title: Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase.
abstract: UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.
authors.affiliation: Graduate Program in Genetics and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA.
authors.forename: B B
authors.initials: BB
authors.lastname: Quimby
authors.forename: A
authors.initials: A
authors.lastname: Alano
authors.forename: S
authors.initials: S
authors.lastname: Almashanu
authors.forename: A M
authors.initials: AM
authors.lastname: DeSandro
authors.forename: T M
authors.initials: TM
authors.lastname: Cowan
authors.forename: J L
authors.initials: JL
authors.lastname: Fridovich-Keil
mesh_terms: D000483:Alleles; D002461:Cell Line, Transformed; D002675:Child, Preschool; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D005693:Galactosemias / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D008297:Male; D009243:NAD / Q000378:metabolism; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014534:UDPglucose 4-Epimerase / Q000172:deficiency* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D014534:UDPglucose 4-Epimerase