RESUMEN
Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the productive intoxication of susceptible mammalian cells by Shiga-like toxin-1 (SLTx). Recently, it has been proposed that the observed association of certain ER-directed toxins and viruses with detergent-resistant membranes (DRM) may provide a general mechanism for their retrograde transport to endoplasmic reticulum (ER). Here, we show that DRM recruitment of SLTx bound to its globotriosylceramide (Gb(3)) receptor is mediated by the availability of other glycosphingolipids. Reduction in glucosylceramide (GlcCer) levels led to complete protection against SLTx and a reduced cell surface association of bound toxin with DRM. This reduction still allowed efficient binding and transport of the toxin to the ER. However, toxin sequestration within DRM of the ER was abolished under reduced GlcCer conditions, suggesting that an association of toxin with lipid microdomains or rafts in the ER (where these are defined by detergent insolubility) is essential for a later step leading to or involving retro-translocation of SLTx across the ER membrane. In support of this, we show that a number of ER residents, proteins intimately involved in the process of ER dislocation of misfolded proteins, are present in DRM.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Detergentes/farmacología , Retículo Endoplásmico/metabolismo , Glucosilceramidas/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga I/toxicidad , 1-Desoxinojirimicina/análogos & derivados , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Chlorocebus aethiops , Retículo Endoplásmico/efectos de los fármacos , Glicoesfingolípidos/antagonistas & inhibidores , Glicoesfingolípidos/biosíntesis , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Inhibidores de Proteasoma , Transporte de Proteínas/efectos de los fármacos , Trihexosilceramidas/farmacología , Células VeroRESUMEN
Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agencyapproved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.
Asunto(s)
Linfocitos B/patología , Lisosomas/patología , Enfermedad de Niemann-Pick Tipo C/patología , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapéutico , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Biomarcadores , Trasplante de Médula Ósea , Estudios de Casos y Controles , Niño , Preescolar , Humanos , Lactante , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/terapia , Estudios Prospectivos , Proteínas/genética , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , beta-Ciclodextrinas/uso terapéuticoRESUMEN
Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.
Asunto(s)
Calcio/metabolismo , Lisosomas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Esfingosina/metabolismo , Ácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Colesterol/metabolismo , Curcumina/uso terapéutico , Glicoesfingolípidos/metabolismo , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Mutación/genética , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/clasificación , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Fenotipo , Proteínas/genética , Proteínas/metabolismoRESUMEN
Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.
Asunto(s)
Colorantes Fluorescentes/química , Glicósido Hidrolasas/química , Glicoesfingolípidos/química , Oligosacáridos/análisis , ortoaminobenzoatos/química , Cromatografía Líquida de Alta PresiónRESUMEN
Niemann-Pick disease type C (NP-C) is a hereditary neurovisceral lipid storage disorder. Although traditionally considered a primary cholesterol storage disorder, a variety of glycolipids accumulate in NP-C cells, which resemble those from glycosphingolipidosis patients. Substrate reduction therapy (SRT) with miglustat, an inhibitor of glycosphingolipid biosynthesis, is a novel therapy for the glycosphingolipidoses. We report the use of SRT in a patient with NP-C. We show that depletion of glycosphingolipids by miglustat treatment reduces pathological lipid storage, improves endosomal uptake and normalises lipid trafficking in peripheral blood B lymphocytes. The demonstration that treatment with miglustat, which has no direct effect on cholesterol metabolism, corrects the abnormal lipid trafficking seen in B lymphocytes in NP-C indicates that glycosphingolipid accumulation is the primary pathogenetic event in NP-C. These observations support the use of SRT in patients with this devastating neurodegenerative disease.