Galactose 1‐phosphate accumulates to high levels in galactose‐treated cells due to low GALT activity and absence of product inhibition of GALK

Abstract

Classic Galactosaemia is a genetic disorder, characterised by galactose intolerance in newborns. It occurs due to recessive mutations in the galactose-1-phosphate uridylyltransferase (GALT) gene. One of the main alterations caused by GALT deficiency is the accumulation of galactose 1-phosphate (Gal-1P) in cells. Studies have suggested that Gal-1P exerts cellular toxicity, possibly by inhibiting cellular metabolism. However, the exact significance of Gal-1P in disease pathogenesis remains unclear. In this study, we tested the hypothesis that Gal-1P inhibits cellular glucose utilisation by competing with substrates in the glycolytic pathway. We also investigated the metabolism of both galactose and glucose in GALT-expressing HEK293T and 143B cells to identify critical reactions steps contributing to the metabolic toxicity of galactose. Notably, we found that galactose-treated HEK293T and 143B cells, which express endogenous GALT, accumulate markedly high intracellular Gal-1P concentrations. Despite very high intracellular Gal-1P concentrations, no inhibition of cellular glucose uptake and no significant changes in the intracellular concentrations of glycolytic metabolites were observed. This indicates that Gal-1P does not exert an inhibitory effect on glycolysis in cells and rules out one potential hypothesis for cellular Gal-1P toxicity. We also investigated the mechanism responsible for the observed Gal-1P accumulation. Our results suggest that Gal-1P accumulation is a result of both low GALT activity and the absence of product inhibition by Gal-1P on galactokinase (GALK1), the enzyme responsible for phosphorylating galactose to Gal-1P. These findings provide a better understanding of the disease mechanisms underlying Classic Galactoaemia. K E Y W O R D SClassic Galactosaemia, galactose metabolism, GALT deficiency, Leloir pathway, metabolomics

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“…Our metabolomics data cannot distinguish the hexose-phosphate species unique to each sugar utilization pathway (Figure 6A). Nevertheless, the dramatic increase in the total hexose-phosphate pool during growth on galactose, mannose and fructose (Figure 6B) pointed towards internal bottlenecks, as recently suggested for galactose-grown HEK293T cells 44 . In contrast, the lack of hexose-phosphate accumulation for maltose rather pointed towards transport bottlenecks.…”

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“…Our metabolomics data cannot distinguish the hexose-phosphate species unique to each sugar utilization pathway. Nevertheless, the dramatic increase in the total hexose-phosphate pool during growth Article ll on galactose, mannose, and fructose (Figure 7B) pointed toward internal bottlenecks, as suggested for galactose-grown HEK293T cells (Oh et al, 2020). In contrast, the lack of hexose-phosphate accumulation for maltose pointed toward transport bottlenecks.…”

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“…Galactose is converted into glucose by several enzymes, including GALK , galactose-1-phosphate uridyltransferase ( GALT ), aldose transferase, galactose dehydrogenase ( GALDH ), and UDP-glucose/galactose pyrophosphorylase ( UGP ), and other genes in the body [16, 17]. Lactose intolerance due to an inborn error of metabolism with nonfunctional GALT is known to result in cataracts at a young age.…”