Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human hereditary inclusion body myopathy.
Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional 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 it 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.
Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Susan E
SE
Sparks
Carla
C
Ciccone
Molly
M
Lalor
Eduard
E
Orvisky
Riko
R
Klootwijk
Paul J
PJ
Savelkoul
Marinos C
MC
Dalakas
Donna M
DM
Krasnewich
William A
WA
Gahl
Marjan
M
Huizing
D000328:Adult; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D002478:Cells, Cultured; D004252:DNA Mutational Analysis / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D009154:Mutation; D018979:Myositis, Inclusion Body / Q000150:complications / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017853:Phosphotransferases (Alcohol Group Acceptor); C018765:N-acylmannosamine kinase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase