RESUMEN
We have endeavored in this review to summarize our findings, which point to a systemic deficiency of ganglioside GM1 in Parkinson's disease (PD) tissues. These include neuronal tissues well known to be involved in PD, such as substantia nigra of the brain and those of the peripheral nervous system, such as the colon and heart. Moreover, we included skin and fibroblasts in the study as well as peripheral blood mononuclear cells; these are tissues not directly involved in neuronal signaling. We show similar findings for ganglioside GD1a, which is the metabolic precursor to GM1. We discuss the likely causes of these GM1 deficiencies and the resultant biochemical mechanisms underlying loss of neuronal viability and normal functioning. Strong support for this hypothesis is provided by a mouse PD model involving partial GM1 deficiency based on mono-allelic disruption of the B4galnt1 gene. We point out that progressive loss of GM1/GD1a occurs in the periphery as well as the brain, thus obviating the need to speculate PD symptom transfer between these tissues. Finally, we discuss how these findings point to a potential disease-altering therapy for PD:GM1 replacement, as is strongly implicated in animal studies and clinical trials.
Asunto(s)
Gangliósido G(M1) , Enfermedad de Parkinson , Animales , Modelos Animales de Enfermedad , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósido G(M1)/uso terapéutico , Leucocitos Mononucleares/metabolismo , Ratones , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismoRESUMEN
Several signaling processes in the plasma membrane are intensified by ceramides that are formed by sphingomyelinase-mediated hydrolysis of sphingomyelin. These ceramides trigger clustering of signaling-related biomolecules, but how they concentrate such biomolecules remains unclear. Here, the spatiotemporal localization of ganglioside GM1, a glycolipid receptor involved in signaling, during sphingomyelinase-mediated hydrolysis is described. Real-time visualization of the dynamic remodeling of the heterogeneous lipid membrane that occurs due to sphingomyelinase action is used to examine GM1 clustering, and unexpectedly, it is found that it is more complex than previously thought. Specifically, lipid membranes generate two distinct types of condensed GM1: 1) rapidly formed but short-lived GM1 clusters that are formed in ceramide-rich domains nucleated from the liquid-disordered phase; and 2) late-onset yet long-lasting, high-density GM1 clusters that are formed in the liquid-ordered phase. These findings suggest that multiple pathways exist in a plasma membrane to synergistically facilitate the rapid amplification and persistence of signals.
Asunto(s)
Ceramidas/genética , Gangliósido G(M1)/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielinas/genética , Bacillus cereus/enzimología , Membrana Celular/genética , Membrana Celular/metabolismo , Ceramidas/biosíntesis , Ceramidas/química , Análisis por Conglomerados , Gangliósido G(M1)/genética , Hidrólisis , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos/química , Lípidos/genética , Lípidos de la Membrana/química , Lípidos de la Membrana/genética , Transducción de Señal/genética , Esfingomielina Fosfodiesterasa/química , Esfingomielinas/química , Esfingomielinas/metabolismoRESUMEN
SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac[Formula: see text](2-3)Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc) and its precursor, asialo-GM1 (Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the [Formula: see text]-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.
Asunto(s)
Gangliósido G(M1)/química , Gangliósidos/química , Lectinas/ultraestructura , Neoplasias/genética , Animales , Bivalvos/química , Secuencia de Carbohidratos , Gangliósido G(M1)/genética , Gangliósidos/genética , Humanos , Lectinas/química , Lectinas/genética , Neoplasias/patologíaRESUMEN
Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59 cluster rafts and GM1 cluster rafts were stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK and continually recruited Lyn and H-Ras right beneath them in the inner leaflet with dwell lifetimes <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the nonraftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Because GM1 cluster rafts recruited Lyn and H-Ras as efficiently as CD59 cluster rafts, and because the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions and thus serve as a key signal transduction platform.
Asunto(s)
Antígenos CD59/metabolismo , Gangliósido G(M1)/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Imagen Individual de Molécula , Familia-src Quinasas/metabolismo , Antígenos CD59/genética , Gangliósido G(M1)/genética , Células HeLa , Humanos , Microdominios de Membrana/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Familia-src Quinasas/genéticaRESUMEN
It is widely accepted that the abnormal self-association of amyloid ß-protein (Aß) is central to the pathogenesis of Alzheimer's disease, the most common form of dementia. Accumulating evidence, both in vivo and in vitro, suggests that the binding of Aß to gangliosides, especially monosialoganglioside GM1, plays an important role in the aggregation of Aß. This review summarizes the molecular details of the binding of Aß to ganglioside-containing membranes and subsequent structural changes, as revealed by liposomal and cellular studies. Furthermore, mechanisms of cytotoxicity by aggregated Aß are also discussed.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Gangliósido G(M1)/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Gangliósido G(M1)/metabolismo , Humanos , Liposomas/química , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Unión Proteica/efectos de los fármacosRESUMEN
A major characteristic of Alzheimer's disease (AD) is the accumulation of misfolded amyloid-ß (Aß) peptide. Several studies linked AD with type 2 diabetes due to similarities between Aß and human amylin. This study investigates the effect of amylin and pramlintide on Aß pathogenesis and the predisposing molecular mechanism(s) behind the observed effects in TgSwDI mouse, a cerebral amyloid angiopathy (CAA) and AD model. Our findings showed that thirty days of intraperitoneal injection with amylin or pramlintide increased Aß burden in mice brains. Mechanistic studies revealed both peptides altered the amyloidogenic pathway and increased Aß production by modulating amyloid precursor protein (APP) and γ-secretase levels in lipid rafts. In addition, both peptides increased levels of B4GALNT1 enzyme and GM1 ganglioside, and only pramlintide increased the level of GM2 ganglioside. Increased levels of GM1 and GM2 gangliosides play an important role in regulating amyloidogenic pathway proteins in lipid rafts. Increased brain Aß burden by amylin and pramlintide was associated with synaptic loss, apoptosis, and microglia activation. In conclusion, our findings showed amylin or pramlintide increase Aß levels and related pathology in TgSwDI mice brains, and suggest that increased amylin levels or the therapeutic use of pramlintide could increase the risk of AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Microdominios de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Angiopatía Amiloide Cerebral/genética , Angiopatía Amiloide Cerebral/metabolismo , Angiopatía Amiloide Cerebral/patología , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósido G(M2)/genética , Gangliósido G(M2)/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/patología , Ratones , Ratones Transgénicos , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismoRESUMEN
Parkinson's disease (PD) is a major neurodegenerative disorder characterized by a variety of non-motor symptoms in addition to the well-recognized motor dysfunctions that have commanded primary interest. We previously described a new PD mouse model based on heterozygous disruption of the B4galnt1 gene leading to partial deficiency of the GM1 family of gangliosides that manifested several nigrostriatal neuropathological features of PD as well as movement impairment. We now show this mouse also suffers three non-motor symptoms characteristic of PD involving the gastrointestinal, sympathetic cardiac, and cerebral cognitive systems. Treatment of these animals with a synthetic form of GM1 ganglioside, produced by transfected E. coli, proved ameliorative of these symptoms as well as the motor defect. These findings further suggest subnormal GM1 to be a systemic defect constituting a major risk factor in sporadic PD and indicate the B4galnt1(+/-) (HT) mouse to be a true neuropathological model that recapitulates both motor and non-motor lesions of this condition.
Asunto(s)
Modelos Animales de Enfermedad , Gangliósido G(M1)/administración & dosificación , Gangliósido G(M1)/deficiencia , N-Acetilgalactosaminiltransferasas/deficiencia , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Animales , Femenino , Gangliósido G(M1)/genética , Enfermedades Gastrointestinales/tratamiento farmacológico , Enfermedades Gastrointestinales/genética , Enfermedades Gastrointestinales/metabolismo , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Trastornos de la Destreza Motora/tratamiento farmacológico , Trastornos de la Destreza Motora/genética , Trastornos de la Destreza Motora/metabolismo , N-Acetilgalactosaminiltransferasas/genética , Enfermedad de Parkinson/genéticaRESUMEN
Mucopolysaccharidoses (MPS) are a group of inherited metabolic diseases caused by mutations leading to defective degradation of glycosaminoglycans (GAGs) and their accumulation in cells. Among 11 known types and subtypes of MPS, neuronopathy occurs in seven (MPS I, II, IIIA, IIIB, IIIC, IIID, VII). Brain dysfunctions, occurring in these seven types/subtypes include various behavioral disorders. Intriguingly, behavioral symptoms are significantly different between patients suffering from various MPS types. Molecular base of such differences remains unknown. Here, we asked if expression of genes considered as connected to behavior (based on Gene Ontology, GO terms) is changed in MPS. Using cell lines of all MPS types, we have performed transcriptomic (RNA-seq) studies and assessed expression of genes involved in behavior. We found significant differences between MPS types in this regard, with the most severe changes in MPS IIIA (the type considered as the behaviorally most severely affected), while the lowest changes in MPS IVA and MPS VI (types in which little or no behavioral disorders are known). Intriguingly, relatively severe changes were found also in MPS IVB (in which, despite no behavioral disorder noted, the same gene is mutated as in GM1 gangliosidosis, a severe neurodegenerative disease) and MPS IX (in which only a few patients were described to date, thus, behavioral problems are not well recognized). More detailed analyses of expression of certain genes allowed us to propose an association of specific changes in the levels of transcripts in specific MPS types to certain behavioral disorders observed in patients. Therefore, this work provides a principle for further studies on the molecular mechanism of behavioral changes occurring in MPS patients.
Asunto(s)
Trastornos Mentales/genética , Mucopolisacaridosis/genética , Transcriptoma , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Niño , Preescolar , Femenino , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Humanos , Lactante , Masculino , Trastornos Mentales/etiología , Mucopolisacaridosis/complicacionesRESUMEN
The hepatitis B virus (HBV)-induced chronic liver diseases are serious health threats worldwide. There is evidence to display the alterations of salivary N-linked glycans related to the development of HBV-infected liver diseases. Here, we further investigated the alterations of fucosylated N/O-glycans recognized by LTL in saliva from 120 subjects (30 healthy volunteers (HV), 30 patients with hepatitis B (HB), 30 patients with hepatic cirrhosis (HC), and 30 patients with hepatocellular carcinoma (HCC)) using salivary microarrys and MALDI-TOF/TOF-MS. The results showed that the expression level of fucosylated glycans recognized by LTL was significantly increased in HCC compared with other subjects (P < 0.0001). Besides, the fucosylated glycoproteins were isolated from pooled saliva of HV, HB, HC, and HCC by LTL-magnetic particle conjugates. Then, N/O- glycans were released from the isolated glycoproteins with PNGase F and NaClO, and were identified by MALDI-TOF-MS, respectively. Totally, there were 21/20, 25/18, 29/19, and 28/24 N/O-glycan peaks that were identified and annotated with proposed structures in saliva of HV, HB, HC, and HCC. Among the total, there were 8 N-glycan peaks (e.g., m/z 1905.634, 2158.777 and 2905.036) and 15 O-glycan peaks (e.g., 1177.407, 1308.444 and 1322.444) that only presented in patients with HBV-induced liver diseases. One N-glycan peak (m/z 2205.766) was unique in HC, and 9 O-glycan peaks (e.g., m/z 1157.420, 1163.417 and 1193.402) were unique in HCC. This study could facilitate the discovery of biomarkers for HC and HCC based on precise alterations of fucosylated N/O-glycans in saliva.
Asunto(s)
Biomarcadores de Tumor/genética , Virus de la Hepatitis B/genética , Polisacáridos/genética , Análisis por Matrices de Proteínas , Biomarcadores de Tumor/química , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Femenino , Fibrosis/genética , Fibrosis/virología , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Gangliósido G(M1)/genética , Virus de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/patogenicidad , Hepatitis Crónica/genética , Hepatitis Crónica/virología , Humanos , Lectinas/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Masculino , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Saliva/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes.
Asunto(s)
Gangliósido G(M1)/análogos & derivados , N-Acetilgalactosaminiltransferasas/genética , Sialiltransferasas/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Gangliósido G(M1)/biosíntesis , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósidos/genética , Gangliósidos/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicoesfingolípidos/genética , Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Humanos , Ratones , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/metabolismo , Metástasis de la Neoplasia , Sialiltransferasas/metabolismoRESUMEN
Axon-like neuritogenesis in neuroblastoma (NG108-15) cells and primary cerebellar granular neurons is furthered by the presence of ganglioside GM1. We describe here that galectin-1 (Gal-1), a homobivalent endogenous lectin, is an effector by cross-linking the ganglioside and its associated glycoprotein α5 ß1 -integrin. The thereby triggered signaling cascade involves autophosphorylation of focal adhesion kinase and activation of phospholipase Cγ and phosphoinositide-3 kinase. This leads to a transient increase in the intracellular Ca(2+) concentration by opening of TRPC5 channels, which belong to the signal transduction-gated cation channels. Controls with GM1-defective cells (NG-CR72 and neurons from ganglio-series KO mice) were retarded in axonal growth, underscoring the relevance of GM1 as functional counterreceptor for Gal-1. The lectin's presence was detected in the NG108-15 cells, suggesting an autocrine mechanism of action, and in astrocytes in situ. Gal-1, as cross-linking lectin, can thus translate metabolic conversion of ganglioside GD1a to GM1 by neuraminidase action into axon growth. Galectin-1 (Gal-1) was shown an effector of axonogenesis in cerebellar granule neurons (CGNs) and NG108-15 cells by cross-linking GM1 ganglioside and its associated glycoprotein α5 ß1 -integrin. The resulting signaling led to a transient increase in intracellular Ca(2+) by opening TRPC5 channels. CGNs deficient in GM1 showed retarded axonogenesis, underscoring the relevance of GM1 as functional counterreceptor for Gal-1 in this process. This Gal-1/GM1-induced signaling was manifest only at the earliest, initiating stage of axon development.
Asunto(s)
Axones/fisiología , Calcio/metabolismo , Gangliósido G(M1)/metabolismo , Galectina 1/metabolismo , Integrinas/metabolismo , Transducción de Señal/genética , Canales Catiónicos TRPC/metabolismo , Animales , Animales Recién Nacidos , Benzamidas/farmacocinética , Células Cultivadas , Cerebelo/citología , Inhibidores Enzimáticos/farmacología , Gangliósido G(M1)/genética , Galectina 1/genética , Regulación de la Expresión Génica/genética , Integrinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Canales Catiónicos TRPC/genética , Temperatura , Tirosina/análogos & derivados , Tirosina/farmacocinéticaRESUMEN
We tested autoantibodies to neurofascin-186 (NF186) and gliomedin in sera from patients with multifocal motor neuropathy (MMN, n=53) and chronic inflammatory demyelinating polyneuropathy (CIDP, n=95) by ELISA. IgG antibodies to NF186 or gliomedin were found in 62% of MMN and 1% of CIDP sera, and IgM antibodies to the same antigens in 12% of MMN and 1% of CIDP sera. These autoantibodies activated complement. Ten percent of the MMN sera without IgM anti-GM1 reactivity had anti-NF186 antibodies. Because NF186 and gliomedin play a crucial role for salutatory conduction, the autoantibodies may contribute to produce motor nerve conduction block and muscle weakness in MMN.
Asunto(s)
Autoanticuerpos/sangre , Moléculas de Adhesión Celular/inmunología , Proteínas de la Membrana/inmunología , Enfermedad de la Neurona Motora/sangre , Factores de Crecimiento Nervioso/inmunología , Proteínas del Tejido Nervioso/inmunología , Polineuropatías/sangre , Animales , Moléculas de Adhesión Celular/genética , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Femenino , Gangliósido G(M1)/genética , Gangliósido G(M1)/inmunología , Humanos , Masculino , Proteínas de la Membrana/genética , Enfermedad de la Neurona Motora/complicaciones , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Polineuropatías/complicaciones , Isoformas de Proteínas/inmunología , Ratas , TransfecciónRESUMEN
Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2ß1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2ß1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2ß1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2ß1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.
Asunto(s)
Neoplasias Óseas/metabolismo , Gangliósido G(M1)/metabolismo , Integrina alfa2/metabolismo , Neoplasias de la Próstata/metabolismo , Ácidos Siálicos/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Gangliósido G(M1)/genética , Humanos , Integrina alfa2/genética , Masculino , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Ácidos Siálicos/genéticaRESUMEN
Soluble Aß oligomers contribute importantly to synaptotoxicity in Alzheimer's disease, but their dynamics in vivo remain unclear. Here, we found that soluble Aß oligomers were sequestered from brain interstitial fluid onto brain membranes much more rapidly than nontoxic monomers and were recovered in part as bound to GM1 ganglioside on membranes. Aß oligomers bound strongly to GM1 ganglioside, and blocking the sialic acid residue on GM1 decreased oligomer-mediated LTP impairment in mouse hippocampal slices. In a hAPP transgenic mouse model, substantial levels of GM1-bound Aß42 were recovered from brain membrane fractions. We also detected GM1-bound Aß in human CSF, and its levels correlated with Aß42, suggesting its potential as a biomarker of Aß-related membrane dysfunction. Together, these findings highlight a mechanism whereby hydrophobic Aß oligomers become sequestered onto GM1 ganglioside and presumably other lipids on neuronal membranes, where they may induce progressive functional and structural changes.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Gangliósido G(M1)/metabolismo , Fragmentos de Péptidos/metabolismo , Factores de Edad , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Líquido Extracelular/metabolismo , Gangliósido G(M1)/genética , Gangliósidos/genética , Gangliósidos/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/farmacologíaRESUMEN
We have demonstrated previously that n-3 PUFA endogenously produced by fat-1 transgenic mice regulate CD4+ T-cell function by affecting the formation of lipid rafts, liquid-ordered mesodomains in the plasma membrane. In the present study, we tested the effects of dietary sources of n-3 PUFA, i.e. fish oil (FO) or purified DHA, when compared with an n-6 PUFA-enriched maize oil control diet in DO11.10 T-cell receptor transgenic mice. Dietary n-3 PUFA were enriched in CD4+ T-cells, resulting in the increase of the n-3:n-6 ratio. Following antigen-specific CD4+ T-cell activation by B-lymphoma cells pulsed with the ovalbumin 323-339 peptide, the formation of liquid-ordered mesodomains at the immunological synapse relative to the whole CD4+ T-cell, as assessed by Laurdan labelling, was increased (P< 0·05) in the FO-fed group. The FO diet also suppressed (P< 0·05) the co-localisation of PKCθ with ganglioside GM1 (monosialotetrahexosylganglioside), a marker for lipid rafts, which is consistent with previous observations. In contrast, the DHA diet down-regulated (P< 0·05) PKCθ signalling by moderately affecting the membrane liquid order at the immunological synapse, suggesting the potential contribution of the other major n-3 PUFA components of FO, including EPA.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Ácidos Docosahexaenoicos/farmacología , Aceites de Pescado/farmacología , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Membrana Celular , Aceite de Maíz/química , Dieta/veterinaria , Ácidos Docosahexaenoicos/química , Regulación hacia Abajo , Ácidos Grasos Omega-6/química , Ácidos Grasos Omega-6/farmacología , Femenino , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-thetaRESUMEN
Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.
Asunto(s)
Ceramidas/metabolismo , Toxina del Cólera/metabolismo , Toxina del Cólera/farmacología , Gangliósido G(M1)/metabolismo , Transcitosis/efectos de los fármacos , Animales , Ceramidas/genética , Perros , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Endosomas/genética , Endosomas/metabolismo , Gangliósido G(M1)/genética , Células de Riñón Canino Madin DarbyRESUMEN
Hemagglutinating virus of Japan-envelope (HVJ-E) is a drug delivery vector based on inactivated Sendai virus. Recently, antitumor activities were found for HVJ-E itself and clinical trials of HVJ-E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ-E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ-E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK-N-SH was the most sensitive to HVJ-E in vitro and total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK-N-SH cells was achieved by intratumoral injection of HVJ-E in SCID mice and no recurrence was observed for more than 300 days after HVJ-E inoculation. In contrast, NB1 cells expressed the lowest amount of GD1a and SPG and were resistant to HVJ-E in vitro. The expression of GD1a increased by 13-cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ-E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ-E in the SCID mice, but the combination of 13cRA and HVJ-E could achieve partial eradication of the xenograft and also lead to an improved prognosis. In conclusion, HVJ-E is a promising therapeutic modality for neuroblastoma and 13cRA can be used as an adjuvant to HVJ-E.
Asunto(s)
Antineoplásicos/farmacología , Isotretinoína/farmacología , Neuroblastoma/terapia , Neuroblastoma/virología , Viroterapia Oncolítica/métodos , Virus Sendai/fisiología , Proteínas del Envoltorio Viral/farmacología , Animales , Línea Celular Tumoral , Quimioterapia Adyuvante/métodos , Femenino , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Gangliósidos/genética , Gangliósidos/metabolismo , Vectores Genéticos/farmacología , Humanos , Ratones , Ratones SCID , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genéticaRESUMEN
Clostridium perfringens alpha-toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72-93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H SXWY G) that is the same motif seen in botulinum neurotoxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-toxin was only slightly toxic in ß1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed alpha-toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of alpha-toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for alpha-toxin, which can be a potential target for drug developed against this pathogen.
Asunto(s)
Clostridium perfringens , Gangliósido G(M1)/análogos & derivados , Macrófagos Peritoneales/metabolismo , Receptor trkA/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Toxinas Bacterianas , Proteínas de Unión al Calcio , Línea Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , Estructura Terciaria de Proteína , Receptor trkA/genética , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Fosfolipasas de Tipo C , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Shiga-toxigenic Escherichia coli (STEC) O113:H21 strains that lack the locus of enterocyte effacement (LEE) efficiently invade eukaryotic cells in vitro, unlike LEE-positive O157:H7 strains. We used a fliC deletion mutant of the O113:H21 STEC strain 98NK2 (98NK2ΔfliC) to show that invasion of colonic epithelial (HCT-8) cells is heavily dependent on production of flagellin, even though adherence to the cells was actually enhanced in the mutant. Flagellin binds and signals through Toll-like receptor 5 (TLR5), but there was no evidence that either TLR5, the adaptor protein myeloid differentiation primary response gene 88 (MyD88), or the serine kinase interleukin-1 receptor-associated kinase (IRAK) were required for invasion of HCT-8 cells by strain 98NK2, as judged by transfection, RNA knockdown, or inhibitor studies. However, pretreatment of cells with anti-asialo-GM1 significantly decreased 98NK2 invasion (by 40.8%), while neuraminidase treatment (which cleaves terminal sialic acid residues, thus converting GM1 into asialo-GM1) significantly increased invasion (by 70.7%). Pretreatment of HCT-8 cells with either the cholesterol-depleting agent methyl-ß-cyclodextrin (MßCD) or the tyrosine kinase inhibitor genistein significantly decreased invasion by 98NK2, indicating a potential role for lipid rafts in the invasion mechanism. Confocal microscopy also showed invading 98NK2 colocalized with lipid raft markers caveolin-1 and GM1. Interestingly, anti-asialo-GM1, neuraminidase, MßCD, and genistein have similar effects on the vestigial level of STEC invasion seen for STEC strain 98NK2ΔfliC, indicating that lipid rafts mediate a common step in flagellin-dependent and flagellin-independent cellular invasion.
Asunto(s)
Colon/citología , Células Epiteliales/microbiología , Flagelina/metabolismo , Microdominios de Membrana/fisiología , Escherichia coli Shiga-Toxigénica/metabolismo , Línea Celular Tumoral , Células Epiteliales/citología , Flagelina/genética , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/genética , Receptor Toll-Like 5/metabolismoRESUMEN
Several studies have successfully employed GM1 ganglioside to treat animal models of Parkinson's disease (PD), suggesting involvement of this ganglioside in PD etiology. We recently demonstrated that genetically engineered mice (B4galnt1(-/-) ) devoid of GM1 acquire characteristic symptoms of this disorder, including motor impairment, depletion of striatal dopamine, selective loss of tyrosine hydroxylase-expressing neurons, and aggregation of α-synuclein. The present study demonstrates similar symptoms in heterozygous mice (HTs) that express only partial GM1 deficiency. Symptoms were alleviated by administration of L-dopa or LIGA-20, a membrane-permeable analog of GM1 that penetrates the blood-brain barrier and accesses intracellular compartments. Immunohistochemical analysis of paraffin sections from PD patients revealed significant GM1 deficiency in nigral dopaminergic neurons compared with age-matched controls. This was comparable to the GM1 deficiency of HT mice and suggests that GM1 deficiency may be a contributing factor to idiopathic PD. We propose that HT mice with partial GM1 deficiency constitute an especially useful model for PD, reflecting the actual pathophysiology of this disorder. The results point to membrane-permeable analogs of GM1 as holding promise as a form of GM1 replacement therapy.