Glycobiology Advance Access published online on June 29, 2005
Glycobiology, doi:10.1093/glycob/cwi100
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1 Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD
* To whom correspondence should be addressed. Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in UDP-N-acetylglucosamine 2-epimerase (GNE)/N-acetylmannosamine kinase (MNK), the bi-functional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients with HIBM harboring missense mutations in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription-translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and revealed that mutations in one enzymatic domain (in GNE, G135V, V216A, and R246W; in MNK, A631V, M712T) affected not only that domain’s enzyme activity, but also the activity of the other domain. Moreover, studies of the residual enzyme activity associated with specific mutations revealed a discrepancy between the fibroblasts and the cell-free systems. Fibroblasts exhibited higher residual activities of both GNE and MNK than the cell-free system. These findings add complexity to the tightly regulated system of sialic acid biosynthesis. This cell-free approach can be applied to other glycosylation pathway enzymes that are difficult to evaluate in whole cells because their substrate specificities overlap with those of ancillary enzymes.
Received May 2, 2005
Revised June 17, 2005
Accepted June 20, 2005
Article
Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human Hereditary Inclusion Body Myopathy
2 Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC
3 Neuromuscular Diseases Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda
4 Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD; Office of Rare Diseases, Intramural Program, Office of the Director, National Institutes of Health, Bethesda, MD
Marjan Huizing, E-mail: mhuizing{at}mail.nih.gov
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