Ros U, Pedrera L, Martínez D, Tejuca M, Pazos F, Lanio ME, Alvarez C

Language: English
References: 19
Page: 312-316
PDF size: 176.54 Kb.

ABSTRACT

The Caribbean Sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (StI and StII). To clarify the contribution of the first thirty (StII) or thirty-one (StI) N-terminal amino acid residues to the activity of the toxins, four peptides spanning residues 1-31 of StI (StI_1-31, StI_12-31) and 1-30 of StII (StII_1-30, StII_11-30) were synthesized. StII_1-30 was the most active peptide, followed by StI_1-31 and the shortest ones. The difference between the hemolytic activities of the largest peptides qualitatively reproduces that found between the respective toxins StI and StII. The results suggest the importance of continuity of the 1-10 hydrophobic amino acid sequence in StII_1-30 for displaying higher membrane binding and activity. Thus, the different peptide membranotropic action is explained in terms of the differences in their hydrophobic and electrostatic properties. Furthermore, we also demonstrated that StII_1-30 forms pores of similar radius to that of the protein (around 1 nm), with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins. Altogether these results demonstrate that synthetic peptides that reproduce sticholysins’ N-terminus are not only a good model of these toxins structure and function but, and due to its reduced molecular size, could also be useful biotechnological tools instead of their larger parental proteins. This research won the 2012 Award of the Cuban National Academy of Sciences.

REFERENCES

1. Lanio ME, Morera V, Alvarez C, Tejuca M, Gomez T, Pazos F, et al. Purifi cation and characterization of two hemolysins from Stichodactyla helianthus. Toxicon. 2001;39(2-3):187-94.

2. Tejuca M, Dalla Serra M, Potrich C, Alvarez C, Menestrina G. Sizing the radius of the pore formed in erythrocytes and lipid vesicles by the toxin sticholysin I from the sea anemone Stichodactyla helianthus. J Membr Biol. 2001;183(2):125-35.

3. Kem WR. Sea anemones toxins: structure and action. In: Hessinger DA, Lenhoff HM, editors. The biology of nematocysts. San Diego: Academic Press; 1988. p. 375-405.

4. Tejuca M, Serra MD, Ferreras M, Lanio ME, Menestrina G. Mechanism of membrane permeabilization by sticholysin I, a cytolysin isolated from the venom of the sea anemone Stichodactyla helianthus. Biochemistry. 1996;35(47):14947-57.

5. Valcarcel CA, Dalla Serra M, Potrich C, Bernhart I, Tejuca M, Martinez D, et al. Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus. Biophys J. 2001;80(6):2761-74.

6. Anderluh G, Macek P. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon. 2002;40(2):111-24.

7. Alvarez C, Mancheño JM, Martinez D, Tejuca M, Pazos F, Lanio ME. Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes. Toxicon. 2009;54(8):1135-47.

8. Huerta V, Morera V, Guanche Y, Chinea G, Gonzalez LJ, Betancourt L, et al. Primary structure of two cytolysin isoforms from Stichodactyla helianthus differing in their hemolytic activity. Toxicon. 2001; 39(8):1253-6.

9. Martinez D, Campos AM, Pazos F, Alvarez C, Lanio ME, Casallanovo F, et al. Properties of St I and St II, two isotoxins isolated from Stichodactyla helianthus: a comparison. Toxicon. 2001;39(10):1547-60.

10. Mancheño JM, Martin-Benito J, Martinez- Ripoll M, Gavilanes JG, Hermoso JA. Crystal and electron microscopy structures of sticholysin II actinoporin reveal insights into the mechanism of membrane pore formation. Structure. 2003;11(11):1319-28.

11. Castrillo I, Alegre-Cebollada J, Martínez del Pozo A, Gavilanes JG, Santoro J, Bruix M. 1H, 13C, and 15N NMR assignments of the actinoporin Sticholysin I. Biomol NMR Assign. 2009;3(1):5-7.

12. Atherton E, Sheppard RC. Solid phase peptide synthesis: A practical approach; Oxford: Oxford University Press; 1989.

13. Casallanovo F, de Oliveira FJ, de Souza FC, Ros U, Martinez Y, Penton D, et al. Model peptides mimic the structure and function of the N-terminus of the poreforming toxin sticholysin II. Biopolymers. 2006;84(2):169-80.

14. Renkin EM. Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physiol. 1954; 38(2):225-43.

15. Lakowicz JR. Principles of Fluorescence Spectroscopy. 3rd ed. New York: Springer; 2006.

16. Lau SY, Taneja AK, Hodges RS. Synthesis of a model protein of defi ned secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils. J Biol Chem. 1984;259(21):13253-61.

17. 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;254(1):174-80.

18. Brockman H. Lipid monolayers: why use half a membrane to characterize protein-membrane interactions? Curr Opin Struct Biol. 1999;9:438-43.

19. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157(1): 105-32.