Abstract
Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing –SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO−). At low ONOO−/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in –SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.
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Abbreviations
- DPPC:
-
1,2-Palmitoyl-sn-glycero-3-phosphatidylcholine
- SM:
-
Sphingomyelin
- DTNB:
-
5,5′-Dithiobis-(2-nitrobenzoic acid)
- HA:
-
Hemolytic activity
- H:
-
Hemolysis percentage
- E:
-
Permeation percentage
- RBC:
-
Red blood cells
- t20Hs:
-
Time required to produce 20 % hemolysis for non-treated toxins
- t20Hm:
-
Time required to produce 20 % hemolysis for treated toxins with peroxynitrite
- t20Es:
-
Time required to produce 20 % of liposomes with pores for non-treated toxins
- t20Em:
-
Time required to produce 20 % of liposomes with pores for treated toxins with peroxynitrite
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Acknowledgments
Thanks are given to FONDECYT (Project 10702859). LL also acknowledges a doctoral Fellowship. This work was supported by the Iberian American Network CYTED 212RT0467 Biotox.
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León, L., Lissi, E.A., Celedón, G. et al. Inactivation of the Pore-Forming Toxin Sticholysin I by Peroxynitrite: Protection by Cys Groups Incorporated in the Toxin. Protein J 33, 493–501 (2014). https://doi.org/10.1007/s10930-014-9582-x
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DOI: https://doi.org/10.1007/s10930-014-9582-x