One of the fundamental questions in human genetics concerns the normal function of proteins associated with genetic disease, and the impact of patient mutations on those proteins and their activities. The long-term goal of this project is to elucidate the natural structure/ function relationships and interactions of enzymes that mediate galactose metabolism in humans, and the impact of mutations in the corresponding genes that result in galactosemia, a potentially lethal inborn error of metabolism that affects - 1145,000 liveborns. Galactosemia is characterized by extraordinary allelic, biochemical, and clinical heterogeneity. The proposed experiments address the basis of that heterogeneity, and focus on galactose- 1-phosphate uridylyltransferase (GALT), impaired in patients with classic galactosemia, and UDP-galactose-4'-epimerase (GALE), impaired in patients with epimerase-deficiency galactosemia. These enzymes normally catalyze successive steps in the Leloir pathway of galactose metabolism. Human GALE further catalyzes the interconversion of UDP-gaINAc/UDPglcNAc, thereby regulating substrate pools essential for all 0-linked glycosylation reactions in humans. Prior studies have demonstrated marked allelic heterogeneity in both galactosemias, and have suggested a relationship between genotype, biochemical impairment, and clinical outcome for both disorders. The basis for why some mutations or allelic combinations result in greater impairment than others, however, remains largely obscure, and is a principle focus for the proposed work. The short-term goals of this project are to: (1) define structure/function relationships and interactions for both wild-type and mutant forms of human GALT, (2) define structure/function relationships and interactions for both wild-type and mutant forms of human GALE, and (3) define the role of human GALE in determining metabolic and glycosylation defects in mammalian cells with transferase-deficiency vs. epimerase-deficiency galactosemia. The results of these studies will be significant in that they will provide basic science advances and challenge existing paradigms regarding human GALT and GALE activities, structures, interactions, coordination, and metabolic influence. They will also result in the production of useful reagents, including mammalian cell lines specifically deficient in 0-linked protein glycosylation. Finally, they will offer improved diagnostic and prognostic tools for both classic and epimerase-deficiency galactosemia, and ultimately should help to inform a rational approach to the generation of novel treatments for patients with these disorders.
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