Schulz Jenny M, Ross Kerry L, Malmstrom Kerstin, Krieger Monty, Fridovich-Keil Judith L
Graduate Program in Nutrition and Health Sciences, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2005 Apr 8;280(14):13493-502. doi: 10.1074/jbc.M414045200. Epub 2005 Feb 8.
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.
UDP-半乳糖4'-表异构酶（GALE）催化半乳糖代谢的勒洛伊尔途径的最后一步，使UDP-半乳糖和UDP-葡萄糖相互转化。与大肠杆菌中的对应物不同，哺乳动物的GALE还能使UDP-N-乙酰半乳糖胺和UDP-N-乙酰葡糖胺相互转化。鉴于这四种UDP-糖在糖基化过程中都发挥着关键作用，因此人类GALE不仅对勒洛伊尔途径有贡献，还充当着一个把关者，监督糖基化大分子合成所需重要底物池的比例。人类GALE的缺陷会导致表异构酶缺乏性半乳糖血症。为了探究哺乳动物细胞中GALE活性、底物特异性、代谢平衡和半乳糖敏感性之间的关系，我们采用了先前描述的中国仓鼠卵巢细胞的GALE基因敲除系ldlD。通过转染方案，我们构建了表达不同水平野生型人类GALE或大肠杆菌GALE的ldlD衍生细胞系，并比较了在有半乳糖和无半乳糖条件下培养的这些细胞系的表型和代谢谱。我们发现，在有半乳糖的情况下，GALE基因敲除细胞中Gal-1-P和UDP-Gal积累异常高，而UDP-Glc和UDP-GlcNAc积累异常低，并且人类GALE的表达纠正了所有这些缺陷。比较表达人类GALE和大肠杆菌GALE的细胞，我们发现虽然恢复代谢平衡需要GALE对两种底物的活性，但在存在抑制细胞生长浓度的半乳糖的情况下，细胞增殖并不需要UDP-GalNAc活性。最后，我们发现补充尿苷基本上纠正了UDP-Glc以及在较小程度上UDP-GlcNAc的消耗，使得ldlD细胞在有半乳糖的情况下能够增殖，尽管Gal-1-P和UDP-Gal仍持续积累。这些数据为表异构酶受损细胞中半乳糖敏感性的机制提供了重要见解，并为表异构酶缺乏性半乳糖血症患者提出了一种潜在的新疗法。
