Correlation assessment among clinical phenotypes, expression analysis and molecular modeling of 14 novel variations in the human galactose-1-phosphate uridylyltransferase gene

Abstract

Galactose-1-phosphate uridylyltransferase (GALT) catalyses the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by deleterious variations in the GALT gene can cause a potentially lethal disease called Classic Galactosemia. In this study, we selected 14 novel nucleotide sequence changes in the GALT genes found in galactosemic patients for expression analysis and molecular modeling. Several variants showed decreased levels of expression and decreased abundance in the soluble fraction of the Escherichia coli cell extracts, suggesting altered stability and solubility. Only six variant GALT enzymes had detectable enzymatic activities. Kinetic studies showed that their Vmax decreased significantly. To further characterize the variants at molecular level, we performed static and dynamic molecular modeling studies. Effects of variations on local and/or global structural features of the enzyme were anticipated for the majority of variants. In-depth studies with molecular dynamic simulations on selected variants predicted the alteration of the protein structure even when static models apparently did not highlight any perturbation. Overall, these studies offered new insights on the molecular properties of GALT enzyme, with the aim of correlating them with the clinical outcome.

Mutation spectrum in the French cohort of galactosemic patients and structural simulation of 27 novel missense variations

“…In fact, even those variations with no apparent effects on the static model could have caused perturbations in the protein's structure and dynamics. One of the variations with no predicted structural effects (p.Val168Met) is very similar to another one (p.Val168Leu) that showed no effects in the static model, but showed alterations when submitted to a molecular dynamics simulation protocol [23]. Probably, a similar effect could be argued also for the variations characterized in this work.…”

“…The high percentage of variations predicted to have a negative impact on the overall stability of GALT enzyme is in line with our previous observations [13] (to date, only 1 variation included in GALT Prot database was predicted not to affect protein stability, and only 4 variations are predicted to increase the stability of the protein). Moreover, in a recent characterization of other novel GALT variations [23], mutant proteins were expressed in E. coli and purified, and most of them showed solubility problems and/or reduced expression, thus supporting the idea that stability is a main issue in GALT variants.…”

“…In three of these cases (p.Arg67His, pSer143Trp, pVal168Met), variations affect residues that were previously analyzed for the impact of other protein changes on the structure and function of GALT enzyme [13,23], but also in these cases, no effects were predicted from simple static modeling.…”

Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase

“…It is of note that various cofactors and modifications can alter a metabolic enzyme’s turnover and stability, with functional association to disease (38). As renewed interest in the mechanistic basis of galactosemia has shown that disease-associated mutants exert their effects on misfolding through affecting expression (39), solubility (33,40), stability (31,40,41) and aggregation tendency (31) of hGALT, it is critical to understand the contribution of these factors. The previous lack of consideration of the effects of covalent modification by UMP and divalent metal binding has been in part due to the absence of a crystal structure of the human protein.…”

Hypothalamic Hypothyroidism in a Newborn With Classic Galactosemia: Case Report

“…Most patients present with feeding difficulties, failure to thrive, liver disease, cataracts, encephalopathy and sepsis. Early diagnosis and treatment improve the prognosis in patients with classic galactosemia (1)(2)(3). Regarding endocrine abnormalities in classic galactosemia, female hypergonadotropic hypogonadism has often been reported (4)(5)(6)(7).…”