Elution of Lipopolysaccharides from Polyacrylamide Gels

NRC publication: Ye

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 cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Contribución al mecanismo de formación de poros de sticholisina I, una proteína formadora de poros de la anémona Stichodactyla helianthus, mediante el empleo de mutantes de Cys en zonas funcionalmente relevantes de la proteína

Sticholisina I (St I) es una citolisina producida por la anémona marina Stichodactyla helianthus, que se caracteriza por formar poros oligoméricos en membranas naturales y artificiales. En este trabajo se describe la obtención por vía recombinante en Escherichia coli (E. coli) de una variante recombinante de St I (St Ir), así como su caracterización conformacional y funcional. Estos trabajos permitieron reducir el impacto ecológico que provoca la obtención de St I a partir de las anémonas como fuente natural y además realizar mutagénesis sitio-específica para su caracterización funcional. La ausencia de Cys en St I facilitó la introducción de este residuo aminoacídico, por mutagénesis dirigida, en zonas funcionalmente importantes de la toxina. En el trabajo se sustituyeron por Cys en St Ir de forma independiente, los aminoácidos Glu2 y Phe15 (localizados en el segmento de los primeros treinta aminoácidos que participan en la formación del canal) y la Arg52, Pro80 y Trp111 (en la región de interacción con las membranas). Así, se obtuvieron y purificaron de E. coli cinco proteínas mutadas: StI E2C, StI F15C, StI R52C, StI P80C y StI W111C. Los estudios de caracterización espectroscópicos permitieron establecer que las sustituciones aminoacídicas no provocaron cambios conformacionales en los mutantes con respecto a St Ir. Los monómeros de StI E2C, StI R52C y StI P80C mostraron capacidades similares de unión a las membranas en comparación con la variante recombinante. StI F15C mostró un ligero incremento en su capacidad de interacción con las membranas mientras que StI W111C resultó la de menor capacidad de unión. La capacidad formadora de poros de los monómeros, medida en vesículas liposomales y membrana eritrocitaria, resultó similar entre StI E2C, StI F15C y St Ir. Sin embargo, la actividad lítica de StI R52C, StI P80C y StI W111C disminuyó con respecto a St Ir. Los agregados diméricos por enlace disulfuro en StI R52C y StI W111C disminuyeron la actividad biológica de las proteínas. Una de las novedades científicas más importante del presente trabajo radica en que se demuestra que la presencia de agregados diméricos estabilizados por enlace disulfuro, en StI E2C, incrementa la actividad biológica formadora de poros tanto en vesículas liposomales como en eritrocitos. Estos resultados demuestran, por primera vez, que la unión covalente de los extremos aminos de St I potencia la formación de poros funcionales por un mecanismo hasta ahora desconocido. En la investigación se aportan nuevos elementos sobre la importancia de los residuos Glu2, Phe15, Arg52, Pro80 y Trp111 en las etapas de unión inicial a las membranas y de formación de poros durante el mecanismo lítico. Por último, la obtención de los mutantes de Cys abre las posibilidades para el marcaje de St I con sondas de espín o sondas fluorescentes para estudiar el mecanismo de formación de poros mediante las espectroscopias de resonancia paramagnética electrónica (EPR) y de fluorescencia, y sustentan otras aplicaciones nanobiotecnológicas actualmente en desarrollo en nuestro grupo de trabajo.Sticholisin I (St I) is a cytolysin produced by the sea anemone Stichodactyla helianthus, which is characterized by forming oligomeric pores in natural and artificial membranes. This work describes the recombinant production in Escherichia coli (E. coli) of a recombinant variant of St I (St Ir), as well as its conformational and functional characterization. These works allowed us to reduce the ecological impact caused by obtaining St I from anemones as a natural source and also perform site-specific mutagenesis for its functional characterization. The absence of Cys in St I facilitated the introduction of this amino acid residue, by directed mutagenesis, into functionally important areas of the toxin. In the work, the amino acids Glu2 and Phe15 (located in the segment of the first thirty amino acids that participate in the formation of the channel) and Arg52, Pro80 and Trp111 (in the interaction region) were independently replaced by Cys in St Ir. with the membranes). Thus, five mutated proteins were obtained and purified from E. coli: StI E2C, StI F15C, StI R52C, StI P80C and StI W111C. Spectroscopic characterization studies allowed us to establish that amino acid substitutions did not cause conformational changes in the mutants with respect to St Ir. The monomers of StI E2C, StI R52C and StI P80C showed similar binding capacities to membranes compared to the recombinant variant. StI F15C showed a slight increase in its interaction capacity with membranes while StI W111C had the lowest binding capacity. The pore-forming capacity of the monomers, measured in liposomal vesicles and erythrocyte membrane, was similar between StI E2C, StI F15C and St Ir. However, the lytic activity of StI R52C, StI P80C and StI W111C decreased with respect to St Ir The dimeric aggregates by disulfide bond in StI R52C and StI W111C decreased the biological activity of the proteins. One of the most important scientific novelties of the present work is that it is demonstrated that the presence of dimeric aggregates stabilized by disulfide bond, in StI E2C, increases the pore-forming biological activity in both liposomal vesicles and erythrocytes. These results demonstrate, for the first time, that covalent attachment of the amino termini of St I enhances the formation of functional pores by a previously unknown mechanism. The research provides new elements on the importance of residues Glu2, Phe15, Arg52, Pro80 and Trp111 in the stages of initial binding to membranes and pore formation during the lytic mechanism. Finally, obtaining the Cys mutants opens the possibilities for labeling St I with spin probes or fluorescent probes to study the mechanism of pore formation through electron paramagnetic resonance (EPR) and fluorescence spectroscopies, and support other nanobiotechnological applications currently under development in our working group.Universidad Nacional, Costa RicaAcademia de Ciencias de Cuba, CubaEscuela de Ciencias Biológica