Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

The use of pore-forming toxins from sea anemones(actinoporins) in the construction of immunotoxins(ITs) against tumour cells is an alternative for cancertherapy. However, the main disadvantage of actino-porin-based ITs obtained so far has been the poor cel-lular specificity associated with the toxin’s ability tobind and exert its activity in almost any cell mem-brane. Our final goal is the construction of tumourproteinase-activated ITs using a cysteine mutant at themembrane binding region of sticholysin-I (StI), a cyto-lysin isolated from the sea anemone Stichodactylahelianthus. The mutant and the ligand moiety wouldbe linked by proteinase-sensitive peptides through theStI cysteine residue blocking the toxin binding regionand hence the IT non-specific killing activity. Toaccomplish this objective the first step was to obtainthe mutant StI W111C, and to evaluate the impact ofmutating tryptophan 111 by cysteine on the toxinpore-forming capacity. After proteolysis of the cleavagesequence, a short peptide would remain attached tothe toxin. The next step was to evaluate whether thismutant is able to form pores even with a residualpeptide linked to cysteine 111. In this work we demon-strated that (i) StI W111C shows pore-forming capacityin a nanomolar range, although it is 8-fold less activethan the wild-type recombinant StI, corroborating thepreviously reported importance of residue 111 for thebinding of StI to membranes, and (ii) the mutant isable to form pores even with a residual seven-residuepeptide linked to cysteine 111. In addition, it wasdemonstrated that binding of a large molecule tocysteine 111 renders an inactive toxin that is no longerable to bind to the membrane. These results validatethe mutant StI W111C for its use in the constructionof tumour proteinase-activated ITs.Keywords: actinoporin/immunotoxin/pore-forming toxin/sea anemone cytolysin/sticholysinsIntroductionImmunotoxins (ITs) are chimeric molecules in which a cellbinding ligand, such as a monoclonal antibody (mAb) or agrowth factor, is coupled to a killer toxin to direct its activitytowards a specific cell. The targets can be tumour cells,virally infected cells, subsets of normal cells or parasitic cells.Most of the ITs currently in use or under investigation containa toxic component that acts intracellularly to inactivateprotein synthesis. Among the most clinically active are thosetargeted to tumours, specifically those directed against haema-tological tumours. Obstacles to successful treatment of solidtumours include poor penetration into tumour masses, toxicityand the immune response to the toxin component. Exploringother classes of toxins to create new ITs is underway toimprove the treatment of cancer. Relatively few membrane-acting toxins have been investigated for their use in immuno-conjugates, in spite of some inherent advantages of their use.Specifically, the pore-forming toxins constitute an attrac-tive target in the construction of membrane-acting ITsbecause, alternatively to their killer effect, this type of immu-noconjugate could just strengthen the effects of chemothera-peutic agents and common anti-cancer drugs by facilitatingtheir access into the cytosol. Belonging to this group, toxinsfrom sea anemones (Avila et al., 1988, 1989, 2007;Pederzolli et al., 1995; Tejuca et al., 1999, 2004; Potrichet al., 2000), the bacteria Bacillus thuringiensis d-endotoxin(Al yahyaee and Ellar, 1996; Gurkan and Ellar, 2003a,b;Cohen et al., 2007) and the N-terminal fragment of humanperforin (Wan et al., 2006;Zeng et al., 2006) have been usedin IT construction.Interest in the use of pore-forming cytolysins from seaanemones (actinoporins) in this field has been recentlyrenewed. It has been shown that toxin Bc2 and EquinotoxinII (EqTII), pore-forming toxins from the anemonesBunodosoma caissarium and Actinia equina, respectively,potentiate in a significant way the cytotoxicity induced bylow-dose concentrations of the chemotherapeutic agentscytosine arabinoside, doxorubicin and vincristine (Solettiet al., 2008). The induction of a more effective cytotoxicityfor these classical drugs when combined with actinoporinscould allow the reduction of their therapeutic doses and, con-sequently, their undesirable side effects. On the other hand,the actinoporins’ cytotoxic mechanism has been poorlyinvestigated; nevertheless, it was shown recently that the