RESUMEN
Verotoxin (VT-1) is a cytotoxin, produced by Shigella dysenteriae type 1 or by Shiga toxin-producing Escherichia coli, which binds specifically to globotriaosylceramide (Gb3). This glycosphingolipid is a B cell differentiation antigen (Gb3/CD77) strongly expressed on Burkitt's lymphoma cells. We have previously shown that, in these cells, VT-1 induces apoptosis via a caspase- and mitochondria-dependent pathway. In this report, we provide new insights into this signal transduction pathway. First, we demonstrate that VT-1-induced apoptosis requires degradation of the caspase-8 inhibitory molecule c-FLIPL and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we show that mitochondrial activation is mainly due to i) cleavage and activation of the pro-apoptotic Bcl-2 family member Bid by caspase-8 and ii) Bax relocalization to mitochondrial membranes which lead to cytochrome c release. However, tBid is not involved in Bax relocalization, and relocalization is most likely controlled by the extent of Bax phosphorylation: in non-treated BL cells, p38 MAPK participates in the retention of Bax in the cytoplasm in an inactive form whereas in VT-1 treated cells, protein phosphatase 2A is activated and induces Bax relocalization to mitochondria.
Asunto(s)
Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Linfoma de Burkitt/metabolismo , Caspasa 8/metabolismo , Proteína Fosfatasa 2/metabolismo , Toxina Shiga I/farmacología , Proteína X Asociada a bcl-2/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular , Citocromos c/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Shigella dysenteriae/metabolismo , Transducción de Señal , Trihexosilceramidas/metabolismo , Ubiquitina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.
Asunto(s)
Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/fisiología , Globósidos , Lectinas/química , Lectinas/metabolismo , Estructura Cuaternaria de Proteína , Pseudomonas aeruginosa/metabolismo , Trihexosilceramidas , Adhesinas Bacterianas/genética , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Linfoma de Burkitt , Conformación de Carbohidratos , Secuencia de Carbohidratos , Línea Celular , Cristalografía por Rayos X , Disacáridos/química , Disacáridos/metabolismo , Globósidos/química , Globósidos/metabolismo , Humanos , Enlace de Hidrógeno , Lectinas/genética , Análisis por Micromatrices , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por Sustrato , Trihexosilceramidas/química , Trihexosilceramidas/metabolismo , Agua/químicaRESUMEN
Globotriasosylceramide (Gb3), a neutral glycosphingolipid, is the B-cell differentiation antigen CD77 and acts as the receptor for most Shiga toxins, including verotoxin-1 (VT-1). We have shown that both anti-Gb3/CD77 mAb and VT-1 induce apoptosis in Burkitt's lymphoma cells. We compared the apoptotic pathways induced by these two molecules by selecting cell lines sensitive to only one of these inducers or to both. In all these cell lines (including the apoptosis-resistant line), VT-1 was transported to the endoplasmic reticulum and inhibited protein synthesis similarly, suggesting that VT-1-induced apoptosis is dissociated from these processes. VT-1 triggered a caspase- and mitochondria-dependent pathway (rapid activation of caspases 8 and 3 associated with a loss of mitochondrial membrane potential (Deltapsim) and the release of cytochrome c from mitochondria). In contrast, the anti-Gb3/CD77 mAb-induced pathway was caspase-independent and only involved partial depolarization of mitochondria. Antioxidant compounds had only marginal effects on VT-1-induced apoptosis but strongly protected cells from anti-Gb3/CD77 mAb-induced apoptosis. VT-1- and anti-Gb3/CD77 mAb-treated cells displayed very different features on electron microscopy. These results clearly indicate that the binding of different ligands to Gb3/CD77 triggers completely different apoptotic pathways.