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Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

ABSTRACT: Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. It has been reported that GFPT1 mutations lead to a distinct sub-class of congenital myasthenic syndromes (CMS) termed "limb-girdle CMS with tubular aggregates". CMS are hereditary neuromuscular transmission disorders in which neuromuscular junctions are impaired. To investigate whether alterations in protein glycosylation at the neuromuscular junction might be involved in this impairment, we have employed mass spectrometric strategies to study the N-glycomes of myoblasts and myotubes derived from two healthy controls, three GFPT1 patients, and four patients with other muscular diseases, namely CMS caused by mutations in DOK7, myopathy caused by mutations in MTND5, limb girdle muscular dystrophy type 2A (LGMD2A), and Pompe disease. A comparison of the relative abundances of bi-, tri-, and tetra-antennary N-glycans in each of the cell preparations revealed that all samples exhibited broadly similar levels of branching. Moreover, although some differences were observed in the relative abundances of some of the N-glycan constituents, these variations were modest and were not confined to the GFPT1 samples. Therefore, GFPT1 mutations in CMS patients do not appear to compromise global N-glycosylation in muscle cells.

SUBMITTER: Chen Q

PROVIDER: S-EPMC4693256 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Chen Qiushi Q Müller Juliane S JS Pang Poh-Choo PC Laval Steve H SH Haslam Stuart M SM Lochmüller Hanns H Dell Anne A

Biomolecules 20151016 4

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway. It transfers an amino group from glutamine to fructose-6-phosphate to yield glucosamine-6-phosphate, thus providing the precursor for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is an essential substrate for all mammalian glycosylation biosynthetic pathways and N-glycan branching is especially sensitive to alterations in the concentration of this sugar ...[more]

PMID: 26501342

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Project description:Epithelial-mesenchymal transition (EMT) is a diverse and dynamic biological process which is involved in cancer progression. It is important for carcinoma cells during invasion and metastasis. EMT has been in the spotlight for cancer cells to disseminate to distant organs by gaining partial EMT phenotype. Cancer cells with partial EMT are believed to be more cancerogenic/invasive than cells that have undergone complete EMT. The proteomic changes that occur following EMT in breast epithelial cells and how these relate to changes in cellular metabolism are incompletely understood. To study metabolic reprogramming in different mesenchymal states, we analyzed proteomic changes following EMT in the breast epithelial cell model D492, with single-shot LFQ supported by SILAC proteomic approach. The D492 EMT cell model contains three isogenic cell lines: epithelial D492 cells, mesenchymal D492M cells, and partial mesenchymal, HER2 overexpressing, tumorigenic D492HER2 cells. Proteomic analysis positioned the D492 and D492M cells as basal-like while D492HER2 as claudin-low. Further comparison of the non-tumorigenic D492 and D492M cells to tumorigenic D492HER2 differentiated the metabolic EMT markers of migration from those of invasion. Among these were markers of glycan metabolism. We identified glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2) as the top dysregulated enzyme in glycan metabolism and found increased GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA knockdown of GFPT2 influenced both cell growth and invasion in vitro and was accompanied by lowered flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway which regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 expression was regulated by the level of reduced glutathione (GSH) and suppressed by the oxidative stress regulator, GSK3-β. Our results demonstrate that GFPT2 is a marker for oxidative stress. It is upregulated and controls growth and invasion in the D492 EMT model and is associated with claudin-low and poor prognosis in breast cancer.

Dataset Information

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

Publications

Global N-linked Glycosylation is Not Significantly Impaired in Myoblasts in Congenital Myasthenic Syndromes Caused by Defective Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1).

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