| Abstract
| - Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutationsin UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialicacid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported tocause the disease. Ten of them, representing mutations in both functional domains of GNE, wererecombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reductionin the two known GNE activities, thus revealing that mutations may also influence the function of thedomain not harboring them. The extent of reduction strongly differs among the point mutants, rangingfrom only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activityin D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined bystructural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis,respectively. We therefore generated models of the three-dimensional structures of the epimerase and thekinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-epimerase and glucokinase,respectively, and determined the localization of the HIBM mutations within these proteins. Whereas inthe kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerasedomain are mostly located at the protein surface. Otherwise, the different mutations result in differentenzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role ofthe enzymatic function of GNE in the disease mechanism.
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