Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Abstract

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1, 2). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2, 3).Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30 -50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2 and 3), the biochemical mechanism of pathophysiology in galactosemia remains unknown.

Newborn Screening for Galactosemia in the United States: Looking Back, Looking Around, and Looking Ahead

“…Blood spots from affected newborns show very low to absent GALT activity (Beutler and Baluda 1966) and elevated total galactose (Table 1), especially if the infant consumed milk before the NBS blood spot was collected. However, not all mutations in GALT are functional nulls; many are "hypomorphs," mutations that leave some residual activity intact (e.g., (Riehman et al 2001)). Indeed, one extremely mild variant called the Duarte (D or D2) allele is associated with about half of the normal level of GALT activity (Carney et al 2009;Elsas et al 2001;Greber et al 1995;Levy et al 1978;Mellman et al 1968;Podskarbi et al 1996;Tighe et al 2004;Trbusek et al 2001;Tyfield 2000).…”

Structural and molecular biology of type I galactosemia: Disease‐associated mutations

“…In agreement with this, GALT null yeast were not rescued by expression of human K285N GALT and showed no detectable activity. The amount of GALT was also found to be lower in comparison to wild-type levels, but the main cause of its decreased activity is unknown (8,25). The change from lysine to asparagine may cause increased susceptibility to proteolysis, inability to fold correctly, and/or global changes to the structure.…”

“…However, if the yeast strain is then transferred to media containing galactose as the sole energy source, it rapidly induces the genes encoding Leloir pathway enzymes. Human GALT substitutes well for the yeast's own enzyme (Gal7p) (8). However, many of the disease-associated mutations result in impaired growth on this sugar (see below).…”

“…Homozygotes for Q188R show very little to no GALT activity in their erythrocytes and this variant of the enzyme shows no activity when expressed in GALT-null yeast (8,11,12). In transformed COS cells, however, the enzyme retained 10% of its activity, but it is thought that endogenous GALT in the cell line contributed to this discrepancy (11,13,14).…”

“…The change from lysine to asparagine may cause increased susceptibility to proteolysis, inability to fold correctly, and/or global changes to the structure. Molecular modeling has suggested that residue Lys-285 is on the protein's surface (8). Several other mutations associated with reduced protein abundance (V151A, R231H, and R259W) also occur in residues near the surface [ Fig.…”