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GALT deficiency causes UDP-hexose deficit in human galactosemic cells
K Lai1, S D Langley, F W Khwaja
1Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA. klai@med.miami.edu
Glycobiology
|March 11, 2003
Summary
Galactose toxicity in galactosemia is caused by excess galactose-1-phosphate (Gal-1-P) accumulation, which inhibits UDP-hexose pyrophosphorylases and depletes essential UDP-hexose levels. Restoring these levels via gene transfection can rescue cells and normalize glycoprotein synthesis.
Area of Science:
- Biochemistry
- Cell Biology
- Metabolic Disorders
Background:
- Galactosemia is a metabolic disorder characterized by the inability to metabolize galactose, leading to toxic accumulation of galactose-1-phosphate (Gal-1-P).
- Previous studies in yeast indicated that human UDP-glucose pyrophosphorylase (hUGP2) could alleviate "galactose toxicity."
- The precise mechanisms underlying galactose toxicity in human cells, including the role of UDP-hexose depletion, remain incompletely understood.
Purpose of the Study:
- To investigate the hypothesis that galactose toxicity in human cell lines results from Gal-1-P accumulation, hUGP2 inhibition, and subsequent UDP-hexose deficiency.
- To assess the impact of GALT and hUGP2 gene transfection on Gal-1-P levels, UDP-hexose concentrations, and cell growth in galactosemic cells.
- To determine the inhibitory effect of Gal-1-P on UDP-hexose pyrophosphorylases and its consequences for glycoprotein synthesis.
Main Methods:
- Cultured SV40-transformed fibroblasts from a galactosemic patient and control fibroblasts were exposed to glucose or galactose.
- Cellular Gal-1-P and UDP-hexose (UDP-glucose, UDP-galactose) levels were quantified.
- Galactosemic cells were transfected with either the GALT or hUGP2 gene, and their metabolic and growth characteristics were analyzed.
- Enzyme kinetics of purified hUGP2 and UDP-N-acetylglucosamine pyrophosphorylase were assessed in the presence of varying Gal-1-P concentrations.
Main Results:
- Galactosemic fibroblasts accumulated significantly higher levels of Gal-1-P and exhibited growth arrest when cultured in galactose compared to controls.
- Transfection with GALT or hUGP2 genes restored UDP-glucose and UDP-galactose levels in galactosemic cells, enabling growth in galactose.
- Gal-1-P was found to be a potent inhibitor of purified hUGP2 and UDP-N-acetylglucosamine pyrophosphorylase, with Ki values of 0.47 mM and 2.5 mM respectively.
- Increased Gal-1-P levels correlated with reduced Sambucus nigra agglutinin binding to cellular glycoproteins, indicating impaired glycosylation.
Conclusions:
- Galactose toxicity in galactosemia is mediated by the accumulation of Gal-1-P, which inhibits key UDP-hexose pyrophosphorylases.
- This inhibition leads to a deficiency in intracellular UDP-hexose pools, disrupting essential metabolic pathways including glycoprotein synthesis.
- Restoration of UDP-hexose levels through gene therapy targeting GALT or hUGP2 holds promise for treating galactosemia.
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