Mediators of galactose sensitivity in UDP-galactose 4'-epimerase-impaired mammalian cells
| Autor: | Jenny M. Schulz, Monty Krieger, Kerstin Malmstrom, Judith L. Fridovich-Keil, Kerry L. Ross |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Galactosemias
Glycosylation CHO Cells Biology Biochemistry Substrate Specificity chemistry.chemical_compound UDPglucose 4-Epimerase Cricetinae medicine Animals Humans Molecular Biology Uridine Chinese hamster ovary cell Escherichia coli Proteins Galactosemia Galactose Cell Biology Transfection medicine.disease Uridine Diphosphate Sugars carbohydrates (lipids) Leloir pathway chemistry Bromodeoxyuridine Cell culture |
| Zdroj: | The Journal of biological chemistry. 280(14) |
| ISSN: | 0021-9258 |
| Popis: | UDP-galactose 4′-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia. |
| Databáze: | OpenAIRE |
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