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1.
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
2.
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
3.
Am J Hum Genet ; 93(1): 29-41, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-23768512

RESUMO

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.


Assuntos
Distroglicanas/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação de Sentido Incorreto , Nucleotidiltransferases/metabolismo , Animais , Pré-Escolar , Análise Mutacional de DNA/métodos , Distroglicanas/genética , Anormalidades do Olho/patologia , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Estudos de Associação Genética/métodos , Glicosilação , Guanosina Difosfato Manose/metabolismo , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Nucleotidiltransferases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Hum Mol Genet ; 22(9): 1746-54, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23359570

RESUMO

Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker-Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the ß-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.


Assuntos
Mutação de Sentido Incorreto , N-Acetilglucosaminiltransferases/genética , Síndrome de Walker-Warburg/genética , Animais , Linhagem Celular Tumoral , Mapeamento Cromossômico , Estudos de Coortes , Distroglicanas/metabolismo , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicosilação , Homozigoto , Humanos , Lactente , Laminina/metabolismo , Masculino , Distrofia Muscular do Cíngulo dos Membros/genética , N-Acetilglucosaminiltransferases/metabolismo , Linhagem , Fenótipo , Ligação Proteica , Síndrome de Walker-Warburg/patologia , Peixe-Zebra/genética
5.
PLoS Genet ; 7(12): e1002427, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22242004

RESUMO

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5-13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations.


Assuntos
Cardiomiopatia Dilatada/genética , Distroglicanas/metabolismo , Genes Recessivos , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adolescente , Cardiomiopatia Dilatada/metabolismo , Criança , Pré-Escolar , Fosfatos de Dolicol/metabolismo , Feminino , Expressão Gênica , Glicosilação , Haplótipos , Homozigoto , Humanos , Masculino , Linhagem , Saccharomyces cerevisiae/genética , Sarcolema/metabolismo
6.
Ann Neurol ; 72(4): 550-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23109149

RESUMO

OBJECTIVE: Congenital disorders of glycosylation (CDG) are a group of metabolic diseases due to defects in protein and lipid glycosylation. We searched for the primary defect in 3 children from 2 families with a severe neurological phenotype, including profound developmental delay, intractable epilepsy, progressive microcephaly, severe hypotonia with elevated blood creatine kinase levels, and early fatal outcome. There was clinical evidence of a muscular dystrophy-dystroglycanopathy syndrome, supported by deficient O-mannosylation by muscle immunohistochemistry. METHODS: Biochemical and molecular methods were combined to pinpoint the defect in the glycosylation pathway in the endoplasmic reticulum. RESULTS: Metabolic investigations revealed CDG-I, pointing to a defect in protein N-glycosylation in the endoplasmic reticulum. Analysis of lipid-linked oligosaccharides in fibroblasts showed accumulation of Dol-PP-GlcNAc(2) -Man(5) . DNA analysis revealed mutations in DPM2, 1 of the subunits of the dolichol-phosphate-mannose (DPM) synthase; the patient in the first family is compound heterozygous for 2 mutations (c.68A>G, predicting a missense mutation p.Y23C and c.4-1G>C, a splice mutation), whereas the patients in the second family are homozygous for the same missense mutation (c.68A>G, p.Y23C). INTERPRETATION: We describe a new CDG, due to a deficiency of DPM2. Hence, mutations have now been described in the genes for the 3 subunits of DPM: DPM1, DPM2, and DPM3, whereby DPM2-CDG links the congenital disorders of glycosylation to the congenital muscular dystrophies.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Epilepsia/genética , Manosiltransferases/genética , Distrofias Musculares/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Transtornos de Proteínas de Coagulação/genética , Defeitos Congênitos da Glicosilação/complicações , Análise Mutacional de DNA , Resistência a Medicamentos , Distroglicanas/metabolismo , Eletromiografia , Retículo Endoplasmático , Epilepsia/etiologia , Feminino , Fibroblastos/metabolismo , Glicosilação , Humanos , Lactente , Focalização Isoelétrica , Hepatopatias/complicações , Hepatopatias/genética , Masculino , Manose/metabolismo , Microcefalia/genética , Microcefalia/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Distrofias Musculares/complicações , Mutação/genética , Mutação/fisiologia , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Gravidez , Transtornos da Visão/genética , Transtornos da Visão/patologia , Adulto Jovem
7.
Eur J Hum Genet ; 25(11): 1195-1201, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28853722

RESUMO

Dilated cardiomyopathy (DCM) is extremely heterogeneous with a large proportion due to dominantly inherited disease-causing variants in sarcomeric genes. Recessive metabolic diseases may cause DCM, usually with onset in childhood, and in the context of systemic disease. Whether metabolic defects can also cause adult-onset DCM is currently unknown. Therefore, we performed an extensive metabolic screening in 36 consecutive adult-onset DCM patients. Diagnoses were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Measurement of propionyl-CoA carboxylase (PCC) activity was done in fibroblasts. Whole exome sequencing (WES) data of 157 additional DCM patients were analyzed for genetic defects. We found a metabolic profile characteristic for propionic acidemia in a patient with severe DCM from 55 years of age. Genetic analysis demonstrated compound heterozygous variants in PCCA. Enzymatic activity of PCC in fibroblasts was markedly reduced. A targeted analysis of the PCCA and PCCB genes using available WES data from 157 further DCM patients subsequently identified another patient with propionic acidemia. This patient had compound heterozygous variants in PCCB, and developed severe DCM from 42 years of age. Adult-onset DCM can be caused by propionic acidemia, an autosomal recessive inheritable metabolic disorder usually presenting as neonatal or childhood disease. Current guidelines advise a low-protein diet to ameliorate or prevent detrimental aspects of the disease. Long-term follow-up of a larger group of patients may show whether this diet would also ameliorate DCM. Our results suggest that diagnostic metabolic screening to identify propionic acidemia and related disorders in DCM patients is justified.


Assuntos
Cardiomiopatia Dilatada/genética , Metilmalonil-CoA Descarboxilase/genética , Acidemia Propiônica/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/urina , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Heterozigoto , Humanos , Masculino , Metilmalonil-CoA Descarboxilase/metabolismo , Pessoa de Meia-Idade , Mutação , Linhagem , Acidemia Propiônica/genética
8.
Genome Med ; 9(1): 118, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273094

RESUMO

BACKGROUND: The phenotypic severity of congenital muscular dystrophy-dystroglycanopathy (MDDG) syndromes associated with aberrant glycosylation of α-dystroglycan ranges from the severe Walker-Warburg syndrome or muscle-eye-brain disease to mild, late-onset, isolated limb-girdle muscular dystrophy without neural involvement. However, muscular dystrophy is invariably found across the spectrum of MDDG patients. METHODS: Using linkage mapping and whole-exome sequencing in two families with an unexplained neurodevelopmental disorder, we have identified homozygous and compound heterozygous mutations in B3GALNT2. RESULTS: The first family comprises two brothers of Dutch non-consanguineous parents presenting with mild ID and behavioral problems. Immunohistochemical analysis of muscle biopsy revealed no significant aberrations, in line with the absence of a muscular phenotype in the affected siblings. The second family includes five affected individuals from an Iranian consanguineous kindred with mild-to-moderate intellectual disability (ID) and epilepsy without any notable neuroimaging, muscle, or eye abnormalities. Complementation assays of the compound heterozygous mutations identified in the two brothers had a comparable effect on the O-glycosylation of α-dystroglycan as previously reported mutations that are associated with severe muscular phenotypes. CONCLUSIONS: In conclusion, we show that mutations in B3GALNT2 can give rise to a novel MDDG syndrome presentation, characterized by ID associated variably with seizure, but without any apparent muscular involvement. Importantly, B3GALNT2 activity does not fully correlate with the severity of the phenotype as assessed by the complementation assay.


Assuntos
Deficiência Intelectual/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Fenótipo , Síndrome de Walker-Warburg/genética , Adolescente , Adulto , Linhagem Celular , Criança , Feminino , Genes Recessivos , Genótipo , Humanos , Deficiência Intelectual/patologia , Masculino , N-Acetilgalactosaminiltransferases/metabolismo , Linhagem , Síndrome de Walker-Warburg/patologia
9.
Nat Commun ; 7: 11491, 2016 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-27173435

RESUMO

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.


Assuntos
Cílios/metabolismo , Ciliopatias/genética , Nanismo/genética , Hipotonia Muscular/genética , Mapas de Interação de Proteínas , Proteínas/metabolismo , Coluna Vertebral/anormalidades , Transporte Biológico/fisiologia , Cromatografia de Afinidade/métodos , Ciliopatias/patologia , Ciliopatias/terapia , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Nanismo/patologia , Nanismo/terapia , Fibroblastos , Células HEK293 , Humanos , Espectrometria de Massas , Terapia de Alvo Molecular/métodos , Hipotonia Muscular/patologia , Hipotonia Muscular/terapia , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , Proteínas/isolamento & purificação , Proteômica/métodos , Coluna Vertebral/patologia , Análise de Sistemas
10.
ACS Chem Biol ; 10(10): 2353-63, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26258433

RESUMO

Sialoglycans play a vital role in physiology, and aberrant sialoglycan expression is associated with a broad spectrum of diseases. Since biosynthesis of sialoglycans is only partially regulated at the genetic level, chemical tools are crucial to study their function. Here, we report the development of propargyloxycarbonyl sialic acid (Ac5NeuNPoc) as a powerful tool for sialic acid glycoengineering. Ac5NeuNPoc showed strongly increased labeling efficiency and exhibited less toxicity compared to those of widely used mannosamine analogues in vitro and was also more efficiently incorporated into sialoglycans in vivo. Unlike mannosamine analogues, Ac5NeuNPoc was exclusively utilized in the sialoglycan biosynthesis pathway, allowing a genetic defect in sialic acid biosynthesis to be specifically detected. Furthermore, Ac5NeuNPoc-based sialic acid glycoengineering enabled the on-cell synthesis of high-affinity Siglec-7 ligands and the identification of a novel Siglec-2 ligand. Thus, Ac5NeuNPoc glycoengineering is a highly efficient, nontoxic, and selective approach to study and modulate sialoglycan interactions on living cells.


Assuntos
Ácido N-Acetilneuramínico/química , Polissacarídeos/química , Engenharia de Proteínas , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Animais , Western Blotting , Sequência de Carboidratos , Feminino , Citometria de Fluxo , Glicoproteínas/química , Humanos , Células Jurkat , Ligantes , Glicoproteínas de Membrana/química , Camundongos , Microscopia Confocal , Ligação Proteica
11.
Neurology ; 84(21): 2177-82, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25934851

RESUMO

OBJECTIVE: To identify the underlying genetic defect in 5 patients from a consanguineous family with a Walker-Warburg phenotype, together with intracranial calcifications. METHODS: Homozygosity mapping and exome sequencing, followed by Sanger sequencing of the obtained candidate gene, was performed. Expression of the candidate gene was tested by reverse transcription PCR. Patient fibroblasts were converted to myotubes, and the expression and function of dystroglycan was tested by Western blotting. RESULTS: We detected a homozygous loss-of-function frameshift mutation in the DAG1 gene and showed that this mutation results in a complete absence of both α- and ß-dystroglycan. CONCLUSIONS: A loss-of-function mutation in DAG1 can result in Walker-Warburg syndrome and is not embryonic lethal.


Assuntos
Distroglicanas/deficiência , Distroglicanas/genética , Síndrome de Walker-Warburg/genética , Árabes/genética , Consanguinidade , Feminino , Mutação da Fase de Leitura , Humanos , Lactente , Recém-Nascido , Israel , Síndrome de Walker-Warburg/patologia
12.
Chem Biol ; 22(12): 1643-52, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26687144

RESUMO

A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.


Assuntos
Distroglicanas/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Células Cultivadas , Colina-Fosfato Citidililtransferase/química , Cristalografia por Raios X , Distroglicanas/química , Fibroblastos , Técnicas de Inativação de Genes , Glicosilação , Humanos , Nucleotidiltransferases/química
13.
Science ; 340(6131): 479-83, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23519211

RESUMO

Glycosylated α-dystroglycan (α-DG) serves as cellular entry receptor for multiple pathogens, and defects in its glycosylation cause hereditary Walker-Warburg syndrome (WWS). At least eight proteins are critical to glycosylate α-DG, but many genes mutated in WWS remain unknown. To identify modifiers of α-DG, we performed a haploid screen for Lassa virus entry, a hemorrhagic fever virus causing thousands of deaths annually that hijacks glycosylated α-DG to enter cells. In complementary screens, we profiled cells for absence of α-DG carbohydrate chains or biochemically related glycans. This revealed virus host factors and a suite of glycosylation units, including all known Walker-Warburg genes and five additional factors critical for the modification of α-DG. Our findings accentuate the complexity of this posttranslational feature and point out genes defective in dystroglycanopathies.


Assuntos
Distroglicanas/metabolismo , Interações Hospedeiro-Patógeno/genética , Febre Lassa/genética , Vírus Lassa/fisiologia , Proteínas de Membrana/genética , Proteoma/metabolismo , Internalização do Vírus , Síndrome de Walker-Warburg/genética , Sequência de Aminoácidos , Linhagem Celular , Feminino , Glicosilação , Haploidia , Humanos , Lactente , Febre Lassa/virologia , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Pentosiltransferases
14.
Genome Biol ; 13(2): R12, 2012 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-22356826

RESUMO

BACKGROUND: Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs. RESULTS: We have developed an iterative orthology prediction method, Ortho-Profile, that uses reciprocal best hits at the level of sequence profiles to infer orthology. It increases ortholog detection by 20% compared to sequence-to-sequence comparisons. Ortho-Profile predicts 598 human orthologs of mitochondrial proteins from Saccharomyces cerevisiae and Schizosaccharomyces pombe with 94% accuracy. Of these, 181 were not known to localize to mitochondria in mammals. Among the predictions of the Ortho-Profile method are 11 human cytochrome c oxidase (COX) assembly proteins that are implicated in mitochondrial function and disease. Their co-expression patterns, experimentally verified subcellular localization, and co-purification with human COX-associated proteins support these predictions. For the human gene C12orf62, the ortholog of S. cerevisiae COX14, we specifically confirm its role in negative regulation of the translation of cytochrome c oxidase. CONCLUSIONS: Divergent homologs can often only be detected by comparing sequence profiles and profile-based hidden Markov models. The Ortho-Profile method takes advantage of these techniques in the quest for orthologs.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Proteínas de Membrana , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Biologia Computacional , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética
15.
Nat Genet ; 44(5): 581-5, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22522421

RESUMO

Walker-Warburg syndrome (WWS) is an autosomal recessive multisystem disorder characterized by complex eye and brain abnormalities with congenital muscular dystrophy (CMD) and aberrant a-dystroglycan glycosylation. Here we report mutations in the ISPD gene (encoding isoprenoid synthase domain containing) as the second most common cause of WWS. Bacterial IspD is a nucleotidyl transferase belonging to a large glycosyltransferase family, but the role of the orthologous protein in chordates is obscure to date, as this phylum does not have the corresponding non-mevalonate isoprenoid biosynthesis pathway. Knockdown of ispd in zebrafish recapitulates the human WWS phenotype with hydrocephalus, reduced eye size, muscle degeneration and hypoglycosylated a-dystroglycan. These results implicate ISPD in a-dystroglycan glycosylation in maintaining sarcolemma integrity in vertebrates.


Assuntos
Distroglicanas/metabolismo , Mutação/genética , Síndrome de Walker-Warburg/genética , Peixe-Zebra/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Pré-Escolar , Embrião não Mamífero , Olho/metabolismo , Olho/patologia , Glicosilação , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Peixe-Zebra/embriologia
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