RESUMO
Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.
Assuntos
Cistos do Sistema Nervoso Central/genética , Defeitos Congênitos da Glicosilação/genética , Hamartoma/genética , Deficiência Intelectual/genética , Oligossacarídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Polimicrogiria/genética , alfa-Manosidase/genética , Adolescente , Alelos , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Linhagem Celular Tumoral , Cistos do Sistema Nervoso Central/metabolismo , Cistos do Sistema Nervoso Central/patologia , Vermis Cerebelar/metabolismo , Vermis Cerebelar/patologia , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Feminino , Feto , Glicosilação , Hamartoma/metabolismo , Hamartoma/patologia , Humanos , Hipotálamo/metabolismo , Hipotálamo/patologia , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Manose/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polimicrogiria/metabolismo , Polimicrogiria/patologia , Língua/metabolismo , Língua/patologia , alfa-Manosidase/deficiênciaRESUMO
Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.
Assuntos
Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Extratos Vegetais/química , Arabidopsis/química , Arabidopsis/enzimologia , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Plantas/metabolismo , Polissacarídeos/metabolismoRESUMO
Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.
Assuntos
Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Extratos Vegetais/química , Sequência de Aminoácidos , Arabidopsis/química , Cromatografia Líquida/métodos , Corantes Fluorescentes/química , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Solanum lycopersicum/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/isolamento & purificação , Folhas de Planta/química , Plantas Geneticamente Modificadas , Especificidade por SubstratoRESUMO
Wolfram syndrome is an autosomal recessive neuro-degenerative disorder associated with juvenile onset non-autoimmune diabetes mellitus and progressive optic atrophy. The disease has been attributed to mutations in the WFS1 gene, which codes for a protein predicted to possess 9-10 transmembrane segments. Little is known concerning the function of the WFS1 protein (wolframin). Endoglycosidase H digestion, immunocytochemistry, and subcellular fractionation studies all indicated that wolframin is localized to the endoplasmic reticulum in rat brain hippocampus and rat pancreatic islet beta-cells, and after ectopic expression in Xenopus oocytes. Reconstitution of wolframin from oocyte membranes into planar lipid bilayers demonstrated that the protein induced a large cation-selective ion channel that was blocked by Mg2+ or Ca2+. Inositol triphosphate was capable of activating channels in the fused bilayers that were similar to channel components induced by wolframin expression. Expression of wolframin also increased cytosolic calcium levels in oocytes. Wolframin thus appears to be important in the regulation of intracellular Ca2+ homeostasis. Disruption of this function may place cells at risk to suffer inappropriate death decisions, thus accounting for the progressive beta-cell loss and neuronal degeneration associated with the disease.