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1.
J Med Genet ; 57(1): 11-17, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31391289

RESUMO

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are novel therapeutics for reducing low-density lipoprotein cholesterol (LDLc). While serious side-effects have not been observed in short-term clinical trials, there remain concerns that long-term PCSK9 inhibition may cause neurocognitive side-effects. METHODS AND RESULTS: An adult male with childhood-onset global developmental delay, cerebellar atrophy and severe hypolipidaemia underwent extensive biochemical and genetic investigations. Initial testing revealed low circulating PCSK9 levels and a common loss-of-function PCSK9 polymorphism, but these findings did not fully account for severe hypolipidaemia. Whole-exome sequencing was subsequently performed and identified two pathogenic phosphomannose mutase 2 (PMM2) variants (p.Arg141His and p.Pro69Ser) known to cause PMM2-associated congenital disorder of glycosylation (PMM2-CDG). A diagnosis of PMM2-CDG was consistent with the proband's neurological symptoms and severe hypolipidaemia. Given that PMM2-CDG is characterised by defective protein N-glycosylation and that PCSK9 is a negative regulator of LDLc, we postulated that loss of PCSK9 N-glycosylation mediates hypolipidaemia among patients with PMM2-CDG. First, in an independent cohort of patients with PMM2-CDG (N=8), we verified that circulating PCSK9 levels were significantly lower in patients than controls (p=0.0006). Second, we conducted in vitro experiments in hepatocyte-derived cells to evaluate the effects of PCSK9 N-glycosylation loss on LDL receptor (LDLR) activity. Experimental results suggest that defective PCSK9 N-glycosylation reduces the ability of circulating PCSK9 to degrade LDLR. CONCLUSION: Life-long exposure to genetically lower PCSK9 per se is unlikely to cause neurocognitive impairment. Both observational and experimental findings suggest that hypolipidaemia in PMM2-CDG may be partially mediated by loss of PCSK9 N-glycosylation and/or its regulators.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Dislipidemias/metabolismo , Fosfotransferases (Fosfomutases)/deficiência , Pró-Proteína Convertase 9/sangue , Receptores de LDL/metabolismo , Adulto , Estudos de Coortes , Defeitos Congênitos da Glicosilação/complicações , Defeitos Congênitos da Glicosilação/metabolismo , Análise Mutacional de DNA , Dislipidemias/etiologia , Regulação da Expressão Gênica , Glicosilação , Células Hep G2 , Humanos , Mutação com Perda de Função , Masculino , Linhagem , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Polimorfismo Genético , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Receptores de LDL/genética , Sequenciamento Completo do Exoma
2.
BMC Med Genet ; 20(1): 181, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727010

RESUMO

BACKGROUND: PMM2-CDG, is the most common N-linked glycosylation disorder and subtype among all CDG syndromes, which are a series of genetic disorders involving the synthesis and attachment of glycoproteins and glycolipid glycans. The mutations of PMM2-CDG might lead to the loss of PMM2, which is responsible for the conversion of mannose 6- phosphate into mannose 1-phosphate. Most patients with PMM2-CDG have central nervous system involvement, abnormal coagulation, and hepatopathy. The neurological symptoms of PMM2-CDG are intellectual disability (ID), cerebellar ataxia, and peripheral neuropathy. Now, over 100 new CDG cases have been reported. However, each type of CDG is very rare, and CDGs are problematic to diagnose. In addition, few CDGs have been reported in the Chinese population. CASE PRESENTATION: Here we present a Hani ethnic minority family including two siblings with congenital glycosylation disorders. Whole-exome sequencing revealed compound heterozygous for one novel mutation (c.241-242 del variant) and previously reported mutation (c.395 T > C) in gene of PMM2. Two mutations were found in proband and her sibling by whole-exome sequencing. The mutations were identified in this family by Sanger sequencing and no mutations were detected in the normal control. CONCLUSIONS: This is the first report to describe mutations in two siblings of Hani ethnic minority which is one of five ethnic groups found only in Yunnan with a population of more than 1 million.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Grupos Étnicos/genética , Irmãos , Sequenciamento Completo do Exoma , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Linhagem , Fosfotransferases (Fosfomutases)/genética
3.
Int J Mol Sci ; 20(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454904

RESUMO

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Glucose-6-Fosfato/análogos & derivados , Mutação , Fosfotransferases (Fosfomutases)/deficiência , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Fosfotransferases (Fosfomutases)/genética , Ligação Proteica
4.
Nat Commun ; 10(1): 3698, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420548

RESUMO

Phosphonates are rare and unusually bioactive natural products. However, most bacterial phosphonate biosynthetic capacity is dedicated to tailoring cell surfaces with molecules like 2-aminoethylphosphonate (AEP). Although phosphoenolpyruvate mutase (Ppm)-catalyzed installation of C-P bonds is known, subsequent phosphonyl tailoring (Pnt) pathway steps remain enigmatic. Here we identify nucleotidyltransferases in over two-thirds of phosphonate biosynthetic gene clusters, including direct fusions to ~60% of Ppm enzymes. We characterize two putative phosphonyl tailoring cytidylyltransferases (PntCs) that prefer AEP over phosphocholine (P-Cho) - a similar substrate used by the related enzyme LicC, which is a virulence factor in Streptococcus pneumoniae. PntC structural analyses reveal steric discrimination against phosphocholine. These findings highlight nucleotidyl activation as a predominant chemical logic in phosphonate biosynthesis and set the stage for probing diverse phosphonyl tailoring pathways.


Assuntos
Ácido Aminoetilfosfônico/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/fisiologia , N-Acilneuraminato Citidililtransferase/metabolismo , Organofosfonatos/metabolismo , Actinobacteria , Bactérias/genética , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Cristalização , Cristalografia por Raios X , Escherichia coli , N-Acilneuraminato Citidililtransferase/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Fosfolipídeos/metabolismo , Fosforilcolina/metabolismo , Fosfotransferases (Fosfomutases) , Polissacarídeos/metabolismo , Especificidade por Substrato
5.
Int J Mol Med ; 44(1): 262-272, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115488

RESUMO

Congenital disorder of glycosylation (CDG) type Ia is a multisystem disorder that occurs due to mutations in the phosphomannomutase 2 (PMM2) gene, which encodes for an enzyme involved in the N­glycosylation pathway. Mutated PMM2 leads to the reduced conversion of mannose­6­P to mannose­1­P, which results in low concentration levels of guanosine 5'­diphospho­D­mannose (GDP­Man), a nucleotide­activated sugar essential for the construction of protein oligosaccharide chains. In the present study, an in vitro therapeutic approach was used, based on GDP­Man­loaded poly (D,L­lactide­co­glycolide) (PLGA) nanoparticles (NPs), which were used to treat CDG­Ia fibroblast cultures, thus bypassing the glycosylation pathway reaction catalysed by PMM2. To assess the degree of hypoglycosylation in vitro, the present study examined the activities of α­mannosidase, ß­glucoronidase and ß­galactosidase in defective and normal fibroblasts. GDP­Man (30 µg/ml GDP­Man PLGA NPs) was incubated for 48 h with the cells and the specific activities of α­mannosidase and ß­galactosidase were estimated at 69 and 92% compared with healthy controls. The residual activity of ß­glucoronidase increased from 6.5 to 32.5% and was significantly higher compared with that noted in the untreated CDG­Ia fibroblasts. The glycosylation process of fibroblasts was also analysed by two­dimensional electrophoresis. The results demonstrated that treatment caused the reappearance of several glycosylated proteins. The data in vitro showed that GDP­Man PLGA NPs have desirable efficacy and warrant further evaluation in a preclinical validation animal model.


Assuntos
Defeitos Congênitos da Glicosilação/tratamento farmacológico , Portadores de Fármacos , Guanosina Difosfato Manose , Nanopartículas , Fosfotransferases (Fosfomutases)/deficiência , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Células Cultivadas , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Fibroblastos , Glicosilação/efeitos dos fármacos , Guanosina Difosfato Manose/química , Guanosina Difosfato Manose/farmacologia , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia
7.
Environ Microbiol ; 21(11): 3969-3978, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30938049

RESUMO

Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for twitching motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria.


Assuntos
Fímbrias Bacterianas/genética , Lysobacter/enzimologia , Proteínas de Membrana/genética , Fosfoglucomutase/genética , Fosfotransferases (Fosfomutases)/genética , Proteínas de Bactérias/genética , Lysobacter/genética , Proteínas de Membrana/metabolismo , Família Multigênica , Domínios Proteicos/genética , Pseudomonas aeruginosa/genética , Xanthomonas campestris/genética
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(4): 314-317, 2019 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-30950015

RESUMO

OBJECTIVE: To identify potential mutation of PMM2 gene in an infant with congenital disorders of glycosylation type 1a (CDG-1a). METHODS: Genomic DNA was extracted from peripheral blood sample of the patient. All coding exons (exons 1-8) and splicing sites of the PMM2 gene were amplified with PCR. Potential variants were detected by direct sequencing of the PCR products and comparing the results against the ESP and SNP human gene databases. A protein BLAST system was employed to analyze cross-species conservation of the variants amino acid. A PubMed BLAST CD-search system was employed to identify functional domains damaged by variants of the PMM2 gene. Impact of potential variants was analyzed using software including PolyPhen-2 SIFT and Mutation Taster. Whole exome sequencing was used to identify additional variants of the PMM2 gene which may explain the condition of the patient. RESULTS: The child was found to carry compound heterozygous variants (c.458_462delTAAGA and c.395T>C) of the PMM2 gene, which were inherited respectively from his father and mother. The c.458_462delTAAGA has not been reported previously and may result in disruption of 10 functional domains within the PMM2 protein. The c.395T>C mutation has been recorded by a SNP database with frequency unknown. Both mutations were predicted as "probably damaging". Whole exome sequencing has identified no additional disease-causing variant which can explain the patient's condition. CONCLUSION: The patient's condition may be attributed to the compound heterozygous variants c.458_462delTAAGA and c.395T>C of the PMM2 gene. Above results has facilitated molecular diagnosis for the patient.


Assuntos
Defeitos Congênitos da Glicosilação , Fosfotransferases (Fosfomutases)/genética , Defeitos Congênitos da Glicosilação/genética , Éxons , Humanos , Lactente , Mutação
9.
Ann Neurol ; 85(5): 740-751, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30873657

RESUMO

OBJECTIVE: Phosphomannomutase deficiency (PMM2 congenital disorder of glycosylation [PMM2-CDG]) causes cerebellar syndrome and strokelike episodes (SLEs). SLEs are also described in patients with gain-of-function mutations in the CaV2.1 channel, for which acetazolamide therapy is suggested. Impairment in N-glycosylation of CaV2.1 promotes gain-of-function effects and may participate in cerebellar syndrome in PMM2-CDG. AZATAX was designed to establish whether acetazolamide is safe and improves cerebellar syndrome in PMM2-CDG. METHODS: A clinical trial included PMM2-CDG patients, with a 6-month first-phase single acetazolamide therapy group, followed by a randomized 5-week withdrawal phase. Safety was assessed. The primary outcome measure was improvement in the International Cooperative Ataxia Rating Scale (ICARS). Other measures were the Nijmegen Pediatric CDG Rating Scale (NPCRS), a syllable repetition test (PATA test), and cognitive scores. RESULTS: Twenty-four patients (mean age = 12.3 ± 4.5 years) were included, showing no serious adverse events. Thirteen patients required dose adjustment due to low bicarbonate or asthenia. There were improvements on ICARS (34.9 ± 23.2 vs 40.7 ± 24.8, effect size = 1.48, 95% confidence interval [CI] = 4.0-7.6, p < 0.001), detected at 6 weeks in 18 patients among the 20 responders, on NPCRS (95% CI = 0.3-1.6, p = 0.013) and on the PATA test (95% CI = 0.5-3.0, p = 0.006). Acetazolamide improved prothrombin time, factor X, and antithrombin. Clinical severity, epilepsy, and lipodystrophy predicted greater response. The randomized withdrawal phase showed ICARS worsening in the withdrawal group (effect size = 1.46, 95% CI = 2.65-7.52, p = 0.001). INTERPRETATION: AZATAX is the first clinical trial of PMM2-CDG. Acetazolamide is well tolerated and effective for motor cerebellar syndrome. Its ability to prevent SLEs and its long-term effects on kidney function should be addressed in future studies. Ann Neurol 2019;85:740-751.


Assuntos
Acetazolamida/uso terapêutico , Inibidores da Anidrase Carbônica/uso terapêutico , Doenças Cerebelares/diagnóstico , Doenças Cerebelares/tratamento farmacológico , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Fosfotransferases (Fosfomutases)/deficiência , Acetazolamida/farmacologia , Adolescente , Inibidores da Anidrase Carbônica/farmacologia , Doenças Cerebelares/genética , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/genética , Feminino , Glicosilação/efeitos dos fármacos , Humanos , Masculino , Fosfotransferases (Fosfomutases)/genética , Método Simples-Cego , Resultado do Tratamento , Adulto Jovem
10.
J Inherit Metab Dis ; 42(1): 5-28, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740725

RESUMO

Phosphomannomutase 2 (PMM2-CDG) is the most common congenital disorder of N-glycosylation and is caused by a deficient PMM2 activity. The clinical presentation and the onset of PMM2-CDG vary among affected individuals ranging from a severe antenatal presentation with multisystem involvement to mild adulthood presentation limited to minor neurological involvement. Management of affected patients requires a multidisciplinary approach. In this article, a systematic review of the literature on PMM2-CDG was conducted by a group of international experts in different aspects of CDG. Our managment guidelines were initiated based on the available evidence-based data and experts' opinions. This guideline mainly addresses the clinical evaluation of each system/organ involved in PMM2-CDG, and the recommended management approach. It is the first systematic review of current practices in PMM2-CDG and the first guidelines aiming at establishing a practical approach to the recognition, diagnosis and management of PMM2-CDG patients.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/tratamento farmacológico , Fosfotransferases (Fosfomutases)/deficiência , Seguimentos , Glicosilação , Humanos
11.
J Assist Reprod Genet ; 36(1): 39-45, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30406445

RESUMO

PURPOSE: To investigate the potential genetic etiology of premature ovarian insufficiency (POI). METHODS: Whole-exome sequencing (WES) was done on DNA samples from women diagnosed with POI. Mutations identified were analyzed by in silico tools and were annotated according to the guidelines of the American College of Medical Genetics and Genomics. Plausible variants were confirmed by Sanger sequencing. RESULTS: Four of the 33 individuals (12%) carried pathogenic or likely pathogenic variants, and 6 individuals carried variants of unknown significance. The genes identified with pathogenic or likely pathogenic variants included PMM2, MCM9, and PSMC3IP. CONCLUSIONS: WES is an efficient tool for identifying gene variants in POI women; however, interpretation of variants is hampered by few exome studies involving ovarian disorders and the need for trio sequencing to determine inheritance and to detect de novo variants.


Assuntos
Exoma , Variação Genética , Proteínas de Manutenção de Minicromossomo/genética , Proteínas Nucleares/genética , Fosfotransferases (Fosfomutases)/genética , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/patologia , Transativadores/genética , Sequenciamento Completo do Exoma/métodos , Adulto , Feminino , Humanos
12.
Genet Med ; 21(5): 1181-1188, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30293989

RESUMO

PURPOSE: PMM2-CDG is the most common congenital disorder of glycosylation (CDG), which presents with either a neurologic or multisystem phenotype. Little is known about the longitudinal evolution. METHODS: We performed data analysis on PMM2-CDG patients' clinical features according to the Nijmegen CDG severity score and laboratory data. Seventy-five patients (28 males) were followed up from 11.0 ± 6.91 years for an average of 7.4 ± 4.5 years. RESULTS: On a group level, there was no significant evolution in overall clinical severity. There was some improvement in mobility and communication, liver and endocrine function, and strabismus and eye movements. Educational achievement and thyroid function worsened in some patients. Overall, the current clinical function, the system-specific involvement, and the current clinical assessment remained unchanged. On follow-up there was improvement of biochemical variables with (near) normalization of activated partial thromboplastin time (aPTT), factor XI, protein C, antithrombin, thyroid stimulating hormone, and liver transaminases. CONCLUSION: PMM2-CDG patients show a spontaneous biochemical improvement and stable clinical course based on the Nijmegen CDG severity score. This information is crucial for the definition of endpoints in clinical trials.


Assuntos
Defeitos Congênitos da Glicosilação/epidemiologia , Defeitos Congênitos da Glicosilação/fisiopatologia , Fosfotransferases (Fosfomutases)/deficiência , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Fenótipo , Adulto Jovem
13.
G3 (Bethesda) ; 9(2): 413-423, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30530630

RESUMO

Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast Saccharomyces cerevisiae gene SEC53 Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations - R141H, F119L respectively- and the most common compound heterozygote genotype - PMM2R141H/F119L - observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Mutação com Perda de Função , Fosfotransferases (Fosfomutases)/deficiência , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Humanos , Mutação de Sentido Incorreto , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Neuropediatrics ; 49(6): 408-413, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30304743

RESUMO

Phosphomannomutase deficiency (PMM2-CDG) causes a cerebellar syndrome that has been evaluated using the International Cooperative Ataxia Rating Scale (ICARS). However, no particular dysarthria tests have been used. Speech ICARS subscore subjectively assesses fluency and clarity of speech with two items. Repetition of syllables, traditionally used for characterization of ataxic speech, was validated in early-onset ataxia conditions. We assess the validity of the PATA test (SCA Functional Index [SCAFI]) in PMM2-CDG patients.PATA rates from 20 patients were compared with a control population were and correlated with ICARS and neuroimaging.There was a difference between the PATA rate in patients and controls. PATA rate increased with age in controls. In patients, the improvement of PATA rate with age was not significant. In patients, the PATA rate was negatively correlated with the total ICARS score and the Speech ICARS subscore. Regarding neuroimaging, midsaggital vermis relative diameter was positively correlated with PATA results. These last differences were also significant when the results are corrected by age.PATA rate provides an easy measure for a quantitative assessment of dysarthria that may help clinicians to monitor patients' evolution in a regular consultation. It could also be used in PMM2-CDG clinical trials implementing ICARS speech subscore information.


Assuntos
Doenças Cerebelares/diagnóstico , Defeitos Congênitos da Glicosilação/complicações , Disartria/diagnóstico , Fosfotransferases (Fosfomutases)/deficiência , Índice de Gravidade de Doença , Adolescente , Adulto , Fatores Etários , Doenças Cerebelares/etiologia , Criança , Disartria/etiologia , Feminino , Humanos , Imagem por Ressonância Magnética , Estudos Prospectivos , Adulto Jovem
15.
Int J Mol Sci ; 19(8)2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061496

RESUMO

Type I disorders of glycosylation (CDG), the most frequent of which is phosphomannomutase 2 (PMM2-CDG), are a group of diseases causing the incomplete N-glycosylation of proteins. PMM2-CDG is an autosomal recessive disease with a large phenotypic spectrum, and is associated with mutations in the PMM2 gene. The biochemical analysis of mutants does not allow a precise genotype⁻phenotype correlation for PMM2-CDG. PMM2 is very tolerant to missense and loss of function mutations, suggesting that a partial deficiency of activity might be beneficial under certain circumstances. The patient phenotype might be influenced by variants in other genes associated with the type I disorders of glycosylation in the general population.


Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Mutação , Fosfotransferases (Fosfomutases)/genética , Estudos de Associação Genética , Glicosilação , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Fosfotransferases (Fosfomutases)/química , Conformação Proteica
16.
J Microbiol Biotechnol ; 28(8): 1293-1298, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-29996619

RESUMO

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.


Assuntos
Escherichia coli/genética , Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Glucofosfatos/metabolismo , Concentração de Íons de Hidrogênio , Manosefosfatos/metabolismo , Fosfotransferases (Fosfomutases)/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura
17.
Mol Plant Microbe Interact ; 31(12): 1291-1300, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29953334

RESUMO

Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.


Assuntos
Família Multigênica , Cebolas/microbiologia , Pantoea/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Organofosfonatos/metabolismo , Pantoea/genética , Fosfotransferases (Fosfomutases)/genética , Folhas de Planta/microbiologia
18.
Ann Biol Clin (Paris) ; 76(2): 217-223, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29623892

RESUMO

Congenital disorders of glycosylation (CDG) are rare inborn diseases of glycan component of N-glycosylated proteins. We report here the case of a 28-year-old patient with CDG syndrome type Ia, who presented with a deep venous thrombosis in the left suro-popliteal vein with no known triggers or antecedents. The patient was treated with rivaroxaban for six months. Blood tests performed after discontinuing anticoagulant treatment showed multiple abnormalities affecting the proteins involved in haemostasis (both coagulation factors and inhibitors), i.e. a combined factor XI, antithrombin and protein C deficiency (35%, 41%, and 42% respectively) associated with a moderate increase of FVIII (179%) and VWFAg (163%) without inflammation. Patient results are here discussed with regard to the limited number of articles addressing haemostasis in this rare disease, as the occurrence of deep venous thrombosis remains uncommon in the literature.


Assuntos
Defeitos Congênitos da Glicosilação/tratamento farmacológico , Fosfotransferases (Fosfomutases)/deficiência , Rivaroxabana/uso terapêutico , Trombose Venosa/tratamento farmacológico , Adulto , Defeitos Congênitos da Glicosilação/complicações , Feminino , Humanos , Doenças Raras , Resultado do Tratamento , Trombose Venosa/complicações
19.
Biochemistry ; 57(25): 3480-3492, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29695157

RESUMO

The human phosphomannomutases PMM1 and PMM2 catalyze the interconversion of hexose 6-phosphates and hexose 1-phosphates. The two isoforms share 66% sequence identity and have kinetic properties similar to those of mutases in vitro but differ in their functional roles in vivo. Though the physiological role of PMM2 is catalysis of the mutase reaction that provides the mannose 1-phosphate (Man-1-P) essential for protein glycosylation, PMM1 is thought to provide a phosphohydrolase activity in the presence of inosine monophosphate (IMP), converting glucose 1,6-bisphosphate (Glu-1,6-P2) to glucose 6-phosphate (Glu-6-P), rescuing glycolysis during brain ischemia. To uncover the structural basis of how IMP binding converts PMM1 from a mutase to a phosphatase, the 1.93 Å resolution structure of PMM1 complexed with IMP was determined. The structure reveals IMP bound at the substrate recruitment site, thus inhibiting the mutase activity while simultaneously activating a phosphatase activity (IMP Kact = 1.5 µM) resulting from the hydrolysis of the phospho-enzyme. The bound structure and site-directed mutagenesis confirm that the long-range electrostatic interactions provided by Arg180 and Arg183 conserved in PMM1 are the major contributors to IMP binding, and their oblation removes phosphatase but not mutase activity. These residues are not present in the PMM2 isoform, which consequently lacks significant phosphatase activity in the presence of IMP. T2 relaxation nuclear magnetic resonance and small angle X-ray scattering together support the hypothesis that binding of IMP to PMM1 favors an enzyme conformation that is catalytically competent for water attack at the phosphoaspartyl intermediate. Such a mechanism may be generalizable to other enzymes that act through covalent intermediates.


Assuntos
Inosina Monofosfato/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Isquemia Encefálica/metabolismo , Cristalografia por Raios X , Glicólise , Humanos , Modelos Moleculares , Fosfotransferases (Fosfomutases)/química , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Especificidade por Substrato
20.
Microbiology ; 164(4): 614-624, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29493491

RESUMO

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1- mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1- strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.


Assuntos
Antibacterianos/farmacologia , Vias Biossintéticas , Guanosina Difosfato Manose/metabolismo , Nucleotidiltransferases/genética , Fosfotransferases (Fosfomutases)/genética , Streptomyces coelicolor/efeitos dos fármacos , Streptomyces coelicolor/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Manosiltransferases/genética , Manosiltransferases/metabolismo , Mutação , Nucleotidiltransferases/metabolismo , Fenótipo , Fosfotransferases (Fosfomutases)/metabolismo , Streptomyces coelicolor/virologia
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