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Glycobiology Advance Access published online on January 3, 2003
Glycobiology, doi:10.1093/glycob/cwg033
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Submitted on September 3, 2002
Revised on October 28, 2002
Accepted on November 22, 2002

© 2003 Oxford University Press

ORIGINAL ARTICLES

Galactose-1-phosphate uridyltransferase deficiency causes UDP-hexose deficit in human galactosemic cells

K Lai 1*, SD Langley 1, FW Khwaja 2, EW Schmitt , LJ Elsas 3

1 Division of Medical Genetics, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA
2 Genetics and Molecular Biology Program, Graduate Division of Biological Sciences, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA
3 Division of Medical Genetics, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA; Genetics and Molecular Biology Program, Graduate Division of Biological Sciences, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA

* To whom correspondence should be addressed. E-mail: klai{at}med.miami.edu.

Abstract

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±0.5mM 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±10µmoles UDP-glucose/100g protein and 25±5µmoles UDP-galactose/100g protein when grown in 0.1% glucose. The control cells had 236±25µmoles UDP-glucose/100g protein and 82±10µmoles UDP-galactose/100g 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±28µmoles/100g protein (hUGP2-transfected) and 210±13µmoles/100g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75±12µmoles/100g protein (hUGP2-transfected) and 55±9µmoles/100g protein (GALT-transfected), respectively. While the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2±0.02mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1±0.4mM).

We found that 2.5mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7µM to 169µM, without changes in apparent Vmax. The Ki of the reaction was 0.47mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase (AGX1), 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, 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.


Keywords: Competitive Inhibition, Fibroblast, Galactosemia, Galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase
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