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.
Highlights
Protein glycosylation, the attachment of carbohydrate chains to proteins, is a common post-translational modification occurring ubiquitously in eukaryotic cells
We found that culturing in media containing 15% foetal calf serum (FCS) was optimal for our glycomics experiments
MALDI-TOF N-glycomic profiling was performed on duplicate myoblast preparations with high quality data being acquired in all instances minor glycans were not observed in Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) patient 3 whose myoblasts were difficult to culture
Summary
The attachment of carbohydrate chains to proteins, is a common post-translational modification occurring ubiquitously in eukaryotic cells. Mutations in genes encoding protein glycosylation enzymes, or enzymes synthesising the building blocks for protein glycosylation, have recently been identified to cause neuromuscular transmission defects called congenital myasthenic syndromes (CMS). The first of these genes to be correlated with CMS was GFPT1. GFPT1 converts fructose-6-phosphate and glutamine into glucosamine-6-phosphate and glutamate; the end product of this pathway is uridine diphospho-N-acetylglucosamine (UDP-GlcNAc), which is a basic substrate for protein N- and O-linked glycosylation, and for lipid glycosylation, proteoglycan synthesis, and O-GlcNAc glycosylation [1] It is not clear which of these pathways is affected most by lower levels of UDP-GlcNAc caused by GFPT1 deficiency. It is currently not clear why muscles, or the neuromuscular junctions, are more vulnerable than other tissues to GFPT1 deficiency
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