Abstract
Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.
Highlights
Galactose-1-phosphate uridylyltransferase (GALT)1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose (UDP-Glc) and galactose-1phosphate (Gal-1-P) into glucose-1-phosphate (Glc-1-P) and UDP-galactose (UDP-Gal) (Fig. 1)
We have observed that wild-type human GALT expressed in yeast displays a native banding pattern indistinguishable from that seen in normal human hemolysates, and that the N314D-substituted human enzyme expressed in yeast demonstrates the characteristic “Duarte” shift reported for patient samples [14]
In the studies reported here, we have explored the possibility of covalent heterogeneity of human GALT
Summary
Yeast Strains and Plasmid Manipulation—All recombinant human GALT proteins used in this study were expressed in yJFK1, a haploid strain of Saccharomyces cerevisiae that lacks endogenous GALT and has been described previously [16]. Where indicated, following Western blot analysis, filters were stripped at 50 °C for 30 min in stripping buffer (2% sodium dodecyl sulfate, 0.625 M Tris, pH 6.8, 7.1 l/ml -mercaptoethanol) and stained with 0.1% India ink in TNT (25 mM Tris, pH 7.5, 0.16 M NaCl, 0.05% Tween 20) to reveal a constellation of more than 150 abundant endogenous yeast proteins. Selected subsets of these endogenous proteins were used where indicated to provide markers against which to measure the migration of the hGALT signals. Gels were electroblotted onto nitrocellulose and visualized as described above
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