Abstract
Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) 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 hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI 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 rhMUC1. 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.
Highlights
Galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12), as one of the enzymes in the Leloir pathway, is responsible for the conversion of galactose-1-phosphate to UDP-galactose
Dys-glycosylations of secreted proteins, like transferrin and glycopeptide hormones, were reported [2], no reports are currently available for aberrant N-glycosylation of membrane glycoproteins in classic galactosemia
The simulation of a galactosemic state in a cell affords a partial reduction of GALT activity by appropriate inhibitors. 2-Fluorinated galactose (F-Gal) and 2,20 -difluorinated galactose (F2 -Gal) are expected to exert such effects, as they are taken up by the cells and both are tolerated by the galactose kinase 1 as substrates, which form the 1-phosphates, at significantly reduced rates
Summary
Galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12), as one of the enzymes in the Leloir pathway, is responsible for the conversion of galactose-1-phosphate to UDP-galactose. A GALT deficiency (classic galactosemia, refer to entry #5056 in the Disease Database), which leads to an accumulation of galactose-1-phosphate, is associated with a potentially lethal acute hepatotoxic syndrome, unless affected newborns are kept under a galactose-restricted diet. Synthesized galactose can exert chronic cell toxic effects and worsen the prospects of patients. Besides direct cell-toxic effects exerted by accumulated galactose-1-phosphate, an indirect effect on the glycosylation machinery mediated by reduced provision of the substrate UDP-Gal could lead to altered protein glycosylation with impact on cellular functions in general and on the expression of membrane glycoproteins in particular. Dys-glycosylations of secreted proteins, like transferrin and glycopeptide hormones, were reported [2], no reports are currently available for aberrant N-glycosylation of membrane glycoproteins in classic galactosemia. Our own studies on membrane-bound N-glycoproteins from patient-derived urinary exosomes had indicated altered
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