RESUMEN
The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal , Manosafosfatos , Mucolipidosis , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Dominio Catalítico , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/enzimología , Manosafosfatos/metabolismo , Mucolipidosis/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Mucolipidosis II and III (ML II/III) are caused by a deficiency of uridine-diphosphate N-acetylglucosamine: lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. The process is performed by a sequential two-step process: first, GlcNAc-1-phosphotransferase catalyzes the transfer of GlcNAc-1-phosphate to the selected mannose residues on lysosomal enzymes in the cis-Golgi network. The second step removes GlcNAc from lysosomal enzymes by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (uncovering enzyme) and exposes the mannose 6-phosphate (M6P) residues in the trans-Golgi network, in which the enzymes are targeted to the lysosomes by M6Preceptors. A deficiency of GlcNAc-1-phosphotransferase causes the hypersecretion of lysosomal enzymes out of cells, resulting in a shortage of multiple lysosomal enzymes within lysosomes. Due to a lack of GlcNAc-1-phosphotransferase, the accumulation of cholesterol, phospholipids, glycosaminoglycans (GAGs), and other undegraded substrates occurs in the lysosomes. Clinically, ML II and ML III exhibit quite similar manifestations to mucopolysaccharidoses (MPSs), including specific skeletal deformities known as dysostosis multiplex and gingival hyperplasia. The life expectancy is less than 10 years in the severe type, and there is no definitive treatment for this disease. In this review, we have described the updated diagnosis and therapy on ML II/III.
Asunto(s)
Terapia de Reemplazo Enzimático/métodos , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Lisosomas/metabolismo , Mucolipidosis/diagnóstico , Animales , Transporte Biológico Activo , Modelos Animales de Enfermedad , Glicosaminoglicanos/metabolismo , Humanos , Manosafosfatos/metabolismo , Mucolipidosis/enzimología , Mucolipidosis/fisiopatología , Mucolipidosis/terapiaRESUMEN
Mutations in the galactosidase ß 1 (GLB1) gene cause lysosomal ß-galactosidase (ß-Gal) deficiency and clinical onset of the neurodegenerative lysosomal storage disease, GM1 gangliosidosis. ß-Gal and neuraminidase 1 (NEU1) form a multienzyme complex in lysosomes along with the molecular chaperone, protective protein cathepsin A (PPCA). NEU1 is deficient in the neurodegenerative lysosomal storage disease sialidosis, and its targeting to and stability in lysosomes strictly depend on PPCA. In contrast, ß-Gal only partially depends on PPCA, prompting us to investigate the role that ß-Gal plays in the multienzyme complex. Here, we demonstrate that ß-Gal negatively regulates NEU1 levels in lysosomes by competitively displacing this labile sialidase from PPCA. Chronic cellular uptake of purified recombinant human ß-Gal (rhß-Gal) or chronic lentiviral-mediated GLB1 overexpression in GM1 gangliosidosis patient fibroblasts coincides with profound secondary NEU1 deficiency. A regimen of intermittent enzyme replacement therapy dosing with rhß-Gal, followed by enzyme withdrawal, is sufficient to augment ß-Gal activity levels in GM1 gangliosidosis patient fibroblasts without promoting NEU1 deficiency. In the absence of ß-Gal, NEU1 levels are elevated in the GM1 gangliosidosis mouse brain, which are restored to normal levels following weekly intracerebroventricular dosing with rhß-Gal. Collectively, our results highlight the need to carefully titrate the dose and dosing frequency of ß-Gal augmentation therapy for GM1 gangliosidosis. They further suggest that intermittent intracerebroventricular enzyme replacement therapy dosing with rhß-Gal is a tunable approach that can safely augment ß-Gal levels while maintaining NEU1 at physiological levels in the GM1 gangliosidosis brain.
Asunto(s)
Terapia de Reemplazo Enzimático , Fibroblastos/enzimología , Lisosomas/enzimología , Mucolipidosis , beta-Galactosidasa/uso terapéutico , Animales , Células CHO , Cricetulus , Humanos , Lisosomas/genética , Ratones , Ratones Mutantes , Mucolipidosis/tratamiento farmacológico , Mucolipidosis/enzimología , Mucolipidosis/genética , Neuraminidasa/genética , Neuraminidasa/metabolismoRESUMEN
BACKGROUND: Mucolipidosis alpha/beta is an inborn error of metabolism characterized by deficiency of GlcNAc-1-phosphotransferase, in which essential alpha/beta subunits are encoded by the GNPTAB gene. The autosomal recessive condition is due to disruptions of hydrolase mannose 6-phosphate marker generation, defective lysosomal targeting and subsequent intracellular accumulation of non-degraded material. Clinical severity depends on residual GlcNAc-1-phosphotransferase activity, which distinguishes between the milder type III disease and the severe, neonatal onset type II disease. CASE PRESENTATION: We report the clinical, biochemical and genetic diagnosis of mucolipidosis III alpha/beta in a two-year-old Chinese boy who initially presented with poor weight gain, microcephaly and increased tone. He was confirmed to harbor the common splice site mutation c.2715 + 1G > A and the nonsense variant c.2404C > T (p.Q802*). Clinically, the patient had multiple phenotypic features typical of mucopolysaccharidosis including joint contractures, coarse facial features, kypho-lordosis, pectus carinatum and umbilical hernia. However, the relatively mild developmental delay compared to severe type I and type II mucopolysaccharidosis and the absence of macrocephaly raised the possibility of the less commonly diagnosed mucolipidosis alpha/beta. Critical roles of lysosomal enzyme activity assay, which showed elevated α-iduronidase, iduronate sulfatase, galactose-6-sulphate sulphatase, arylsulfatase B and α-hexosaminidase activities; and genetic study, which confirmed the parental origin of both mutations, were highlighted. CONCLUSIONS: The recently reported nonsense variant c.2404C > T in the GNPTAB gene is further recognized and this contributes to the genotype-phenotype spectrum of mucolipidosis alpha/beta.
Asunto(s)
Codón sin Sentido , Mucolipidosis/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Preescolar , Condroitinsulfatasas/genética , Condroitinsulfatasas/metabolismo , Regulación de la Expresión Génica , Genes Recesivos , Humanos , Iduronato Sulfatasa/genética , Iduronato Sulfatasa/metabolismo , Iduronidasa/genética , Iduronidasa/metabolismo , Lisosomas/enzimología , Lisosomas/patología , Masculino , Mucolipidosis/diagnóstico , Mucolipidosis/enzimología , Mucolipidosis/patología , N-Acetilgalactosamina-4-Sulfatasa/genética , N-Acetilgalactosamina-4-Sulfatasa/metabolismo , Linaje , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Cadena alfa de beta-Hexosaminidasa/genética , Cadena alfa de beta-Hexosaminidasa/metabolismoRESUMEN
The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a Ï-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , Glucosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Secuencias de Aminoácidos , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Glucosiltransferasas/genética , Aparato de Golgi/genética , Células HEK293 , Células HeLa , Humanos , Mucolipidosis/enzimología , Mucolipidosis/genética , Mutación Missense , Dominios ProteicosRESUMEN
Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α/ß-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α/ß-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.
Asunto(s)
Mutación con Pérdida de Función , Mucolipidosis/enzimología , Mucolipidosis/genética , Mutación Missense , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Genotipo , Humanos , Lactante , Masculino , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Adulto JovenRESUMEN
The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver.
Asunto(s)
Hidrolasas/metabolismo , Lisosomas/metabolismo , Manosafosfatos/metabolismo , Mucolipidosis/enzimología , Proteoma/análisis , Animales , Células Cultivadas , Cromatografía Liquida , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hígado/metabolismo , Espectrometría de Masas , Ratones , Mucolipidosis/genética , Fosfotransferasas/deficienciaRESUMEN
Lysosomes are cytoplasmic compartments that contain many acid hydrolases and play critical roles in the metabolism of a wide range of macromolecules. Deficiencies in lysosomal enzyme activities cause genetic diseases, called lysosomal storage disorders (LSDs). Many mutations have been identified in the genes responsible for LSDs, and the identification of mutations is required for the accurate molecular diagnoses. Here, we analyzed cell lines that were derived from two different LSDs, GM1 gangliosidosis and sialidosis. GM1 gangliosidosis is caused by mutations in the GLB1 gene that encodes ß-galactosidase. A lack of ß-galactosidase activity leads to the massive accumulation of GM1 ganglioside, which results in neurodegenerative pathology. Mutations in the NEU1 gene that encodes lysosomal sialidase cause sialidosis. Insufficient activity of lysosomal sialidase progressively increases the accumulation of sialylated molecules, and various clinical symptoms, including mental retardation, appear. We sequenced the entire coding regions of GLB1 and NEU1 in GM1 gangliosidosis and sialidosis patient cells, respectively. We found the novel mutations p.E186A in GLB1 and p.R347Q in NEU1, as well as many other mutations that have been previously reported. We also demonstrated that patient cells containing the novel mutations showed the molecular phenotypes of the corresponding disease. Further structural analysis suggested that these novel mutation sites are highly conserved and important for enzyme activity.
Asunto(s)
Gangliosidosis GM1/enzimología , Gangliosidosis GM1/genética , Mucolipidosis/enzimología , Mucolipidosis/genética , Neuraminidasa/genética , beta-Galactosidasa/genética , Secuencia de Aminoácidos , Animales , Fibroblastos/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Neuraminidasa/química , Neuraminidasa/metabolismo , Alineación de Secuencia , beta-Galactosidasa/química , beta-Galactosidasa/metabolismoRESUMEN
Antigen processing and presentation and cytotoxic targeting depend on the activities of several lysosomal enzymes that require mannose 6-phosphate (M6P) sorting signals for efficient intracellular transport and localization. In this paper, we show that mice deficient in the formation of M6P residues exhibit significant loss of cathepsin proteases in B cells, leading to lysosomal dysfunction with accumulation of storage material, impaired antigen processing and presentation, and subsequent defects in B cell maturation and antibody production. The targeting of lysosomal and granular enzymes lacking M6P residues is less affected in dendritic cells and T cells and sufficient for maintenance of degradative and lytic functions. M6P deficiency also impairs serum immunoglobulin levels and antibody responses to vaccination in patients. Our data demonstrate the critical role of M6P-dependent transport routes for B cell functions in vivo and humoral immunity in mice and human.
Asunto(s)
Linfocitos B/enzimología , Lisosomas/enzimología , Manosafosfatos/metabolismo , Péptido Hidrolasas/metabolismo , Animales , Presentación de Antígeno , Antígenos/metabolismo , Linfocitos B/inmunología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Dendríticas/enzimología , Células Dendríticas/inmunología , Humanos , Ratones Transgénicos , Mucolipidosis/enzimología , Mucolipidosis/inmunología , Fosforilación , Procesamiento Proteico-Postraduccional , Linfocitos T/inmunología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase tags newly synthesized lysosomal enzymes with mannose 6-phosphate recognition markers, which are required for their targeting to the endolysosomal system. GNPTAB encodes the α and ß subunits of GlcNAc-1-phosphotransferase, and mutations in this gene cause the lysosomal storage disorders mucolipidosis II and III αß. Prior investigation of missense mutations in GNPTAB uncovered amino acids in the N-terminal region and within the DMAP domain involved in Golgi retention of GlcNAc-1-phosphotransferase and its ability to specifically recognize lysosomal hydrolases, respectively. Here, we undertook a comprehensive analysis of the remaining missense mutations in GNPTAB reported in mucolipidosis II and III αß patients using cell- and zebrafish-based approaches. We show that the Stealth domain harbors the catalytic site, as some mutations in these regions greatly impaired the activity of the enzyme without affecting its Golgi localization and proteolytic processing. We also demonstrate a role for the Notch repeat 1 in lysosomal hydrolase recognition, as missense mutations in conserved cysteine residues in this domain do not affect the catalytic activity but impair mannose phosphorylation of certain lysosomal hydrolases. Rescue experiments using mRNA bearing Notch repeat 1 mutations in GNPTAB-deficient zebrafish revealed selective effects on hydrolase recognition that differ from the DMAP mutation. Finally, the mutant R587P, located in the spacer between Notch 2 and DMAP, was partially rescued by overexpression of the γ subunit, suggesting a role for this region in γ subunit binding. These studies provide new insight into the functions of the different domains of the α and ß subunits.
Asunto(s)
Lisosomas/metabolismo , Mucolipidosis/enzimología , Mucolipidosis/genética , Mutación Missense/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Humanos , Mucolipidosis/metabolismo , Pez CebraRESUMEN
Mucolipidosis II (MLII) is a lysosomal storage disorder caused by loss of N-acetylglucosamine-1-phosphotransferase, which tags lysosomal enzymes with a mannose 6-phosphate marker for transport to the lysosome. In MLII, the loss of this marker leads to deficiency of multiple enzymes and non-enzymatic proteins in the lysosome, leading to the storage of multiple substrates. Here we present a novel mouse model of MLII homozygous for a patient mutation in the GNPTAB gene. Whereas the current gene knock-out mouse model of MLII lacks some of the characteristic features of the human disease, our novel mouse model more fully recapitulates the human pathology, showing growth retardation, skeletal and facial abnormalities, increased circulating lysosomal enzymatic activities, intracellular lysosomal storage, and reduced life span. Importantly, MLII behavioral deficits are characterized for the first time, including impaired motor function and psychomotor retardation. Histological analysis of the brain revealed progressive neurodegeneration in the cerebellum with severe Purkinje cell loss as the underlying cause of the ataxic gait. In addition, based on the loss of Npc2 (Niemann-Pick type C 2) protein expression in the brain, the mice were treated with 2-hydroxypropyl-ß-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of Niemann-Pick type C disease. No improvement in brain pathology was observed. This indicates that cerebellar degeneration is not primarily triggered by loss of Npc2 function. This study emphasizes the value of modeling MLII patient mutations to generate clinically relevant mouse mutants to elucidate the pathogenic molecular pathways of MLII and address their amenability to therapy.
Asunto(s)
Modelos Animales de Enfermedad , Homocigoto , Mucolipidosis , Mutación , Células de Purkinje , Transferasas (Grupos de Otros Fosfatos Sustitutos) , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Conducta Animal , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Excipientes/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Mucolipidosis/enzimología , Mucolipidosis/genética , Mucolipidosis/patología , Enfermedad de Niemann-Pick Tipo C/enzimología , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Células de Purkinje/enzimología , Células de Purkinje/patología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-Ciclodextrinas/farmacologíaRESUMEN
The lysosomal storage disorder mucolipidosis III αß is caused by mutations in the αß subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase). This Golgi-localized enzyme mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases, and loss of function results in impaired lysosomal targeting of these acid hydrolases and decreased lysosomal degradation. Here we show that two patient missense mutations, Lys4Gln and Ser15Tyr, in the N-terminal cytoplasmic tail of the α subunit of phosphotransferase impair retention of the catalytically active enzyme in the Golgi complex. This results in mistargeting of the mutant phosphotransferases to lysosomes, where they are degraded, or to the cell surface and release into the medium. The finding that mislocalization of active phosphotransferase is the basis for mucolipidosis III αß in a subset of patients shows the importance of single residues in the cytoplasmic tail of a Golgi-resident protein for localization to this compartment.
Asunto(s)
Aparato de Golgi/metabolismo , Mucolipidosis/enzimología , Mucolipidosis/etiología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Electroforesis en Gel de Poliacrilamida , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Microscopía Fluorescente , Mutación Missense/genética , ProteolisisRESUMEN
The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase ß-galactosidase (ß-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and ß-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.
Asunto(s)
Catepsina A/metabolismo , Gangliosidosis GM1/enzimología , Enfermedades por Almacenamiento Lisosomal/enzimología , Lisosomas/enzimología , Mucolipidosis/enzimología , Neuraminidasa/metabolismo , beta-Galactosidasa/metabolismo , Animales , Catepsina A/genética , Modelos Animales de Enfermedad , Gangliosidosis GM1/genética , Gangliosidosis GM1/patología , Regulación de la Expresión Génica , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/genética , Lisosomas/patología , Ratones , Ratones Noqueados , Mucolipidosis/genética , Mucolipidosis/patología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Neuraminidasa/genética , Transducción de Señal , beta-Galactosidasa/genéticaRESUMEN
The GlcNAc-1-phosphotransferase catalyzes the first step in the formation of mannose 6-phosphate (M6P) residues on lysosomal acid hydrolases that is essential for the efficient transport of newly synthesized lysosomal enzymes to lysosomes and the maintenance of lysosomal functions. Mutations in the GlcNAc-1-phosphotransferase cause the lysosomal storage disease mucolipidosis type II (MLII), resulting in mistargeting and hypersecretion of multiple lysosomal hydrolases and subsequent lysosomal accumulation of nondegraded material in several tissues. To describe cell-type specificity, compositional differences, and subcellular distribution of the stored material we performed an in-depth ultrastructural analysis of lysosomal storage in brain and retina of MLII knock-in mice using electron microscopy. Massive vacuoles filled with heterogeneous storage material have been found in the soma, swollen axons, and dendrites of Purkinje, and granular cells in 9-month-old MLII mice. In addition, non-neuronal cells, such as microglial, astroglial, and endothelial cells, exhibit storage material. Fucose-specific lectin histochemistry demonstrated the accumulation of fucose-containing oligosaccharides, indicating that targeting of the lysosomal α-fucosidase is strongly impaired in all cerebellar cell types. The data suggest that the accumulation of storage material might affect neuronal function and survival in a direct cell-autonomous manner, as well as indirectly by disturbed metabolic homeostasis between glial and neuronal cells or by cerebrovascular complications.
Asunto(s)
Cerebelo/ultraestructura , Lisosomas/ultraestructura , Mucolipidosis/patología , Neuronas/ultraestructura , Retina/ultraestructura , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Biomarcadores/metabolismo , Supervivencia Celular , Cerebelo/enzimología , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Predisposición Genética a la Enfermedad , Lectinas/metabolismo , Lisosomas/enzimología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucolipidosis/enzimología , Mucolipidosis/genética , Mutación , Neuronas/enzimología , Fenotipo , Retina/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) is an α2ß2γ2 heterohexamer that mediates the initial step in the formation of the mannose 6-phosphate recognition signal on lysosomal acid hydrolases. We previously reported that the specificity of the reaction is determined by the ability of the α/ß subunits to recognize a conformation-dependent protein determinant present on the acid hydrolases. We now present evidence that the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit functions in this recognition process. First, GST-DMAP pulled down several acid hydrolases, but not nonlysosomal glycoproteins. Second, recombinant GlcNAc-1-phosphotransferase containing a missense mutation in the DMAP interaction domain (Lys732Asn) identified in a patient with mucolipidosis II exhibited full activity toward the simple sugar α-methyl d-mannoside but impaired phosphorylation of acid hydrolases. Finally, unlike the WT enzyme, expression of the K732N mutant in a zebrafish model of mucolipidosis II failed to correct the phenotypic abnormalities. These results indicate that the DMAP interaction domain of the α subunit functions in the selective recognition of acid hydrolase substrates and provides an explanation for the impaired phosphorylation of acid hydrolases in a patient with mucolipidosis II.
Asunto(s)
Anomalías Múltiples/metabolismo , Lisosomas/enzimología , Mucolipidosis/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Proteínas de Pez Cebra/metabolismo , Anomalías Múltiples/enzimología , Acetilglucosamina/metabolismo , Animales , Femenino , Células HEK293 , Células HeLa , Humanos , Hidrolasas/metabolismo , Masculino , Manosafosfatos/metabolismo , Ratones , Mucolipidosis/enzimología , Mutagénesis Sitio-Dirigida , Mutación Missense , Fosforilación/fisiología , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Mucolipidosis II alpha/beta (ML II alpha/beta; I-cell disease) is a rare, inherited, metabolic disease and has often been clinically misdiagnosed. ML II alpha/beta results from a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-PT), which causes the lysosomal enzymes to accumulate in plasma. We identified two new Chinese patients with ML II alpha/beta by lysosomal enzyme assay. Using targeted next-generation sequencing genetic analysis, we located two homozygous nonsense mutations in the GNPTAB gene, c.1071G>A (p.W357X) and c.1090C>T (p.R364X). These results were confirmed by Sanger sequencing. To our knowledge, the c.1071G>A mutation has not been previously reported. Our findings add to the number of reported cases of this rare illness and to the GNPTAB pathogenic mutation database. This work also demonstrates the application of lysosomal enzyme assay and targeted next-generation sequencing for the genetic screening analysis and diagnosis of ML II alpha/beta.
Asunto(s)
Codón sin Sentido , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Mucolipidosis/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , China , Femenino , Humanos , Lactante , Lisosomas/enzimología , Masculino , Mucolipidosis/enzimología , Mucolipidosis/patología , Análisis de Secuencia de ADN , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated 'knock-in' mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, ß-hexosaminidase, α-mannosidase or Niemann-Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease.
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Lisosomas/genética , Mucolipidosis/genética , Degeneración Nerviosa/genética , Animales , Atrofia , Autofagia , Encéfalo/enzimología , Encéfalo/patología , Modelos Animales de Enfermedad , Lisosomas/enzimología , Lisosomas/patología , Ratones , Ratones Transgénicos , Mucolipidosis/enzimología , Mucolipidosis/patología , Degeneración Nerviosa/enzimología , Degeneración Nerviosa/patología , Proteínas de Transporte Vesicular/metabolismo , alfa-L-Fucosidasa/metabolismo , alfa-Manosidasa/metabolismo , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Stuttering is a common but poorly understood speech disorder. Consistent evidence for the involvement of genetic factors in stuttering has motivated studies aimed at identifying causative genetic variants that could shed light on the underlying molecular and cellular deficits in this disorder. Such studies have begun to identify causative genes. The purpose of this review is to summarize the gene discoveries to date, and to cover the subsequent functional studies that are beginning to provide insights into how these gene mutations might cause stuttering. Surprisingly, the first variant genes to be associated with stuttering are those encoding the lysosomal targeting system, GNPTAB, GNPTG, and NAGPA. Although mutations in NAGPA have not been associated with a disorder in humans, mutations in GNPTAB and GNPTG cause mucolipidosis types II and III, which are rare autosomal recessive lysosomal storage disorders, associated with pathology of bone, connective tissue, liver, spleen, and brain. Analysis of mutations in these genes has so far identified predominantly missense mutations in stuttering, in contrast to the truncating and other mutations that result in very low GNPTAB/G enzyme activity and are historically associated with mucolipidosis. Genetic evidence for the role of lysosomal targeting mutations in stuttering has now been buttressed by biochemical studies of the mutant enzymes found in this disorder. While data on the GlcNAc-phosphotransferase encoded by GNPTAB/G remains limited and only suggestive, a study of the enzyme encoded by NAGPA has shown that the mutations found in stuttering reduce the overall cellular activity of this enzyme by about half, and that they result in deficits in intracellular processing and trafficking that lead to a reduced cellular half life. How these deficits result in the presumed speech-specific neuropathology associated with stuttering is not yet known. However these findings have opened several new lines of inquiry, including studies in mice carrying human stuttering mutations, that represent promising approaches to this disorder.
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Lisosomas/enzimología , Mucolipidosis/enzimología , Hidrolasas Diéster Fosfóricas/genética , Tartamudeo/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Animales , Transporte Biológico , Modelos Animales de Enfermedad , Humanos , Lisosomas/patología , Ratones , Mucolipidosis/complicaciones , Mucolipidosis/genética , Mutación , Hidrolasas Diéster Fosfóricas/metabolismo , Tartamudeo/complicaciones , Tartamudeo/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Vocalización AnimalAsunto(s)
Mucolipidosis/enzimología , Mucolipidosis/patología , Neuraminidasa/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Capa Delgada , Femenino , Humanos , India , Lactante , Datos de Secuencia Molecular , Mutación/genética , Neuraminidasa/deficiencia , Oligosacáridos/orina , Análisis de Secuencia de ADNRESUMEN
We report undescribed pulmonary findings in a child with mucolipidosis II (ML-II). Children with ML-II bear significant pulmonary morbidity that may include extensive pulmonary fibrosis, persistent hemosiderosis as well as pulmonary airway excrescences as they reach preschool age.