Abstract
Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional–structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural–functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggregation, particularly striking for the p.Q188R variant, resulting from the most frequent (∼60%) allele at a worldwide scale. The absence of major effects at the secondary and tertiary structure levels suggests that the disturbed aggregation results from subtle perturbations causing a higher and/or longer exposure of hydrophobic residues in the variants as compared to WT GALT. The results herein described indicate a possible benefit from introducing proteostasis regulators and/or chemical/pharmacological chaperones to prevent the accumulation of protein aggregates, in new avenues of therapeutic research for classic galactosemia.
Highlights
Classic galactosemia (OMIM #230400) is an autosomal recessive disorder caused by mutations in the Galactose-1-phosphate uridylyltransferase (GALT) gene, resulting in deficient activity of galactose-1-phosphate uridylyltransferase (GALT, EC 2.7.7.12), a key enzyme in galactose metabolism (Fridovich-Keil and Walter 2008)
Namely p.Q188R, p.S135L, p.N314D, and p.G175D, displayed lower T1⁄2 than that of the WT GALT, with ΔT1⁄2 ranging from À8.1°C to À19.9°C (Table 1)
With the exception of p.R231H, all variants displayed slightly higher Tm values than WT GALT, all the DTm Æ SD fell below the 2°C threshold
Summary
Classic galactosemia (OMIM #230400) is an autosomal recessive disorder caused by mutations in the GALT gene, resulting in deficient activity of galactose-1-phosphate uridylyltransferase (GALT, EC 2.7.7.12), a key enzyme in galactose metabolism (Fridovich-Keil and Walter 2008). Acute symptoms generally appear soon after birth upon exposure to milk, and include the following: vomiting, diarrhea, excessive weight loss, lethargy, hypotonia, liver dysfunction, and, in the absence of intervention, can escalate to cataracts, Escherichia (E.) coli sepsis, and eventually to neonatal death (Holton et al 2001; Bosch 2006; Suchy et al 2007; Fridovich-Keil and Walter 2008). These symptoms generally self-resolve once the patient is placed on a stringent lifelong dietary restriction of galactose, which is the current standard of care (Fridovich-Keil 2006).
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