Biochemical characterization of the M712T-mutation of the UDP-N-acetylglucosamine 2-epimerase/N-acetyl-mannosaminekinase in hereditary inclusion body myopathy

Hereditary inclusion body myopathy is a neuromuscular disorder characterized by muscle weakness with a late onset and slow progression. It is caused by mutations of the gene encoding UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE). One of the most frequent mutations is an exchange of methionine to threonine at position 712 (M712T). Here we analyzed wildtype (wt) and M712T-mutated (M712T) GNE. We identified threonine 712 as an additional possible phosphorylation site and found by two-dimensional gel-electrophoresis a lower isoelectric point compared to wt-GNE. This lower isoelectric point could be partially reversed back to the wildtype isoelectric point after treatment with protein phosphatase. Furthermore, in contrast to wt-GNE, a significant fraction of M712T-GNE was in the insoluble fraction. Finally, by using bimolecular fluorescence complementation we demonstrate that the M712T mutation does not disrupt the formation of GNE-oligomers.

► GNE is the key enzyme of the sialic acid biosynthesis. ► The M712T mutation of the GNE is responsible for the hereditary inclusion body myopathy. ► GNE were localized in the water-soluble, cytosolic fraction. ► M712T GNE was also found in significant amounts in the water-insoluble fraction. ► GNE has an isoelectric point between 5.0 and 5.5. ► M712T GNE has an isoelectric point between 4.8 and 5.4. ► The M712T mutation of the GNE is not involved in the oligomerization of the GNE.