CHARACTERIZATION OF 2 MUTATIONS ASSOCIATED WITH EPIMERASE-DEFICIENCY GALACTOSEMIA, BY USE OF A YEAST EXPRESSION SYSTEM FOR HUMAN UDP-GALACTOSE-4-EPIMERASE

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that cat alyzes the interconversion of UDP-galactose and UDP-glucose. Impairmen t of this enzyme in humans results in one of two clinically distinct f orms of epimerase-deficiency galactosemia-one benign, the other severe . The molecular and biochemical distinction between these disorders re mains unknown. To enable structural and functional studies of both wil d-type and patient-derived alleles of human GALE (hGALE), we have deve loped and applied a null-background yeast expression system for the hu man enzyme, We have demonstrated that wild-type hGALE sequences phenot ypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Fu rthermore, we have expressed and characterized two mutant alleles, L18 3P-hGALE and N34S-hGALE, both derived from a patient: with no detectab le GALE activity in red blood cells but with similar to 14% activity i n cultured lymphoblasts, Analyses of crude extracts of Beast expressin g L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abun dance. Extracts of yeast expressing N34S-hGALE demonstrated similar to 70% wild-type activity and normal abundance. However, yeast-coexpress ing both L183P-hGALE and N34S-hGALE exhibited only similar to 7% wild- type levels of activity, thereby confirming the functional impact of b oth substitutions and raising the intriguing possibility that same for m of dominant-negative interaction may exist between the mutant allele s found in this patient. The results reported here establish the utili ty of the yeast-based hGALE-expression system and set the stage for mo re-detailed studies of this important enzyme and its role in epimerase -deficiency galactosemia.