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Inactivation of the Pore-Forming Toxin Sticholysin I by Peroxynitrite: Protection by Cys Groups Incorporated in the Toxin

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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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Authors and Affiliations

  1. Department of Chemistry, Chemistry and Biology Faculty, Universidad de Santiago de Chile (USACH), Santiago, Chile

    L. León & E. A. Lissi

  2. Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile

    G. Celedón & G. Gonzalez

  3. Facultad de Biology, Universidad de La Habana, Havana, Cuba

    F. Pazos, C. Alvarez & M. E. Lanio

Authors
  1. L. León
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  2. E. A. Lissi
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  3. G. Celedón
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  4. G. Gonzalez
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  5. F. Pazos
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  6. C. Alvarez
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  7. M. E. Lanio
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Correspondence to L. León.

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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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