into a pore-forming ring-shaped complex on the surface of human erythrocytes. FEBS Lett., 410, 333-7.
59. McInerney, T.L., El Ahmar, W., Kemp, B.E. and Poumbourios, P. (1998) Mutation-directed chemical cross-linking of human immunodeficiency virus type 1 gp41 oligomers. J. Virol., 72, 1523-33.
60. Nygard, O. and Nika, H. (1982) Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes. EMBO J., 1, 357-62.
61. Holmberg, L. and Nygard, O. (1994) Interaction sites of ribosome-bound eukaryotic elongation factor 2 in 18S and 28S rRNA. Biochemistry, 33, 15159-67.
62. Chang, C.T., Wu, C.S. and Yang, J.T. (1978) Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal. Biochem., 91, 13-31.
63. Yang, J.T., Wu, C.S. and Martinez, H.M. (1986) Calculation of protein conformation from circular dichroism. Methods Enzymol., 130, 208-69.
64. Tetin, S.Y., Prendergast, F.G. and Venyaminov, S.Y. (2003) Accuracy of protein secondary structure determination from circular dichroism spectra based on immunoglobulin examples. Anal. Biochem., 321, 183-7.
65. Hennessey, J.P., Jr. and Johnson, W.C., Jr. (1981) Information content in the circular dichroism of proteins. Biochemistry, 20, 1085-94.
66. Du, J., Knowles, B.H., Li, J. and Ellar, D.J. (1999) Biochemical characterization of Bacillus thuringiensis cytolytic toxins in association with a phospholipid bilayer. Biochem. J., 338, 185-93.
67. Schagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 166, 368-79.
68. Quertenmont, P., Wattiez, R., Falmagne, P., Ruysschaert, J.M. and Cabiaux, V. (1996) Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study. Mol. Microbiol., 21, 1283-96.
69. Lin, W.H., Hung, C.H., Hsu, C.I. and Lin, J.Y. (1997) Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J. Biol. Chem., 272, 20044-8.
70. Chen, J.K., Hung, C.H., Liaw, Y.C. and Lin, J.Y. (1997) Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis. Protein Eng., 10, 827-33.
71. Chong, S., Shao, Y., Paulus, H., Benner, J., Perler, F.B. and Xu, M.Q. (1996) Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J. Biol. Chem., 271, 22159-68.
72. Chong, S. and Xu, M.Q. (1997) Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J. Biol. Chem., 272, 15587-90.
73. Zhou, J.Z., Li, X.H., Zhang, H.G., Tang, Y. and Wang, X.Q. (2003) Cloning and gene expression of G protein competitive inhibitory polypeptide and its prophylactic effects on myocardial hypertrophy in vitro. Acta Pharmacol. Sin., 24, 1108-12.
74. Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene, 192, 271-81.
75. Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M.Q. and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic Acids Res., 26, 5109-15.
76. Martin, G.A., Kawaguchi, R., Lam, Y., DeGiovanni, A., Fukushima, M. and Mutter, W. (2001) High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/ translation system. Biotechniques, 31, 948-50, 952-3.
77. Sangha, N., Kaur, S., Sharma, V. and Krishnasastry, M.V. (1999) Importance of the carboxyl terminus in the folding and function of alpha-hemolysin of Staphylococcus aureus. J. Biol. Chem., 274, 9193-9.
78. Eschbach, E., Scharsack, J.P., John, U. and Medlin, L.K. (2001) Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae. J. Appl. Toxicol., 21, 513-9.
79. Kim, Y.M., Chung, H.T., Simmons, R.L. and Billiar, T.R. (2000) Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition. J. Biol. Chem., 275, 10954-61.
80. Fang, J., Lu, F. and Chen, C.Q. (2001) Dual function of human necrosis factor receptor 75 in cytotoxicity induced by human tumor necrosis factor alpha. Acta Pharmacol. Sin., 22, 1039-44.
81. Fang, L., Fang, J. and Chen, C.Q. (2001) TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death. Cell Res., 11, 217-22.
82. Karlsson, O.P. and Lofas, S. (2002) Flow-mediated on-surface reconstitution of G-protein coupled receptors for applications in surface plasmon resonance biosensors. Anal. Biochem., 300, 132-8.
83. Erb, E.M., Chen, X., Allen, S., Roberts, C.J., Tendler, S.J., Davies, M.C. and Forsen, S. (2000) Characterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chip. Anal. Biochem., 280, 29-35.
84. Huang, P.J. (1993) The complete amino acid sequence and gene cloning of volvatoxin A2 isolated from Volvariella volvacea. Institute of Biology, National Taiwan Normal University, Taipei, Master thesis.
85. Weng, Y.P. (2000) Volvatoxin A2, a Pore-Forming Cardiotoxic Protein from the edible mushroom, Volvariella volvacea. Proceedings of Taiwan Society for Biochemistry and Molecular Biology, Taipei, Taiwan, pp. 49.
86. Li, J., Koni, P.A. and Ellar, D.J. (1996) Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J. Mol. Biol., 257, 129-52.
87. Promdonkoy, B. and Ellar, D.J. (2000) Membrane pore architecture of a cytolytic toxin from Bacillus thuringiensis. Biochem. J., 350 Pt 1, 275-82.
88. Rost, B. and Sander, C. (1994) Combining evolutionary information and neural networks to predict protein secondary structure. Proteins, 19, 55-72.
89. Rost, B. and Sander, C. (1993) Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol., 232, 584-99.
90. Linke, T., Wilkening, G., Sadeghlar, F., Mozcall, H., Bernardo, K., Schuchman, E. and Sandhoff, K. (2001) Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins. J. Biol. Chem., 276, 5760-8.
91. Liang, P.H. and Anderson, K.S. (1998) Kinetic reaction scheme for the dihydrofolate reductase domain of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major. Biochemistry, 37, 12206-12.
92. Eisenberg, D., Weiss, R.M. and Terwilliger, T.C. (1982) The helical hydrophobic moment: a measure of the amphiphilicity of a helix. Nature, 299, 371-4.
93. Watanabe, H., Narai, A. and Shimizu, M. (1999) Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco-2 cell monolayer. Eur. J. Biochem., 262, 850-7.
94. Miles, G., Cheley, S., Braha, O. and Bayley, H. (2001) The staphylococcal leukocidin bicomponent toxin forms large ionic channels. Biochemistry, 40, 8514-22.
95. Lin, Y.P. (2000) Studies on the expression of the C-terminal fragment of Volvatoxin A2, and its structure and function. Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, master thesis.
96. Heuck, A.P., Tweten, R.K. and Johnson, A.E. (2003) Assembly and topography of the prepore complex in cholesterol-dependent cytolysins. J. Biol. Chem., 278, 31218-25.
97. Vecsey-Semjen, B., Knapp, S., Mollby, R. and van der Goot, F.G. (1999) The staphylococcal alpha-toxin pore has a flexible conformation. Biochemistry, 38, 4296-302.
98. Fang, Y., Cheley, S., Bayley, H. and Yang, J. (1997) The heptameric prepore of a staphylococcal alpha-hemolysin mutant in lipid bilayers imaged by atomic force microscopy. Biochemistry, 36, 9518-22.
99. Miller, C.J., Elliott, J.L. and Collier, R.J. (1999) Anthrax protective antigen: prepore-to-pore conversion. Biochemistry, 38, 10432-41.
100. Hong, Q., Gutierrez-Aguirre, I., Barlic, A., Malovrh, P., Kristan, K., Podlesek, Z., Macek, P., Turk, D., Gonzalez-Manas, J.M., Lakey, J.H. and Anderluh, G. (2002) Two-step membrane binding by Equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix. J. Biol. Chem., 277, 41916-24.
101. Venema, R.C., Sayegh, H.S., Arnal, J.F. and Harrison, D.G. (1995) Role of the enzyme calmodulin-binding domain in membrane association and phospholipid inhibition of endothelial nitric oxide synthase. J. Biol. Chem., 270, 14705-11.
102. Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J.A., Blum, H.E., Penin, F. and Moradpour, D. (2002) An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem., 277, 8130-9.
103. Lee, H.J., Choi, C. and Lee, S.J. (2002) Membrane-bound alpha-synuclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form. J. Biol. Chem., 277, 671-8.
104. Eskes, R., Desagher, S., Antonsson, B. and Martinou, J.C. (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol., 20, 929-35.
105. Selkoe, D.J. (2002) Alzheimer's disease is a synaptic failure. Science, 298, 789-91.
106. Johnstone, R.W., Ruefli, A.A. and Smyth, M.J. (2000) Multiple physiological functions for multidrug transporter P-glycoprotein? Trends Biochem. Sci., 25, 1-6.
107. Johnstone, R.W., Cretney, E. and Smyth, M.J. (1999) P-glycoprotein protects leukemia cells against caspase-dependent, but not caspase-independent, cell death. Blood, 93, 1075-85.
108. Johnstone, R.W., Tainton, K.M., Ruefli, A.A., Froelich, C.J., Cerruti, L., Jane, S.M. and Smyth, M.J. (2001) P-glycoprotein does not protect cells against cytolysis induced by pore-forming proteins. J. Biol. Chem., 276, 16667-73.
109. Yamanaka, H., Satoh, T., Katsu, T. and Shinoda, S. (1987) Mechanism of haemolysis by Vibrio vulnificus haemolysin. J. Gen. Microbiol., 133, 2859-64.
110. Iverius, P.H. (1971) Coupling of glycosaminoglycans to agarose beads (Sepharose 4B). Biochem. J., 124, 677-83.

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