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. 2000 Aug 1;349(Pt 3):835–841. doi: 10.1042/bj3490835

The membrane insertion of trichosanthin is membrane-surface-pH dependent.

X F Xia 1, S F Sui 1
PMCID: PMC1221212  PMID: 10903146

Abstract

Trichosanthin (TCS) is the active component extracted from Tianhuafen, a traditional herbal medicine that has been used for abortion in China for centuries. It belongs to the type-I ribosome-inactivating protein (RIP) family and can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. Recent studies have shown TCS to be multifunctional, its pharmacological properties including immunomodulatory, anti-tumour and anti-HIV activities. The membrane-insertion property of TCS is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In this paper, the membrane-insertion mechanism of TCS was studied. The monolayer experiment revealed that TCS's membrane-insertion ability was dependent on low pH. Fluorescence spectroscopy using 1-anilinonaphthalene-8-sulphonic acid as a probe showed that low pH may induce the conformational change of TCS that leads to the hydrophobic-site exposure, and the CD result showed that this conformational change did not alter its secondary structure. Such conformational change leads to an intermediate state, called the 'molten globular state' by previous investigators. The pH-dependent membrane insertion and conformational change were related by the fact that the optimal membrane-surface pH needed was the same for the two events. From these and other results, a membrane-insertion model was proposed.

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Selected References

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  1. Beaumelle B., Alami M., Hopkins C. R. ATP-dependent translocation of ricin across the membrane of purified endosomes. J Biol Chem. 1993 Nov 5;268(31):23661–23669. [PubMed] [Google Scholar]
  2. Beaumelle B., Alami M., Hopkins C. R. ATP-dependent translocation of ricin across the membrane of purified endosomes. J Biol Chem. 1993 Nov 5;268(31):23661–23669. [PubMed] [Google Scholar]
  3. Beaumelle B., Bensammar L., Bienvenüe A. Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes. J Biol Chem. 1992 Jun 5;267(16):11525–11531. [PubMed] [Google Scholar]
  4. Blewitt M. G., Chung L. A., London E. Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration. Biochemistry. 1985 Sep 24;24(20):5458–5464. doi: 10.1021/bi00341a027. [DOI] [PubMed] [Google Scholar]
  5. Byers V. S., Levin A. S., Malvino A., Waites L., Robins R. A., Baldwin R. W. A phase II study of effect of addition of trichosanthin to zidovudine in patients with HIV disease and failing antiretroviral agents. AIDS Res Hum Retroviruses. 1994 Apr;10(4):413–420. doi: 10.1089/aid.1994.10.413. [DOI] [PubMed] [Google Scholar]
  6. Byers V. S., Levin A. S., Waites L. A., Starrett B. A., Mayer R. A., Clegg J. A., Price M. R., Robins R. A., Delaney M., Baldwin R. W. A phase I/II study of trichosanthin treatment of HIV disease. AIDS. 1990 Dec;4(12):1189–1196. doi: 10.1097/00002030-199012000-00002. [DOI] [PubMed] [Google Scholar]
  7. Chang C. T., Wu C. S., Yang J. T. Circular dichroic analysis of protein conformation: inclusion of the beta-turns. Anal Biochem. 1978 Nov;91(1):13–31. doi: 10.1016/0003-2697(78)90812-6. [DOI] [PubMed] [Google Scholar]
  8. Collins E. J., Robertus J. D., LoPresti M., Stone K. L., Williams K. R., Wu P., Hwang K., Piatak M. Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin. J Biol Chem. 1990 May 25;265(15):8665–8669. [PubMed] [Google Scholar]
  9. Demel R. A., London Y., Geurts van Kessel W. S., Vossenberg F. G., van Deenen L. L. The specific interaction of myelin basic protein with lipids at the air-water interface. Biochim Biophys Acta. 1973 Jul 18;311(4):507–519. doi: 10.1016/0005-2736(73)90126-0. [DOI] [PubMed] [Google Scholar]
  10. Demel R. A. Monolayers--description of use and interaction. Methods Enzymol. 1974;32:539–544. doi: 10.1016/0076-6879(74)32052-6. [DOI] [PubMed] [Google Scholar]
  11. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eisenberg M., Gresalfi T., Riccio T., McLaughlin S. Adsorption of monovalent cations to bilayer membranes containing negative phospholipids. Biochemistry. 1979 Nov 13;18(23):5213–5223. doi: 10.1021/bi00590a028. [DOI] [PubMed] [Google Scholar]
  13. Ewbank J. J., Creighton T. E. The molten globule protein conformation probed by disulphide bonds. Nature. 1991 Apr 11;350(6318):518–520. doi: 10.1038/350518a0. [DOI] [PubMed] [Google Scholar]
  14. Ferrari P., Trabaud M. A., Rommain M., Mandine E., Zalisz R., Desgranges C., Smets P. Toxicity and activity of purified trichosanthin. AIDS. 1991 Jul;5(7):865–870. doi: 10.1097/00002030-199107000-00011. [DOI] [PubMed] [Google Scholar]
  15. Gao B., Ma X. Q., Wang Y. P., Chen S. Z., Wu S., Dong Y. C. Refined structure of trichosanthin at 1.73 A resolution. Sci China B. 1994 Jan;37(1):59–73. [PubMed] [Google Scholar]
  16. Geisow M. J., Evans W. H. pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis. Exp Cell Res. 1984 Jan;150(1):36–46. doi: 10.1016/0014-4827(84)90699-2. [DOI] [PubMed] [Google Scholar]
  17. Goto Y., Takahashi N., Fink A. L. Mechanism of acid-induced folding of proteins. Biochemistry. 1990 Apr 10;29(14):3480–3488. doi: 10.1021/bi00466a009. [DOI] [PubMed] [Google Scholar]
  18. Hu V. W., Holmes R. K. Evidence for direct insertion of fragments A and B of diphtheria toxin into model membranes. J Biol Chem. 1984 Oct 10;259(19):12226–12233. [PubMed] [Google Scholar]
  19. Kahn J. O., Kaplan L. D., Gambertoglio J. G., Bredesen D., Arri C. J., Turin L., Kibort T., Williams R. L., Lifson J. D., Volberding P. A. The safety and pharmacokinetics of GLQ223 in subjects with AIDS and AIDS-related complex: a phase I study. AIDS. 1990 Dec;4(12):1197–1204. doi: 10.1097/00002030-199012000-00003. [DOI] [PubMed] [Google Scholar]
  20. Kuo-Fen C. Midtrimester abortion induced by Radix trichosanthis: morphologic observations in placenta and fetus. Obstet Gynecol. 1982 Apr;59(4):494–498. [PubMed] [Google Scholar]
  21. Law L. K., Tam P. P., Yeung H. W. Effects of alpha-trichosanthin and alpha-momorcharin on the development of peri-implantation mouse embryos. J Reprod Fertil. 1983 Nov;69(2):597–604. doi: 10.1530/jrf.0.0690597. [DOI] [PubMed] [Google Scholar]
  22. Li M. X., Yeung H. W., Pan L. P., Chan S. I. Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro. Nucleic Acids Res. 1991 Nov 25;19(22):6309–6312. doi: 10.1093/nar/19.22.6309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mayer R. A., Sergios P. A., Coonan K., O'Brien L. Trichosanthin treatment of HIV-induced immune dysregulation. Eur J Clin Invest. 1992 Feb;22(2):113–122. doi: 10.1111/j.1365-2362.1992.tb01944.x. [DOI] [PubMed] [Google Scholar]
  25. McGrath M. S., Hwang K. M., Caldwell S. E., Gaston I., Luk K. C., Wu P., Ng V. L., Crowe S., Daniels J., Marsh J. GLQ223: an inhibitor of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2844–2848. doi: 10.1073/pnas.86.8.2844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Menestrina G., Forti S., Gambale F. Interaction of tetanus toxin with lipid vesicles. Effects of pH, surface charge, and transmembrane potential on the kinetics of channel formation. Biophys J. 1989 Mar;55(3):393–405. doi: 10.1016/S0006-3495(89)82833-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Merion M., Schlesinger P., Brooks R. M., Moehring J. M., Moehring T. J., Sly W. S. Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5315–5319. doi: 10.1073/pnas.80.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pan K. Z., Lin Y. J., Zhou K. J., Fu Z. J., Chen M. H., Huang D. R., Huang D. H. The crystal and molecular structure of trichosanthin at 2.6 A resolution. Sci China B. 1993 Sep;36(9):1069–1081. [PubMed] [Google Scholar]
  29. Parker M. W., Pattus F., Tucker A. D., Tsernoglou D. Structure of the membrane-pore-forming fragment of colicin A. Nature. 1989 Jan 5;337(6202):93–96. doi: 10.1038/337093a0. [DOI] [PubMed] [Google Scholar]
  30. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  31. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shaw P. C., Chan W. L., Yeung H. W., Ng T. B. Minireview: trichosanthin--a protein with multiple pharmacological properties. Life Sci. 1994;55(4):253–262. doi: 10.1016/0024-3205(94)00727-6. [DOI] [PubMed] [Google Scholar]
  33. Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965 Sep;13(2):482–495. doi: 10.1016/s0022-2836(65)80111-5. [DOI] [PubMed] [Google Scholar]
  34. Wang S. X., Cai G. P., Sui S. F. The insertion of human apolipoprotein H into phospholipid membranes: a monolayer study. Biochem J. 1998 Oct 15;335(Pt 2):225–232. doi: 10.1042/bj3350225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Winiski A. P., McLaughlin A. C., McDaniel R. V., Eisenberg M., McLaughlin S. An experimental test of the discreteness-of-charge effect in positive and negative lipid bilayers. Biochemistry. 1986 Dec 16;25(25):8206–8214. doi: 10.1021/bi00373a013. [DOI] [PubMed] [Google Scholar]
  36. Zhang J. S., Liu W. Y. The mechanism of action of trichosanthin on eukaryotic ribosomes--RNA N-glycosidase activity of the cytotoxin. Nucleic Acids Res. 1992 Mar 25;20(6):1271–1275. doi: 10.1093/nar/20.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang X. J., Wang J. H. Homology of trichosanthin and ricin A chain. 1986 May 29-Jun 4Nature. 321(6069):477–478. doi: 10.1038/321477b0. [DOI] [PubMed] [Google Scholar]
  38. Zhang Y., Li W., Hao Q., Yu G., Li Q., Yao Q. Tb(III) and Eu(III) as fluorescent probes to investigate the metal-binding sites of trichosanthin. Biochem Biophys Res Commun. 1993 Dec 15;197(2):407–414. doi: 10.1006/bbrc.1993.2494. [DOI] [PubMed] [Google Scholar]
  39. Zhao J., Ben L. H., Wu Y. L., Hu W., Ling K., Xin S. M., Nie H. L., Ma L., Pei G. Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors. J Exp Med. 1999 Jul 5;190(1):101–111. doi: 10.1084/jem.190.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van der Goot F. G., González-Mañas J. M., Lakey J. H., Pattus F. A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A. Nature. 1991 Dec 5;354(6352):408–410. doi: 10.1038/354408a0. [DOI] [PubMed] [Google Scholar]

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