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1.
Am J Hum Genet ; 108(11): 2130-2144, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34653363

RESUMO

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.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Genes Dominantes , Hexosiltransferases/genética , Proteínas de Membrana/genética , Doenças Musculoesqueléticas/genética , Doenças do Sistema Nervoso/genética , Adolescente , Adulto , Sequência de Aminoácidos , Domínio Catalítico , Pré-Escolar , Feminino , Heterozigoto , Hexosiltransferases/química , Humanos , Masculino , Proteínas de Membrana/química , Pessoa de Meia-Idade , Linhagem , Homologia de Sequência de Aminoácidos
2.
Int J Mol Sci ; 24(9)2023 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-37175952

RESUMO

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.


Assuntos
Hipoglicemia , Fosfoglucomutase , Animais , Camundongos , Galactose/farmacologia , Glucose , Homeostase , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Nucleotídeos , Fosfatos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo
3.
Glycobiology ; 32(3): 239-250, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-34939087

RESUMO

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.


Assuntos
Metabolismo dos Carboidratos , Ácido N-Acetilneuramínico do Monofosfato de Citidina , Cromatografia Líquida , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Glucosamina/metabolismo , Açúcares
4.
J Inherit Metab Dis ; 45(4): 769-781, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35279850

RESUMO

Congenital disorders of glycosylation type 1 (CDG-I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG-I is a partial absence of complete N-glycans on transferrin. However, recent findings of a diagnostic N-tetrasaccharide for ALG1-CDG and increased high-mannose N-glycans for a few other CDG suggested the potential of glycan structural analysis for CDG-I gene discovery. We analyzed the relative abundance of total plasma N-glycans by high resolution quadrupole time-of-flight mass spectrometry in a large cohort of 111 CDG-I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single-molecule molecular inversion probes (smMIPs) for sequencing of CDG-I candidate genes on the basis of specific N-glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N-tetrasaccharide in ALG2-CDG patients and a novel fucosylated N-pentasaccharide as specific glycomarker for ALG1-CDG. Moreover, group-specific high-mannose N-glycan signatures were found in ALG3-, ALG9-, ALG11-, ALG12-, RFT1-, SRD5A3-, DOLK-, DPM1-, DPM3-, MPDU1-, ALG13-CDG, and hereditary fructose intolerance. Further differential analysis revealed high-mannose profiles, characteristic for ALG12- and ALG9-CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG-I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG-I patient cohorts.


Assuntos
Defeitos Congênitos da Glicosilação , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Glicômica , Glicosilação , Humanos , Manose , Manosiltransferases/genética , N-Acetilglucosaminiltransferases , Oligossacarídeos , Polissacarídeos/genética
5.
Hepatology ; 72(6): 1968-1986, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32145091

RESUMO

BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.


Assuntos
Autofagia/genética , Defeitos Congênitos da Glicosilação/genética , Hepatopatias/genética , ATPases Vacuolares Próton-Translocadoras/genética , Adulto , Biópsia , Células Cultivadas , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/patologia , Análise Mutacional de DNA , Fibroblastos , Humanos , Fígado/citologia , Fígado/patologia , Hepatopatias/sangue , Hepatopatias/diagnóstico , Hepatopatias/patologia , Masculino , Mutação de Sentido Incorreto , Linhagem , Cultura Primária de Células
6.
Mol Cell ; 50(2): 295-302, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23562325

RESUMO

Among the different types of protein glycosylation, C-mannosylation of tryptophan residues stands out because of the unique linkage formed between sugar and protein. Instead of the typical O- or N-glycosidic linkage, a C-C bond is used for attachment of a single mannose. C-mannose is characteristically found in thrombospondin type 1 repeats and in the WSXWS motif of type I cytokine receptors. The genetic base of the enzymatic activity catalyzing C-mannosylation was not known. Here we demonstrate that Caenorhabditis elegans DPY-19 is a C-mannosyltransferase. DPY-19 exhibits topological and sequential homology to the N-glycan oligosaccharyltransferase, highlighting an evolutionary link between N- and C-glycosylation. We show that the C. elegans surface receptors MIG-21 and UNC-5 are acceptor substrates of DPY-19 and that C-mannosylation is essential for the secretion of soluble MIG-21. Thereby, our data provide an explanation for the previously described identical Q neuroblast migration phenotypes of dpy-19 and mig-21 mutants.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Manosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Configuração de Carboidratos , Sequência de Carboidratos , Glicosilação , Manose/metabolismo , Manosiltransferases/química , Proteínas de Membrana/química , Anotação de Sequência Molecular , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos , Trombospondinas/química
7.
Hum Mol Genet ; 27(17): 3029-3045, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878199

RESUMO

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.


Assuntos
Doenças do Desenvolvimento Ósseo/etiologia , Calcificação Fisiológica , Defeitos Congênitos da Glicosilação/complicações , Genômica , Glicômica , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Simportadores/genética , Adulto , Animais , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Células Cultivadas , Estudos de Coortes , Exoma , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Glicosilação , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Lactente , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Linhagem , Fenótipo , Transporte Proteico , Simportadores/metabolismo , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
8.
Am J Hum Genet ; 98(2): 322-30, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26833330

RESUMO

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.


Assuntos
Fosfatase Alcalina/metabolismo , Colesterol/metabolismo , Complexo de Golgi/genética , Homeostase , Proteínas de Membrana/deficiência , Transaminases/metabolismo , Adulto , Sequência de Aminoácidos , Ceruloplasmina/metabolismo , Retículo Endoplasmático/metabolismo , Exoma , Fibroblastos/metabolismo , Genótipo , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação , Fenótipo , Adulto Jovem
9.
Am J Hum Genet ; 98(2): 310-21, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26833332

RESUMO

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.


Assuntos
Complexo de Golgi/genética , Homeostase , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Exoma , Feminino , Fibroblastos/citologia , Glicosilação , Complexo de Golgi/metabolismo , Células HeLa , Heterozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
10.
Clin Chem ; 65(10): 1295-1306, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31375477

RESUMO

BACKGROUND: Many muscular dystrophies currently remain untreatable. Recently, dietary ribitol has been suggested as a treatment for cytidine diphosphate (CDP)-l-ribitol pyrophosphorylase A (CRPPA, ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) myopathy, by raising CDP-ribitol concentrations. Thus, to facilitate fast diagnosis, treatment development, and treatment monitoring, sensitive detection of CDP-ribitol is required. METHODS: An LC-MS method was optimized for CDP-ribitol in human and mice cells and tissues. RESULTS: CDP-ribitol, the product of CRPPA, was detected in all major human and mouse tissues. Moreover, CDP-ribitol concentrations were reduced in fibroblasts and skeletal muscle biopsies from patients with CRPPA myopathy, showing that CDP-ribitol could serve as a diagnostic marker to identify patients with CRPPA with severe Walker-Warburg syndrome and mild limb-girdle muscular dystrophy (LGMD) phenotypes. A screen for potentially therapeutic monosaccharides revealed that ribose, in addition to ribitol, restored CDP-ribitol concentrations and the associated O-glycosylation defect of α-dystroglycan. As the effect occurred in a mutation-dependent manner, we established a CDP-ribitol blood test to facilitate diagnosis and predict individualized treatment response. Ex vivo incubation of blood cells with ribose or ribitol restored CDP-ribitol concentrations in a patient with CRPPA LGMD. CONCLUSIONS: Sensitive detection of CDP-ribitol with LC-MS allows fast diagnosis of patients with severe and mild CRPPA myopathy. Ribose offers a readily testable dietary therapy for CRPPA myopathy, with possible applicability for patients with FKRP and FKTN myopathy. Evaluation of CDP-ribitol in blood is a promising tool for the evaluation and monitoring of dietary therapies for CRPPA myopathy in a patient-specific manner.


Assuntos
Monitoramento de Medicamentos/métodos , Distrofias Musculares/sangue , Distrofias Musculares/tratamento farmacológico , Açúcares de Nucleosídeo Difosfato/sangue , Animais , Cromatografia Líquida , Suplementos Nutricionais , Distroglicanas , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Distrofias Musculares/patologia , Mutação , Açúcares de Nucleosídeo Difosfato/análise , Nucleotidiltransferases/genética , Ribitol/farmacologia , Ribose/farmacologia
11.
J Inherit Metab Dis ; 42(3): 553-564, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30746764

RESUMO

SLC35A2-CDG is caused by mutations in the X-linked SLC35A2 gene encoding the UDP-galactose transporter. SLC35A2 mutations lead to hypogalactosylation of N-glycans. SLC35A2-CDG is characterized by severe neurological symptoms and, in many patients, early-onset epileptic encephalopathy. In view of the diagnostic challenges, we studied the clinical, neuroradiological, and biochemical features of 15 patients (11 females and 4 males) with SLC35A2-CDG from various centers. We describe nine novel pathogenic variations in SLC35A2. All affected individuals presented with a global developmental delay, and hypotonia, while 70% were nonambulatory. Epilepsy was present in 80% of the patients, and in EEG hypsarrhythmia and findings consistent with epileptic encephalopathy were frequently seen. The most common brain MRI abnormality was cerebral atrophy with delayed myelination and multifocal inhomogeneous abnormal patchy white matter hyperintensities, which seemed to be nonprogressive. Thin corpus callosum was also common, and all the patients had a corpus callosum shorter than normal for their age. Variable dysmorphic features and growth deficiency were noted. Biochemically, normal mucin type O-glycosylation and lipid glycosylation were found, while transferrin mass spectrometry was found to be more specific in the identification of SLC35A2-CDG, as compared to routine screening tests. Although normal glycosylation studies together with clinical variability and genetic results complicate the diagnosis of SLC35A2-CDG, our data indicate that the combination of these three elements can support the pathogenicity of mutations in SLC35A2.


Assuntos
Encefalopatias/patologia , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/patologia , Proteínas de Transporte de Monossacarídeos/genética , Espasmos Infantis/patologia , Adolescente , Atrofia , Criança , Pré-Escolar , Feminino , Glicosilação , Humanos , Lactente , Internacionalidade , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Mutação , Adulto Jovem
12.
J Allergy Clin Immunol ; 142(2): 517-529, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29248493

RESUMO

BACKGROUND: Anti-TNF inhibitors successfully improve the quality of life of patients with inflammatory disease. Unfortunately, not all patients respond to anti-TNF therapy, and some patients show paradoxical immune side effects, which are poorly understood. Surprisingly, anti-TNF agents were shown to promote IL-17A production with as yet unknown clinical implications. OBJECTIVE: We sought to investigate the molecular mechanism underlying anti-TNF-driven IL-17A expression and the clinical implications of this phenomenon. METHODS: Fluorescence-activated cell sorting, RNA sequencing, quantitative real-time PCR, Western blotting, small interfering RNA interference, and kinase inhibitors were used to study the molecular mechanisms in isolated human CD4+ T cells from healthy donors. The clinical implication was studied in blood samples of patients with inflammatory bowel disease (IBD) receiving anti-TNF therapy. RESULTS: Here we show that anti-TNF treatment results in inhibition of the anti-inflammatory molecule TNF-α-induced protein 3 (TNFAIP3)/A20 in memory CD4+ T cells. We found an inverse relationship between TNFAIP3/A20 expression levels and IL-17A production. Inhibition of TNFAIP3/A20 promotes kinase activity of p38 mitogen-activated protein kinase and protein kinase C, which drives IL-17A expression. Regulation of TNFAIP3/A20 expression and cognate IL-17A production in T cells are specifically mediated through TNF receptor 2 signaling. Ex vivo, in patients with IBD treated with anti-TNF, we found further evidence for an inverse relationship between TNFAIP3/A20 expression levels and IL-17A-producing T cells. CONCLUSION: Anti-TNF treatment interferes in the TNFAIP3/A20-mediated anti-inflammatory feedback loop in CD4+ T cells and promotes kinase activity. This puts TNFAIP3/A20, combined with IL-17A expression, on the map as a potential tool for predicting therapy responsiveness or side effects of anti-TNF therapy. Moreover, it provides novel targets related to TNFAIP3/A20 activity for superior therapeutic regimens in patients with IBD.


Assuntos
Biomarcadores Farmacológicos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-17/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Interleucina-17/genética , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Proc Natl Acad Sci U S A ; 112(1): 291-6, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25535363

RESUMO

The formation of glycoconjugates depends on nucleotide sugars, which serve as donor substrates for glycosyltransferases in the lumen of Golgi vesicles and the endoplasmic reticulum (ER). Import of nucleotide sugars from the cytosol is an important prerequisite for these reactions and is mediated by nucleotide sugar transporters. Here, we report the identification of REPRESSOR OF CYTOKININ DEFICIENCY 1 (ROCK1, At5g65000) as an ER-localized facilitator of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) transport in Arabidopsis thaliana. Mutant alleles of ROCK1 suppress phenotypes inferred by a reduced concentration of the plant hormone cytokinin. This suppression is caused by the loss of activity of cytokinin-degrading enzymes, cytokinin oxidases/dehydrogenases (CKXs). Cytokinin plays an essential role in regulating shoot apical meristem (SAM) activity and shoot architecture. We show that rock1 enhances SAM activity and organ formation rate, demonstrating an important role of ROCK1 in regulating the cytokinin signal in the meristematic cells through modulating activity of CKX proteins. Intriguingly, genetic and molecular analysis indicated that N-glycosylation of CKX1 was not affected by the lack of ROCK1-mediated supply of UDP-GlcNAc. In contrast, we show that CKX1 stability is regulated in a proteasome-dependent manner and that ROCK1 regulates the CKX1 level. The increased unfolded protein response in rock1 plants and suppression of phenotypes caused by the defective brassinosteroid receptor bri1-9 strongly suggest that the ROCK1 activity is an important part of the ER quality control system, which determines the fate of aberrant proteins in the secretory pathway.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Arabidopsis/ultraestrutura , Transporte Biológico , Meristema/metabolismo , Meristema/ultraestrutura , Fenótipo
14.
Glycobiology ; 27(1): 87-98, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27496760

RESUMO

Cryptococcus neoformans, an opportunistic fungal pathogen, produces a glycan capsule to evade the immune system during infection. This definitive virulence factor is composed mainly of complex polysaccharides, which are made in the secretory pathway by reactions that utilize activated nucleotide sugar precursors. Although the pathways that synthesize these precursors are known, the identity and the regulation of the nucleotide sugar transporters (NSTs) responsible for importing them into luminal organelles remain elusive. The UDP-galactose transporter, Ugt1, was initially identified by homology to known UGTs and glycan composition analysis of ugt1Δ mutants. However, sequence is an unreliable predictor of NST substrate specificity, cells may express multiple NSTs with overlapping specificities, and NSTs may transport multiple substrates. Determining NST activity thus requires biochemical demonstration of function. We showed that Ugt1 transports both UDP-galactose and UDP-N-acetylgalactosamine in vitro. Deletion of UGT1 resulted in growth and mating defects along with altered capsule and cellular morphology. The mutant was also phagocytosed more readily by macrophages than wild-type cells and cleared more quickly in vivo and in vitro, suggesting a mechanism for the lack of virulence observed in mouse models of infection.


Assuntos
Criptococose/genética , Cryptococcus neoformans/imunologia , Proteínas de Transporte de Monossacarídeos/imunologia , Uridina Difosfato Galactose/imunologia , Sequência de Aminoácidos/genética , Animais , Transporte Biológico/genética , Criptococose/enzimologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Galactose/química , Galactose/genética , Humanos , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Polissacarídeos/genética , Polissacarídeos/imunologia , Especificidade por Substrato , Uridina Difosfato Galactose/genética
15.
Hum Mol Genet ; 24(8): 2241-6, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25552652

RESUMO

Binding of cellular α-dystroglycan (α-DG) to its extracellular matrix ligands is fully dependent on a unique O-mannose-linked glycan. Disrupted O-mannosylation is the hallmark of the muscular dystrophy-dystroglycanopathy (MDDG) syndromes. SLC35A1, encoding the transporter of cytidine 5'-monophosphate-sialic acid, was recently identified as MDDG candidate gene. This is surprising, since sialic acid itself is dispensable for α-DG-ligand binding. In a novel SLC35A1-deficient cell model, we demonstrated a lack of α-DG O-mannosylation, ligand binding and incorporation of sialic acids. Removal of sialic acids from HAP1 wild-type cells after incorporation or preventing sialylation during synthesis did not affect α-DG O-mannosylation or ligand binding but did affect sialylation. Lentiviral-mediated complementation with the only known disease mutation p.Q101H failed to restore deficient O-mannosylation in SLC35A1 knockout cells and partly restored sialylation. These data indicate a role for SLC35A1 in α-DG O-mannosylation that is distinct from sialic acid metabolism. In addition, human SLC35A1 deficiency can be considered as a combined disorder of α-DG O-mannosylation and sialylation, a novel variant of the MDDG syndromes.


Assuntos
Distroglicanas/metabolismo , Manose/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Síndrome de Walker-Warburg/genética , Síndrome de Walker-Warburg/metabolismo , Linhagem Celular , Monofosfato de Citidina/metabolismo , Humanos , Mutação , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo
16.
J Med Genet ; 50(11): 733-9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24031089

RESUMO

BACKGROUND: The heritability of autism spectrum disorder is currently estimated at 55%. Identification of the molecular basis of patients with syndromic autism extends our understanding of the pathogenesis of autism in general. The objective of this study was to find the gene mutated in eight patients from a large kindred, who suffered from autism spectrum disorder, arthrogryposis and epilepsy. METHODS AND RESULTS: By linkage analysis and exome sequencing, we identified deleterious mutations in SLC35A3 in these patients. SLC35A3 encodes the major Golgi uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter. In Golgi vesicles isolated from patient fibroblasts the transport of the respective nucleotide sugar was significantly reduced causing a massive decrease in the content of cell surface expressed highly branched N-glycans and a concomitant sharp increase of lower branched glycoforms. CONCLUSIONS: Spontaneous mutation in SLC35A3 has been discovered in cattle worldwide, recapitulating the human phenotype with arthrogryposis and additional skeletal defects known as Complex Vertebral Malformation syndrome. The skeletal anomalies in the mutant cattle and in our patients, and perhaps even the neurological symptoms are likely the consequence of the lack of high-branched N-glycans and the concomitant abundance of lower-branched glycoforms at the cell surface. This pattern has previously been associated with growth arrest and induction of differentiation. With this study, we add SLC35A3 to the gene list of autism spectrum disorders, and underscore the crucial importance of UDP-GlcNAc in the regulation of the N-glycan branching pathway in the Golgi apparatus.


Assuntos
Artrogripose/genética , Transtornos Globais do Desenvolvimento Infantil/genética , Epilepsia/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Animais , Artrogripose/metabolismo , Bovinos , Células Cultivadas , Transtornos Globais do Desenvolvimento Infantil/metabolismo , Epilepsia/metabolismo , Feminino , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Linhagem
17.
J Biol Chem ; 287(4): 2739-48, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22117070

RESUMO

The extracellular domain of Notch contains epidermal growth factor (EGF) repeats that are extensively modified with different O-linked glycans. O-Fucosylation is essential for receptor function, and elongation with N-acetylglucosamine, catalyzed by members of the Fringe family, modulates Notch activity. Only recently, genes encoding enzymes involved in the O-glucosylation pathway have been cloned. In the Drosophila mutant rumi, characterized by a mutation in the protein O-glucosyltransferase, Notch signaling is impaired in a temperature-dependent manner, and a mouse knock-out leads to embryonic lethality. We have previously identified two human genes, GXYLT1 and GXYLT2, encoding glucoside xylosyltransferases responsible for the transfer of xylose to O-linked glucose. The identity of the enzyme further elongating the glycan to generate the final trisaccharide xylose-xylose-glucose, however, remained unknown. Here, we describe that the human gene C3ORF21 encodes a UDP-xylose:α-xyloside α1,3-xylosyltransferase, acting on xylose-α1,3-glucoseß1-containing acceptor structures. We have, therefore, renamed it XXYLT1 (xyloside xylosyltransferase 1). XXYLT1 cannot act on a synthetic acceptor containing an α-linked xylose alone, but requires the presence of the underlying glucose. Activity on Notch EGF repeats was proven by in vitro xylosylation of a mouse Notch1 fragment recombinantly produced in Sf9 insect cells, a bacterially expressed EGF repeat from mouse Notch2 modified in vitro by Rumi and Gxylt2 and in vivo by co-expression of the enzyme with the Notch1 fragment. The enzyme was shown to be a typical type II membrane-bound glycosyltransferase localized in the endoplasmic reticulum.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Pentosiltransferases/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosilação , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Pentosiltransferases/genética , Estrutura Secundária de Proteína , Receptor Notch1/genética , Receptor Notch2/genética , UDP Xilose-Proteína Xilosiltransferase
18.
Glycobiology ; 23(3): 303-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23135544

RESUMO

LARGE (like-glycosyltransferase) and LARGE2 (glycosyltransferase-like 1B (GYLTL1B)) are homologous Golgi glycosyltransferases possessing two catalytic domains with homology to members of glycosyltransferase families GT8 and GT49. Mutations in human and mouse Large result in muscular dystrophy due to underglycosylation of dystroglycan. The systemic function of LARGE2 is unknown, but at a cellular level the enzyme can substitute for LARGE in glycosylating dystroglycan. Here, we show that LARGE2 catalyzes the same glycosylation reaction as LARGE. It is a bifunctional glycosyltransferase using uridine diphosphate (UDP)-xylose (Xyl) and UDP-glucuronic acid (GlcA) as donor sugars to produce a xyloglucuronan with alternating Xyl and GlcA residues.


Assuntos
Glicosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato Xilose/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Glicosilação , Camundongos
19.
Chembiochem ; 14(15): 1936-42, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-24014346

RESUMO

CMP-sialic acid transporter: We report an in-depth, multidisciplinary, structural study that has identified the amino acid residues intimately involved in CMP-sialic acid transporter (CST) substrate specificity. Our data provide a significant contribution towards a better understanding the structure-function relationship of this important family of transporters and the rational design of CST inhibitors.


Assuntos
Monofosfato de Citidina/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Simportadores/química , Simportadores/genética
20.
N Engl J Med ; 360(1): 32-43, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19118303

RESUMO

BACKGROUND: The main features of severe congenital neutropenia are the onset of severe bacterial infections early in life, a paucity of mature neutrophils, and an increased risk of leukemia. In many patients, the genetic causes of severe congenital neutropenia are unknown. METHODS: We performed genomewide genotyping and linkage analysis on two consanguineous pedigrees with a total of five children affected with severe congenital neutropenia. Candidate genes from the linkage interval were sequenced. Functional assays and reconstitution experiments were carried out. RESULTS: All index patients were susceptible to bacterial infections and had very few mature neutrophils in the bone marrow; structural heart defects, urogenital abnormalities, and venous angiectasia on the trunk and extremities were additional features. Linkage analysis of the two index families yielded a combined multipoint lod score of 5.74 on a linkage interval on chromosome 17q21. Sequencing of G6PC3, the candidate gene encoding glucose-6-phosphatase, catalytic subunit 3, revealed a homozygous missense mutation in exon 6 that abolished the enzymatic activity of glucose-6-phosphatase in all affected children in the two families. The patients' neutrophils and fibroblasts had increased susceptibility to apoptosis. The myeloid cells showed evidence of increased endoplasmic reticulum stress and increased activity of glycogen synthase kinase 3beta (GSK-3beta). We identified seven additional, unrelated patients who had severe congenital neutropenia with syndromic features and distinct biallelic mutations in G6PC3. CONCLUSIONS: Defective function of glucose-6-phosphatase, catalytic subunit 3, underlies a severe congenital neutropenia syndrome associated with cardiac and urogenital malformations.


Assuntos
Anormalidades Múltiplas/genética , Glucose-6-Fosfatase/genética , Cardiopatias Congênitas/genética , Mutação de Sentido Incorreto , Neutropenia/genética , Anormalidades Urogenitais/genética , Adolescente , Apoptose/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudo de Associação Genômica Ampla , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/genética , Humanos , Lactente , Escore Lod , Masculino , Neutropenia/congênito , Neutropenia/fisiopatologia , Neutrófilos/fisiologia , Linhagem , Síndrome , Telangiectasia/genética
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