Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells

• Published in: Cells (Basel, Switzerland) Vol. 9; no. 3; p. 607
• Main Authors: Janes, Verena, Grabany, Simona, Delbrouck, Julien, Vincent, Stephane P, Gottschalk, Johannes, Elling, Lothar, Hanisch, Franz-Georg
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 03.03.2020; MDPI AG
• Subjects: classic galactosemia; Galactose - metabolism; galactose-1-phosphate uridyltransferase (galt); Galactosemias - genetics; glycoprotein; Glycosylation; HEK293 Cells; Humans; membrane rafts; Phenotype; UTP-Hexose-1-Phosphate Uridylyltransferase - metabolism; membrane rafts; galactose-1-phosphate uridyltransferase (GALT); glycosylation; glycoprotein; classic galactosemia
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9030607

Genetic defects of human galactose-1-phosphate uridyltransferase ( GALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by GALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant GALT with a K of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and r MUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.