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1.
Cell ; 142(2): 203-17, 2010 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-20637498

RESUMEN

N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.


Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Anomalías Múltiples/metabolismo , Dolicoles/metabolismo , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Animales , Butadienos/metabolismo , Consanguinidad , Embrión de Mamíferos/metabolismo , Estudio de Asociación del Genoma Completo , Glicosilación , Hemiterpenos/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Pentanos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Respuesta de Proteína Desplegada
2.
Am J Hum Genet ; 108(7): 1342-1349, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34143952

RESUMEN

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.


Asunto(s)
Proteínas de Unión al Calcio/genética , Trastornos Congénitos de Glicosilación/genética , Retículo Endoplásmico/genética , alfa-Manosidasa/genética , Adolescente , Alelos , Proteínas de Unión al Calcio/deficiencia , Línea Celular , Niño , Preescolar , Trastornos Congénitos de Glicosilación/sangre , Discapacidades del Desarrollo/genética , Femenino , Glicoproteínas/sangre , Glicosilación , Humanos , Lactante , Discapacidad Intelectual/genética , Masculino , Mutación , Linaje , Polisacáridos/sangre , Deficiencias en la Proteostasis/genética , alfa-Manosidasa/deficiencia
3.
Am J Hum Genet ; 108(11): 2130-2144, 2021 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-34653363

RESUMEN

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.


Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Genes Dominantes , Hexosiltransferasas/genética , Proteínas de la Membrana/genética , Enfermedades Musculoesqueléticas/genética , Enfermedades del Sistema Nervioso/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Dominio Catalítico , Preescolar , Femenino , Heterocigoto , Hexosiltransferasas/química , Humanos , Masculino , Proteínas de la Membrana/química , Persona de Mediana Edad , Linaje , Homología de Secuencia de Aminoácido
4.
Anal Chem ; 96(22): 8956-8964, 2024 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-38776126

RESUMEN

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics has evolved rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of parallel accumulation serial fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, the reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings, showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 min, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics conditions to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data.


Asunto(s)
Glicopéptidos , Proteómica , Proteómica/métodos , Humanos , Glicopéptidos/análisis , Glicopéptidos/química , Glicopéptidos/sangre , Flujo de Trabajo , Glicoproteínas/análisis , Glicoproteínas/química , Glicoproteínas/sangre , Cromatografía Liquida , Espectrometría de Masas en Tándem
5.
Mol Genet Metab ; 142(3): 108511, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38878498

RESUMEN

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.


Asunto(s)
Intrones , ARN Mensajero , Humanos , Masculino , Intrones/genética , ARN Mensajero/genética , ATPasas de Translocación de Protón Vacuolares/genética , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/patología , Mutación , Secuenciación Completa del Genoma , Secuenciación del Exoma , Análisis de Secuencia de ARN , Discapacidad Intelectual/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/patología , Niño , Empalme del ARN/genética , Preescolar
6.
Clin Chem Lab Med ; 62(8): 1626-1635, 2024 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-38332688

RESUMEN

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. METHODS: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. RESULTS: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. CONCLUSIONS: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.


Asunto(s)
Mieloma Múltiple , Humanos , Mieloma Múltiple/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/diagnóstico , Glicosilación , Proteómica/métodos , Espectrometría de Masas en Tándem , Glicoproteínas/metabolismo , Cromatografía Liquida , Proteínas de Mieloma/metabolismo , Proteínas de Mieloma/análisis
7.
Anal Bioanal Chem ; 416(15): 3595-3604, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38676823

RESUMEN

Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-ß-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.


Asunto(s)
Cromatografía de Fase Inversa , Fibroblastos , Espectrometría de Masas en Tándem , Humanos , Fibroblastos/metabolismo , Espectrometría de Masas en Tándem/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía de Fase Inversa/métodos , Nucleótidos/análisis , Nucleótidos/metabolismo , Aniones/análisis , Cromatografía Líquida con Espectrometría de Masas
8.
J Neurochem ; 167(1): 76-89, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37650222

RESUMEN

N-acetylneuraminic acid (sialic acid) is present in large quantities in the brain and plays a crucial role in brain development, learning, and memory formation. How sialic acid contributes to brain development is not fully understood. The purpose of this study was to determine the effects of reduced sialylation on network formation in human iPSC-derived neurons (iNeurons). Using targeted mass spectrometry and antibody binding, we observed an increase in free sialic acid and polysialic acid during neuronal development, which was disrupted by treatment of iNeurons with a synthetic inhibitor of sialic acid biosynthesis. Sialic acid inhibition disturbed synapse formation and network formation on microelectrode array (MEA), showing short but frequent (network) bursts and an overall lower firing rate, and higher percentage of random spikes. This study shows that sialic acid is necessary for neuronal network formation during human neuronal development and provides a physiologically relevant model to study the role of sialic acid in patient-derived iNeurons.


Asunto(s)
Células Madre Pluripotentes Inducidas , Ácido N-Acetilneuramínico , Humanos , Ácido N-Acetilneuramínico/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Encéfalo/metabolismo
9.
Anal Chem ; 95(26): 9787-9796, 2023 07 04.
Artículo en Inglés | MEDLINE | ID: mdl-37341384

RESUMEN

Distinguishing isomeric saccharides poses a major challenge for analytical workflows based on (liquid chromatography) mass spectrometry (LC-MS). In recent years, many studies have proposed infrared ion spectroscopy as a possible solution as the orthogonal, spectroscopic characterization of mass-selected ions can often distinguish isomeric species that remain unresolved using conventional MS. However, the high conformational flexibility and extensive hydrogen bonding in saccharides cause their room-temperature fingerprint infrared spectra to have broad features that often lack diagnostic value. Here, we show that room-temperature infrared spectra of ion-complexed saccharides recorded in the previously unexplored far-infrared wavelength range (300-1000 cm-1) provide well-resolved and highly diagnostic features. We show that this enables distinction of isomeric saccharides that differ either by their composition of monosaccharide units and/or the orientation of their glycosidic linkages. We demonstrate the utility of this approach from single monosaccharides up to isomeric tetrasaccharides differing only by the configuration of a single glycosidic linkage. Furthermore, through hyphenation with hydrophilic interaction liquid chromatography, we identify oligosaccharide biomarkers in patient body fluid samples, demonstrating a generalized and highly sensitive MS-based method for the identification of saccharides found in complex sample matrices.


Asunto(s)
Errores Innatos del Metabolismo , Oligosacáridos , Humanos , Oligosacáridos/química , Isomerismo , Monosacáridos , Espectrofotometría Infrarroja , Biomarcadores , Iones
10.
J Inherit Metab Dis ; 46(5): 956-971, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37340906

RESUMEN

NANS-CDG is a congenital disorder of glycosylation (CDG) caused by biallelic variants in NANS, encoding an essential enzyme in de novo sialic acid synthesis. It presents with intellectual developmental disorder (IDD), skeletal dysplasia, neurologic impairment, and gastrointestinal dysfunction. Some patients suffer progressive intellectual neurologic deterioration (PIND), emphasizing the need for a therapy. In a previous study, sialic acid supplementation in knockout nansa zebrafish partially rescued skeletal abnormalities. Here, we performed the first in-human pre- and postnatal sialic-acid study in NANS-CDG. In this open-label observational study, 5 patients with NANS-CDG (range 0-28 years) were treated with oral sialic acid for 15 months. The primary outcome was safety. Secondary outcomes were psychomotor/cognitive testing, height and weight, seizure control, bone health, gastrointestinal symptoms, and biochemical and hematological parameters. Sialic acid was well tolerated. In postnatally treated patients, there was no significant improvement. For the prenatally treated patient, psychomotor and neurologic development was better than two other genotypically identical patients (one treated postnatally, one untreated). The effect of sialic acid treatment may depend on the timing, with prenatal treatment potentially benefiting neurodevelopmental outcomes. Evidence is limited, however, and longer-term follow-up in a larger number of prenatally treated patients is required.


Asunto(s)
Trastornos Congénitos de Glicosilación , Ácido N-Acetilneuramínico , Animales , Humanos , Proyectos Piloto , Pez Cebra , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Trastornos Congénitos de Glicosilación/genética , Suplementos Dietéticos
11.
J Inherit Metab Dis ; 46(2): 313-325, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36651519

RESUMEN

Congenital disorders of glycosylation (CDG) are a clinically and biochemically heterogeneous subgroup of inherited metabolic disorders. Most CDG with abnormal N-glycosylation can be detected by transferrin screening, however, MOGS-CDG escapes this routine screening. Combined with the clinical heterogeneity of reported cases, diagnosing MOGS-CDG can be challenging. Here, we clinically characterize ten MOGS-CDG cases including six previously unreported individuals, showing a phenotype characterized by dysmorphic features, global developmental delay, muscular hypotonia, and seizures in all patients and in a minority vision problems and hypogammaglobulinemia. Glycomics confirmed accumulation of a Glc3 Man7 GlcNAc2 glycan in plasma. For quantification of the diagnostic Glcα1-3Glcα1-3Glcα1-2Man tetrasaccharide in urine, we developed and validated a liquid chromatography-mass spectrometry method of 2-aminobenzoic acid (2AA) labeled urinary glycans. As an internal standard, isotopically labeled 13 C6 -2AA Glc3 Man was used, while labeling efficiency was controlled by use of 12 C6 -2AA and 13 C6 -2AA labeled laminaritetraose. Recovery, linearity, intra- and interassay coefficients of variability of these labeled compounds were determined. Furthermore, Glc3 Man was specifically identified by retention time matching against authentic MOGS-CDG urine and compared with Pompe urine. Glc3 Man was increased in all six analyzed cases, ranging from 34.1 to 618.0 µmol/mmol creatinine (reference <5 µmol). In short, MOGS-CDG has a broad manifestation of symptoms but can be diagnosed with the use of a quantitative method for analysis of urinary Glc3 Man excretion.


Asunto(s)
Trastornos Congénitos de Glicosilación , Humanos , Trastornos Congénitos de Glicosilación/genética , Espectrometría de Masas/métodos , Oligosacáridos/metabolismo , Polisacáridos , Convulsiones
12.
J Inherit Metab Dis ; 46(1): 66-75, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36088537

RESUMEN

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic cerebrospinal fluid (CSF) profiles from 12 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose, and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.


Asunto(s)
Glucosa , Xilosa , Humanos , Glucosa/metabolismo , Biomarcadores , Encéfalo/metabolismo
13.
Brain ; 145(1): 208-223, 2022 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-34382076

RESUMEN

Subcellular membrane systems are highly enriched in dolichol, whose role in organelle homeostasis and endosomal-lysosomal pathway remains largely unclear besides being involved in protein glycosylation. DHDDS encodes for the catalytic subunit (DHDDS) of the enzyme cis-prenyltransferase (cis-PTase), involved in dolichol biosynthesis and dolichol-dependent protein glycosylation in the endoplasmic reticulum. An autosomal recessive form of retinitis pigmentosa (retinitis pigmentosa 59) has been associated with a recurrent DHDDS variant. Moreover, two recurring de novo substitutions were detected in a few cases presenting with neurodevelopmental disorder, epilepsy and movement disorder. We evaluated a large cohort of patients (n = 25) with de novo pathogenic variants in DHDDS and provided the first systematic description of the clinical features and long-term outcome of this new neurodevelopmental and neurodegenerative disorder. The functional impact of the identified variants was explored by yeast complementation system and enzymatic assay. Patients presented during infancy or childhood with a variable association of neurodevelopmental disorder, generalized epilepsy, action myoclonus/cortical tremor and ataxia. Later in the disease course, they experienced a slow neurological decline with the emergence of hyperkinetic and/or hypokinetic movement disorder, cognitive deterioration and psychiatric disturbances. Storage of lipidic material and altered lysosomes were detected in myelinated fibres and fibroblasts, suggesting a dysfunction of the lysosomal enzymatic scavenger machinery. Serum glycoprotein hypoglycosylation was not detected and, in contrast to retinitis pigmentosa and other congenital disorders of glycosylation involving dolichol metabolism, the urinary dolichol D18/D19 ratio was normal. Mapping the disease-causing variants into the protein structure revealed that most of them clustered around the active site of the DHDDS subunit. Functional studies using yeast complementation assay and in vitro activity measurements confirmed that these changes affected the catalytic activity of the cis-PTase and showed growth defect in yeast complementation system as compared with the wild-type enzyme and retinitis pigmentosa-associated protein. In conclusion, we characterized a distinctive neurodegenerative disorder due to de novo DHDDS variants, which clinically belongs to the spectrum of genetic progressive encephalopathies with myoclonus. Clinical and biochemical data from this cohort depicted a condition at the intersection of congenital disorders of glycosylation and inherited storage diseases with several features akin to of progressive myoclonus epilepsy such as neuronal ceroid lipofuscinosis and other lysosomal disorders.


Asunto(s)
Transferasas Alquil y Aril , Mioclonía , Enfermedades Neurodegenerativas , Retinitis Pigmentosa , Niño , Dolicoles/metabolismo , Humanos , Enfermedades Neurodegenerativas/genética , Retinitis Pigmentosa/genética
14.
Int J Mol Sci ; 24(10)2023 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-37239976

RESUMEN

Heart failure (HF) is a progressive chronic disease that remains a primary cause of death worldwide, affecting over 64 million patients. HF can be caused by cardiomyopathies and congenital cardiac defects with monogenic etiology. The number of genes and monogenic disorders linked to development of cardiac defects is constantly growing and includes inherited metabolic disorders (IMDs). Several IMDs affecting various metabolic pathways have been reported presenting cardiomyopathies and cardiac defects. Considering the pivotal role of sugar metabolism in cardiac tissue, including energy production, nucleic acid synthesis and glycosylation, it is not surprising that an increasing number of IMDs linked to carbohydrate metabolism are described with cardiac manifestations. In this systematic review, we offer a comprehensive overview of IMDs linked to carbohydrate metabolism presenting that present with cardiomyopathies, arrhythmogenic disorders and/or structural cardiac defects. We identified 58 IMDs presenting with cardiac complications: 3 defects of sugar/sugar-linked transporters (GLUT3, GLUT10, THTR1); 2 disorders of the pentose phosphate pathway (G6PDH, TALDO); 9 diseases of glycogen metabolism (GAA, GBE1, GDE, GYG1, GYS1, LAMP2, RBCK1, PRKAG2, G6PT1); 29 congenital disorders of glycosylation (ALG3, ALG6, ALG9, ALG12, ATP6V1A, ATP6V1E1, B3GALTL, B3GAT3, COG1, COG7, DOLK, DPM3, FKRP, FKTN, GMPPB, MPDU1, NPL, PGM1, PIGA, PIGL, PIGN, PIGO, PIGT, PIGV, PMM2, POMT1, POMT2, SRD5A3, XYLT2); 15 carbohydrate-linked lysosomal storage diseases (CTSA, GBA1, GLA, GLB1, HEXB, IDUA, IDS, SGSH, NAGLU, HGSNAT, GNS, GALNS, ARSB, GUSB, ARSK). With this systematic review we aim to raise awareness about the cardiac presentations in carbohydrate-linked IMDs and draw attention to carbohydrate-linked pathogenic mechanisms that may underlie cardiac complications.


Asunto(s)
Cardiomiopatías , Condroitinsulfatasas , Cardiopatías Congénitas , Enfermedades Metabólicas , Humanos , Cardiomiopatías/genética , Enfermedades Metabólicas/complicaciones , Glicosilación , Carbohidratos , Azúcares , Pentosiltransferasa , Manosiltransferasas , Acetiltransferasas
15.
Int J Mol Sci ; 24(3)2023 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-36768261

RESUMEN

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood-CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes.


Asunto(s)
Encéfalo , Transferrina , Humanos , Glicosilación , Transferrina/metabolismo , Encéfalo/metabolismo , Polisacáridos/metabolismo
16.
Int J Mol Sci ; 24(9)2023 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-37175577

RESUMEN

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.


Asunto(s)
Glicopéptidos , Motor de Búsqueda , Secuencia de Aminoácidos , Glicopéptidos/química , Glicosilación , Programas Informáticos , Polisacáridos/química
17.
Int J Mol Sci ; 24(9)2023 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-37175952

RESUMEN

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.


Asunto(s)
Hipoglucemia , Fosfoglucomutasa , Animales , Ratones , Galactosa/farmacología , Glucosa , Homeostasis , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Nucleótidos , Fosfatos , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo
18.
Glycobiology ; 32(3): 239-250, 2022 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-34939087

RESUMEN

Synthetic sugar analogs are widely applied in metabolic oligosaccharide engineering (MOE) and as novel drugs to interfere with glycoconjugate biosynthesis. However, mechanistic insights on their exact cellular metabolism over time are mostly lacking. We combined ion-pair ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry mass spectrometry using tributyl- and triethylamine buffers for sensitive analysis of sugar metabolites in cells and organisms and identified low abundant nucleotide sugars, such as UDP-arabinose in human cell lines and CMP-sialic acid (CMP-NeuNAc) in Drosophila. Furthermore, MOE revealed that propargyloxycarbonyl (Poc)-labeled ManNPoc was metabolized to both CMP-NeuNPoc and UDP-GlcNPoc. Finally, time-course analysis of the effect of antitumor compound 3Fax-NeuNAc by incubation of B16-F10 melanoma cells with N-acetyl-D-[UL-13C6]glucosamine revealed full depletion of endogenous ManNAc 6-phosphate and CMP-NeuNAc within 24 h. Thus, dynamic tracing of sugar metabolic pathways provides a general approach to reveal time-dependent insights into the metabolism of synthetic sugars, which is important for the rational design of analogs with optimized effects.


Asunto(s)
Metabolismo de los Hidratos de Carbono , Ácido N-Acetilneuramínico Citidina Monofosfato , Cromatografía Liquida , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Glucosamina/metabolismo , Azúcares
19.
Mol Vis ; 28: 536-543, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-37089696

RESUMEN

Purpose: A protein quantitative trait locus (pQTL) analysis recently revealed a strong association between hemopexin (HPX) levels and genetic variants at the complement factor H (CFH) locus. In this study, we aimed to determine HPX plasma levels in patients with age-related macular degeneration (AMD) and to compare them with those in controls. We also investigated whether genetic variants at the CFH locus are associated with HPX plasma levels. Methods: HPX levels were quantified in 200 advanced AMD cases and 200 controls using an enzyme-linked immunosorbent assay and compared between the two groups. Furthermore, HPX levels were analyzed per genotype group of three HPX-associated variants (rs61818956, rs10494745, and rs10801582) and four AMD-associated variants (rs794362 [proxy for rs187328863], rs570618, rs10922109, and rs61818924 [proxy for rs61818925]) at the CFH locus. Results: HPX levels were similar in the control group compared with the AMD group. The three variants at the CFH locus, which were previously associated with the HPX levels, showed no association with the HPX levels in our data set. No significant differences in HPX levels were detected between the different genotype groups of AMD-associated variants at the CFH locus. Conclusions: In this study, HPX levels were not associated with AMD or AMD-associated variants at the CFH locus. The finding of a previous pQTL study that variants at the CFH locus were associated with HPX levels was also not confirmed in this study.


Asunto(s)
Hemopexina , Degeneración Macular , Humanos , Hemopexina/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Genotipo , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Factores de Transcripción/genética , Polimorfismo de Nucleótido Simple/genética
20.
J Inherit Metab Dis ; 45(4): 748-758, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35527402

RESUMEN

Messenger RNA (mRNA) has emerged as a novel therapeutic approach for inborn errors of metabolism. Classic galactosemia (CG) is an inborn error of galactose metabolism caused by a severe deficiency of galactose-1-phosphate:uridylyltransferase (GALT) activity leading to neonatal illness and chronic impairments affecting the brain and female gonads. In this proof of concept study, we used our zebrafish model for CG to evaluate the potential of human GALT mRNA (hGALT mRNA) packaged in two different lipid nanoparticles to restore GALT expression and activity at early stages of development. Both one cell-stage and intravenous single-dose injections resulted in hGALT protein expression and enzyme activity in the CG zebrafish (galt knockout) at 5 days post fertilization (dpf). Moreover, the levels of galactose-1-phosphate (Gal-1-P) and galactonate, metabolites that accumulate because of the deficiency, showed a decreasing trend. LNP-packaged mRNA was effectively translated and processed in the CG zebrafish without signs of toxicity. This study shows that mRNA therapy restores GALT protein and enzyme activity in the CG zebrafish model, and that the zebrafish is a suitable system to test this approach. Further studies are warranted to assess whether repeated injections safely mitigate the chronic impairments of this disease.


Asunto(s)
Galactosemias , Animales , Femenino , Galactosa/metabolismo , Galactosemias/diagnóstico , Galactosemias/genética , Galactosemias/terapia , Humanos , Recién Nacido , Liposomas , Nanopartículas , Nucleotidiltransferasas , ARN Mensajero/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo
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