Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Jun;80(6):2761–2774. doi: 10.1016/S0006-3495(01)76244-3

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus.

C A Valcarcel 1, M Dalla Serra 1, C Potrich 1, I Bernhart 1, M Tejuca 1, D Martinez 1, F Pazos 1, M E Lanio 1, G Menestrina 1
PMCID: PMC1301462  PMID: 11371451

Abstract

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA >> PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.

Full Text

The Full Text of this article is available as a PDF (194.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alvarez C., Lanio M. E., Tejuca M., Martínez D., Pazos F., Campos A. M., Encinas M. V., Pertinhez T., Schreier S., Lissi E. A. The role of ionic strength on the enhancement of the hemolytic activity of sticholysin I, a cytolysin from Stichodactyla helianthus. Toxicon. 1998 Jan;36(1):165–178. doi: 10.1016/s0041-0101(97)00069-x. [DOI] [PubMed] [Google Scholar]
  2. Anderluh G., Barlic A., Podlesek Z., Macek P., Pungercar J., Gubensek F., Zecchini M. L., Serra M. D., Menestrina G. Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes. Eur J Biochem. 1999 Jul;263(1):128–136. doi: 10.1046/j.1432-1327.1999.00477.x. [DOI] [PubMed] [Google Scholar]
  3. Anderluh G., Krizaj I., Strukelj B., Gubensek F., Macek P., Pungercar J. Equinatoxins, pore-forming proteins from the sea anemone Actinia equina, belong to a multigene family. Toxicon. 1999 Oct;37(10):1391–1401. doi: 10.1016/s0041-0101(99)00082-3. [DOI] [PubMed] [Google Scholar]
  4. Belmonte G., Menestrina G., Pederzolli C., Krizaj I., Gubensek F., Turk T., Macek P. Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles. Biochim Biophys Acta. 1994 Jun 22;1192(2):197–204. doi: 10.1016/0005-2736(94)90119-8. [DOI] [PubMed] [Google Scholar]
  5. Belmonte G., Pederzolli C., Macek P., Menestrina G. Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes. J Membr Biol. 1993 Jan;131(1):11–22. doi: 10.1007/BF02258530. [DOI] [PubMed] [Google Scholar]
  6. Bernheimer A. W., Avigad L. S. Properties of a toxin from the sea anemone Stoichacis helianthus, including specific binding to sphingomyelin. Proc Natl Acad Sci U S A. 1976 Feb;73(2):467–471. doi: 10.1073/pnas.73.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
  8. Classen J., Haest C. W., Tournois H., Deuticke B. Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane. Biochemistry. 1987 Oct 20;26(21):6604–6612. doi: 10.1021/bi00395a007. [DOI] [PubMed] [Google Scholar]
  9. Connor J., Bucana C., Fidler I. J., Schroit A. J. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci U S A. 1989 May;86(9):3184–3188. doi: 10.1073/pnas.86.9.3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullis P. R., de Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim Biophys Acta. 1979 Dec 20;559(4):399–420. doi: 10.1016/0304-4157(79)90012-1. [DOI] [PubMed] [Google Scholar]
  11. Dalla Serra M., Fagiuoli G., Nordera P., Bernhart I., Della Volpe C., Di Giorgio D., Ballio A., Menestrina G. The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins. Mol Plant Microbe Interact. 1999 May;12(5):391–400. doi: 10.1094/MPMI.1999.12.5.391. [DOI] [PubMed] [Google Scholar]
  12. Edidin M. Lipid microdomains in cell surface membranes. Curr Opin Struct Biol. 1997 Aug;7(4):528–532. doi: 10.1016/s0959-440x(97)80117-0. [DOI] [PubMed] [Google Scholar]
  13. Epand R. M. Lipid polymorphism and protein-lipid interactions. Biochim Biophys Acta. 1998 Nov 10;1376(3):353–368. doi: 10.1016/s0304-4157(98)00015-x. [DOI] [PubMed] [Google Scholar]
  14. Farren S. B., Hope M. J., Cullis P. R. Polymorphic phase preferences of phosphatidic acid: A 31P and 2H NMR study. Biochem Biophys Res Commun. 1983 Mar 16;111(2):675–682. doi: 10.1016/0006-291x(83)90359-5. [DOI] [PubMed] [Google Scholar]
  15. Fivaz M., Abrami L., van der Goot F. G. Landing on lipid rafts. Trends Cell Biol. 1999 Jun;9(6):212–213. doi: 10.1016/s0962-8924(99)01567-6. [DOI] [PubMed] [Google Scholar]
  16. Gascard P., Tran D., Sauvage M., Sulpice J. C., Fukami K., Takenawa T., Claret M., Giraud F. Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane. Biochim Biophys Acta. 1991 Oct 14;1069(1):27–36. doi: 10.1016/0005-2736(91)90100-m. [DOI] [PubMed] [Google Scholar]
  17. Gazit E., Burshtein N., Ellar D. J., Sawyer T., Shai Y. Bacillus thuringiensis cytolytic toxin associates specifically with its synthetic helices A and C in the membrane bound state. Implications for the assembly of oligomeric transmembrane pores. Biochemistry. 1997 Dec 9;36(49):15546–15554. doi: 10.1021/bi9707584. [DOI] [PubMed] [Google Scholar]
  18. Gu L. Q., Braha O., Conlan S., Cheley S., Bayley H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature. 1999 Apr 22;398(6729):686–690. doi: 10.1038/19491. [DOI] [PubMed] [Google Scholar]
  19. Hatakeyama T., Sato T., Taira E., Kuwahara H., Niidome T., Aoyagi H. Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process. J Biochem. 1999 Feb;125(2):277–284. doi: 10.1093/oxfordjournals.jbchem.a022284. [DOI] [PubMed] [Google Scholar]
  20. Ionov R., El-Abed A., Angelova A., Goldmann M., Peretti P. Asymmetrical ion-channel model inferred from two-dimensional crystallization of a peptide antibiotic. Biophys J. 2000 Jun;78(6):3026–3035. doi: 10.1016/S0006-3495(00)76841-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lacy D. B., Stevens R. C. Unraveling the structures and modes of action of bacterial toxins. Curr Opin Struct Biol. 1998 Dec;8(6):778–784. doi: 10.1016/s0959-440x(98)80098-5. [DOI] [PubMed] [Google Scholar]
  22. Langner M., Kubica K. The electrostatics of lipid surfaces. Chem Phys Lipids. 1999 Aug;101(1):3–35. doi: 10.1016/s0009-3084(99)00052-3. [DOI] [PubMed] [Google Scholar]
  23. Lanio M. E., Morera V., Alvarez C., Tejuca M., Gómez T., Pazos F., Besada V., Martínez D., Huerta V., Padrón G. Purification and characterization of two hemolysins from Stichodactyla helianthus. Toxicon. 2001 Feb-Mar;39(2-3):187–194. doi: 10.1016/s0041-0101(00)00106-9. [DOI] [PubMed] [Google Scholar]
  24. Lesieur C., Vécsey-Semjén B., Abrami L., Fivaz M., Gisou van der Goot F. Membrane insertion: The strategies of toxins (review). Mol Membr Biol. 1997 Apr-Jun;14(2):45–64. doi: 10.3109/09687689709068435. [DOI] [PubMed] [Google Scholar]
  25. Lewis R. N., McElhaney R. N. Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids. Biophys J. 2000 Sep;79(3):1455–1464. doi: 10.1016/S0006-3495(00)76397-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li J., Koni P. A., Ellar D. J. Structure of the mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis sp. kyushuensis and implications for membrane pore formation. J Mol Biol. 1996 Mar 22;257(1):129–152. doi: 10.1006/jmbi.1996.0152. [DOI] [PubMed] [Google Scholar]
  27. MacDonald R. C., MacDonald R. I., Menco B. P., Takeshita K., Subbarao N. K., Hu L. R. Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta. 1991 Jan 30;1061(2):297–303. doi: 10.1016/0005-2736(91)90295-j. [DOI] [PubMed] [Google Scholar]
  28. Macek P., Belmonte G., Pederzolli C., Menestrina G. Mechanism of action of equinatoxin II, a cytolysin from the sea anemone Actinia equina L. belonging to the family of actinoporins. Toxicology. 1994 Feb 28;87(1-3):205–227. doi: 10.1016/0300-483x(94)90252-6. [DOI] [PubMed] [Google Scholar]
  29. Macek P., Zecchini M., Pederzolli C., Dalla Serra M., Menestrina G. Intrinsic tryptophan fluorescence of equinatoxin II, a pore-forming polypeptide from the sea anemone Actinia equina L, monitors its interaction with lipid membranes. Eur J Biochem. 1995 Nov 15;234(1):329–335. doi: 10.1111/j.1432-1033.1995.329_c.x. [DOI] [PubMed] [Google Scholar]
  30. Marx U., Lassmann G., Holzhütter H. G., Wüstner D., Müller P., Höhlig A., Kubelt J., Herrmann A. Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach. Biophys J. 2000 May;78(5):2628–2640. doi: 10.1016/S0006-3495(00)76807-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Matsuzaki K. Magainins as paradigm for the mode of action of pore forming polypeptides. Biochim Biophys Acta. 1998 Nov 10;1376(3):391–400. doi: 10.1016/s0304-4157(98)00014-8. [DOI] [PubMed] [Google Scholar]
  32. Matsuzaki K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta. 1999 Dec 15;1462(1-2):1–10. doi: 10.1016/s0005-2736(99)00197-2. [DOI] [PubMed] [Google Scholar]
  33. Mayer L. D., Hope M. J., Cullis P. R. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta. 1986 Jun 13;858(1):161–168. doi: 10.1016/0005-2736(86)90302-0. [DOI] [PubMed] [Google Scholar]
  34. Meinardi E., Florin-Christensen M., Paratcha G., Azcurra J. M., Florin-Christensen J. The molecular basis of the self/nonself selectivity of a coelenterate toxin. Biochem Biophys Res Commun. 1995 Nov 2;216(1):348–354. doi: 10.1006/bbrc.1995.2630. [DOI] [PubMed] [Google Scholar]
  35. Menestrina G., Cabiaux V., Tejuca M. Secondary structure of sea anemone cytolysins in soluble and membrane bound form by infrared spectroscopy. Biochem Biophys Res Commun. 1999 Jan 8;254(1):174–180. doi: 10.1006/bbrc.1998.9898. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Op den Kamp J. A. Lipid asymmetry in membranes. Annu Rev Biochem. 1979;48:47–71. doi: 10.1146/annurev.bi.48.070179.000403. [DOI] [PubMed] [Google Scholar]
  38. Oren Z., Shai Y. Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers. 1998;47(6):451–463. doi: 10.1002/(SICI)1097-0282(1998)47:6<451::AID-BIP4>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  39. Panchal R. G., Cusack E., Cheley S., Bayley H. Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat Biotechnol. 1996 Jul;14(7):852–856. doi: 10.1038/nbt0796-852. [DOI] [PubMed] [Google Scholar]
  40. Pazos I. F., Alvarez C., Lanio M. E., Martinez D., Morera V., Lissi E. A., Campos A. M. Modification of sticholysin II hemolytic activity by free radicals. Toxicon. 1998 Oct;36(10):1383–1393. doi: 10.1016/s0041-0101(98)00016-6. [DOI] [PubMed] [Google Scholar]
  41. Pederzolli C., Belmonte G., Dalla Serra M., Macek P., Menestrina G. Biochemical and cytotoxic properties of conjugates of transferrin with equinatoxin II, a cytolysin from a sea anemone. Bioconjug Chem. 1995 Mar-Apr;6(2):166–173. doi: 10.1021/bc00032a003. [DOI] [PubMed] [Google Scholar]
  42. Rietveld A., Simons K. The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim Biophys Acta. 1998 Nov 10;1376(3):467–479. doi: 10.1016/s0304-4157(98)00019-7. [DOI] [PubMed] [Google Scholar]
  43. Russo M. J., Bayley H., Toner M. Reversible permeabilization of plasma membranes with an engineered switchable pore. Nat Biotechnol. 1997 Mar;15(3):278–282. doi: 10.1038/nbt0397-278. [DOI] [PubMed] [Google Scholar]
  44. Santos N. C., Castanho M. A. Teaching light scattering spectroscopy: the dimension and shape of tobacco mosaic virus. Biophys J. 1996 Sep;71(3):1641–1650. doi: 10.1016/S0006-3495(96)79369-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schneider E., Haest C. W., Plasa G., Deuticke B. Bacterial cytotoxins, amphotericin B and local anesthetics enhance transbilayer mobility of phospholipids in erythrocyte membranes. Consequences for phospholipid asymmetry. Biochim Biophys Acta. 1986 Mar 13;855(3):325–336. doi: 10.1016/0005-2736(86)90078-7. [DOI] [PubMed] [Google Scholar]
  46. Seddon J. M. Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids. Biochim Biophys Acta. 1990 Feb 28;1031(1):1–69. doi: 10.1016/0304-4157(90)90002-t. [DOI] [PubMed] [Google Scholar]
  47. Staudegger E., Prenner E. J., Kriechbaum M., Degovics G., Lewis R. N., McElhaney R. N., Lohner K. X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation. Biochim Biophys Acta. 2000 Sep 29;1468(1-2):213–230. doi: 10.1016/s0005-2736(00)00260-1. [DOI] [PubMed] [Google Scholar]
  48. Tejuca M., Anderluh G., Macek P., Marcet R., Torres D., Sarracent J., Alvarez C., Lanio M. E., Dalla Serra M., Menestrina G. Antiparasite activity of sea-anemone cytolysins on Giardia duodenalis and specific targeting with anti-Giardia antibodies. Int J Parasitol. 1999 Mar;29(3):489–498. doi: 10.1016/s0020-7519(98)00220-3. [DOI] [PubMed] [Google Scholar]
  49. Tejuca M., Serra M. D., Ferreras M., Lanio M. E., Menestrina G. Mechanism of membrane permeabilization by sticholysin I, a cytolysin isolated from the venom of the sea anemone Stichodactyla helianthus. Biochemistry. 1996 Nov 26;35(47):14947–14957. doi: 10.1021/bi960787z. [DOI] [PubMed] [Google Scholar]
  50. Varanda W., Finkelstein A. Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone, Stoichactis helianthus. J Membr Biol. 1980 Aug 7;55(3):203–211. doi: 10.1007/BF01869461. [DOI] [PubMed] [Google Scholar]
  51. Yang L., Weiss T. M., Lehrer R. I., Huang H. W. Crystallization of antimicrobial pores in membranes: magainin and protegrin. Biophys J. 2000 Oct;79(4):2002–2009. doi: 10.1016/S0006-3495(00)76448-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. al-yahyaee S. A., Ellar D. J. Cell targeting of a pore-forming toxin, CytA delta-endotoxin from Bacillus thuringiensis subspecies israelensis, by conjugating CytA with anti-Thy 1 monoclonal antibodies and insulin. Bioconjug Chem. 1996 Jul-Aug;7(4):451–460. doi: 10.1021/bc960030k. [DOI] [PubMed] [Google Scholar]
  53. de los Rios V., Mancheño J. M., Lanio M. E., Oñaderra M., Gavilanes J. G. Mechanism of the leakage induced on lipid model membranes by the hemolytic protein sticholysin II from the sea anemone Stichodactyla helianthus. Eur J Biochem. 1998 Mar 1;252(2):284–289. doi: 10.1046/j.1432-1327.1998.2520284.x. [DOI] [PubMed] [Google Scholar]
  54. de los Ríos V., Mancheño J. M., Martínez del Pozo A., Alfonso C., Rivas G., Oñaderra M., Gavilanes J. G. Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein. FEBS Lett. 1999 Jul 16;455(1-2):27–30. doi: 10.1016/s0014-5793(99)00846-7. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES