| Sumario: |
Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted
as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in
these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins
against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel
actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific
Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from
total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21
Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more
than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino
acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks
Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its
secondary structure is dominated by beta structure (58.5%) with 5.5% of a-helix, and 35% of random
structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as per meabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing
membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and
modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but
several differences were also detectable. Taken together, these results reinforce the notion that Ng is a
novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the
most potent actinoporin so far described, but with peculiar structural characteristics contributing to
expand the understanding of the structure-function relationship in this protein family.
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