title: GALT deficiency causes UDP-hexose deficit in human galactosemic cells.
abstract: Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.
authors.affiliation: Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, GA 30322, USA. klai@med.miami.edu
authors.forename: K
authors.initials: K
authors.lastname: Lai
authors.forename: S D
authors.initials: SD
authors.lastname: Langley
authors.forename: F W
authors.initials: FW
authors.lastname: Khwaja
authors.forename: E W
authors.initials: EW
authors.lastname: Schmitt
authors.forename: L J
authors.initials: LJ
authors.lastname: Elsas
mesh_terms: D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000821:virology; D005690:Galactose / Q000494:pharmacology; D005695:Galactosephosphates / Q000378:metabolism* / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D010641:Phenotype; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000172:deficiency*
chemical_list: D005695:Galactosephosphates; C029973:galactose-1-phosphate; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose; D005690:Galactose