RESUMEN
Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.
Asunto(s)
Glicoesfingolípidos Acídicos/química , Proteínas Anfibias/inmunología , Toxinas Bacterianas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Lisosomas/inmunología , Complejos Multiproteicos/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Factor Trefoil-3/inmunología , Glicoesfingolípidos Acídicos/antagonistas & inhibidores , Glicoesfingolípidos Acídicos/biosíntesis , Aeromonas hydrophila/crecimiento & desarrollo , Aeromonas hydrophila/patogenicidad , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Anuros , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Ceramidas/antagonistas & inhibidores , Ceramidas/biosíntesis , Ceramidas/química , Cerebrósidos/antagonistas & inhibidores , Cerebrósidos/biosíntesis , Cerebrósidos/química , Gangliósidos/antagonistas & inhibidores , Gangliósidos/biosíntesis , Gangliósidos/química , Expresión Génica , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Interleucina-1beta/biosíntesis , Lisosomas/efectos de los fármacos , Lisosomas/microbiología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/inmunología , Microdominios de Membrana/microbiología , Meperidina/análogos & derivados , Meperidina/farmacología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Esfingosina/antagonistas & inhibidores , Esfingosina/biosíntesis , Esfingosina/química , Células THP-1 , Factor Trefoil-3/genética , Factor Trefoil-3/metabolismoRESUMEN
Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.