No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

doi:10.1016/j.bbrc.2004.12.157

Biochemical and Biophysical Research Communications

Volume 328, Issue 1, 4 March 2005, Pages 221-226

https://doi.org/10.1016/j.bbrc.2004.12.157 Get rights and content

Abstract

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.

Section snippets

Materials and methods

Establishment of muscle cell cultures. These studies were approved by the Institutional Review Board of Hadassah Hospital, Jerusalem, and Wolfson Hospital, Holon, Israel. Fresh biopsies were processed as described [9]. In brief, once fat and connective tissue were removed, the muscle was cut into small pieces in 0.05% trypsin/0.02% EDTA solution. Tissue pieces were stirred vigorously for 15 min at 37 °C, debris were allowed to settle for 1 min, and the solution with the dissociated cells was

Establishment of cell cultures

Cell cultures from muscle biopsies of 11 clinically and genetically confirmed HIBM patients with mutations in the GNE gene, and of 9 normal controls, were established as described. Nine of the patients were of Jewish Persian descent, carrying the founder homozygous mutation M712T; one Iranian non Jewish patient carried a homozygous epimerase mutation, V367I [12], and another patient from German origin was a compound heterozygote, with one mutation at the epimerase domain (R11W) and the second

Discussion

HIBM patients worldwide, presenting the same unique phenotype, carry different mutations. The homozygous M712T mutation, occurring in all our Persian Jewish patients (more than 120), leads to the same phenotypic disorder as in all the other identified compound heterozygous mutations in both GNE domains. All the to date recognized mutations in HIBM result in a decrease of the epimerase activity of GNE in recombinant protein experiments ([8], [15], and unpublished data). We have shown here that

Acknowledgments

We are grateful to all patients who made these studies possible. This work was supported by the Fritz-Thyssen-Stiftung, Koln, Germany, the German–Israeli Foundation for Science Research and Development, Jerusalem, Israel, the Mizutani Foundation for Glycoscience, Japan, the Association Francaise contre les Myopathies (AFM), the Fonds der Chemischen Industrie, Frankfurt/Main, Germany, and the Sonnenfeld-Stiftung, Berlin, Germany. Human myoblast cultures 86 and 312 were obtained from the Muscle

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Although hyposialylation has been detected, the correlation between disease severity and reduction in cell surface sialylation is imperfect. Among humans who have GNE myopathy, many display sialic acid levels indistinguishable from unaffected humans (5). Similarly poor correlations between sialic acid levels and symptom severity can be observed in a mouse model based on the disease-associated D176V mutation to GNE (11).
GNE (UDP-GlcNAc 2-epimerase/ManNAc kinase) myopathy is a rare muscle disorder associated with aging and is related to sporadic inclusion body myositis, the most common acquired muscle disease of aging. Although the cause of sporadic inclusion body myositis is unknown, GNE myopathy is associated with mutations in GNE. GNE harbors two enzymatic activities required for biosynthesis of sialic acid in mammalian cells. Mutations to both GNE domains are linked to GNE myopathy. However, correlation between mutation-associated reductions in sialic acid production and disease severity is imperfect. To investigate other potential effects of GNE mutations, we compared sialic acid production in cell lines expressing wild type or mutant forms of GNE. Although we did not detect any differences attributable to disease-associated mutations, lectin binding and mass spectrometry analysis revealed that GNE deficiency is associated with unanticipated effects on the structure of cell-surface glycans. In addition to exhibiting low levels of sialylation, GNE-deficient cells produced distinct N-linked glycan structures with increased branching and extended poly-N-acetyllactosamine. GNE deficiency may affect levels of UDP-GlcNAc, a key metabolite in the nutrient-sensing hexosamine biosynthetic pathway, but this modest effect did not fully account for the change in N-linked glycan structure. Furthermore, GNE deficiency and glucose supplementation acted independently and additively to increase N-linked glycan branching. Notably, N-linked glycans produced by GNE-deficient cells displayed enhanced binding to galectin-1, indicating that changes in GNE activity can alter affinity of cell-surface glycoproteins for the galectin lattice. These findings suggest an unanticipated mechanism by which GNE activity might affect signaling through cell-surface receptors.
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No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Abstract

Section snippets

Materials and methods

Establishment of cell cultures

Discussion

Acknowledgments

J. Neurol. Sci.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Exp. Cell Res.

J. Biol. Chem.

Eur. J. Cell. Biol.

Neuromuscul. Disord.

J. Biol. Chem.