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 μmoles UDP-glucose/100 g protein and 25±5 μmoles; UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236±25 μmoles UDP-glucose/100 g protein and 82±10 μmoles; 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 μmoles/100 g protein (hUGP2-transfected) and 210±13 μmoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75±12 μmoles/100 g protein (hUGP2-transfected) and 55±9 μmoles/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 μM to 169 μM, 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.