Glycobiology Advance Access originally published online on January 3, 2003
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Glycobiology, 2003, Vol. 13, No. 4 285-294
© 2003 Oxford University Press
GALT deficiency causes UDP-hexose deficit in human galactosemic cells
3 Division of Medical Genetics, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA
4 Genetics and Molecular Biology Program, Graduate Division of Biological Sciences, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA
Received on September 3, 2002; revised on October 28, 2002; accepted on November 22, 2002
Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2±0.4 to 5.2±0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157±10 µmoles UDP-glucose/100 g protein and 25±5 µmoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236±25 µmoles UDP- glucose/100 g protein and 82±10 µmoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305±28 µmoles/100 g protein (hUGP2-transfected) and 210±13 µmoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75±12 µmoles/100 g protein (hUGP2-transfected) and 55±9 µmoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2±0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1±0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 µM to 169 µM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.
1 Present address: Department of Pediatrics, University of Miami School of Medicine, P.O. Box 016820 (D-820), Miami, FL 33101, USA
2 To whom correspondence should be addressed; e-mail: klai{at}med.miami.edu
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