Analysis of UDP‐galactose 4′‐epimerase mutations associated with the intermediate form of type III galactosaemia

Abstract

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP-galactose 4'-epimerase (GALE). Traditionally, the condition has been divided into two forms-a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, k (cat)) compared with the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose-sensitivity of gal10Delta cells as fully as was the wild-type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.

A Case Study of Monozygotic Twins Apparently Homozygous for a Novel Variant of UDP-Galactose 4′-epimerase (GALE)

“…The entire hGALE open reading frame of the resulting plasmid (pMM33.hGALE.R220W) was confirmed by dideoxy sequencing. The corresponding positive (wild-type hGALE) and negative (plasmid backbone only) plasmid controls have been reported previously (Chhay et al 2008b;Wohlers et al 1999).…”

“…Plasmids and yeast strains: The R220W substitution was re-created by site-directed mutagenesis of a wild-type human GALE coding sequence within the context of a centromeric yeast expression plasmid (MM33) that has been previously described (Chhay et al 2008b). Mutagenesis was carried out using the Quick-change system (Stratagene, Inc.) according to the manufacturer's instructions using the following primer sequences (lower case letters indicate the mutation to be created): hGALE.R220W.f1 5 0 GTGGCGATCGGGCGAtGGGAGGCCCTGAATGTC 3 0 and hGALE.R220W.r1 5 0 GACATTCAGGGCCTCCCaTCGCCCGATCGCCAC 3 0 .…”

“…All yeast strains used in this study were derived by transformation of JFy3835, a GAL10-null haploid strain of Saccharomyces cerevisiae that lacks endogenous GALE and has been described reviously (Chhay et al 2008b). For in vitro biochemical assays, yeast cultures were maintained in standard synthetic medium containing 2% dextrose (SD) at 28 C. For growth curves, yeast were cultured in standard synthetic medium containing 2% glycerol and 2% ethanol (SGE) at 30 C, with or without the addition of galactose, as indicated.…”

“…More than 20 different, ostensibly causal genetic variants have been identified in the GALE loci of patients with biochemically confirmed epimerase deficiency; a subset of these alleles and the GALE proteins they encode have been studied in vitro and/or in vivo, revealing a range of degrees and mechanisms of impairment (Alano et al 1998;Bang et al 2009;Chhay et al 2008b;Henderson et al 2001;Maceratesi et al 1996Maceratesi et al , 1998Openo et al 2006;Park et al 2005;Quimby et al 1997;Thoden et al 2001b;Timson 2005;Wohlers and Fridovich-Keil 2000;Wohlers et al 1999). Some variant GALE proteins are catalytically impaired, while others demonstrate compromised stability, at least under defined laboratory conditions.…”

Use of Site-Directed Mutagenesis in the Diagnosis, Prognosis and Treatment of Galactosemia

“…All three mutations result in impairment of the kinetic parameters, principally the turnover number, k cat , compared to the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation V94M (Chhay et al, 2008). Studies are limited by the fact the many patients are compound heterozygotes and by the observation that dominant-negative interactions may be involved in some of these cases.…”