In vivo and in vitro function of human UDP-galactose 4'-epimerase variants

Abstract

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.

Fig. 1

Abundance and activity of hGALE proteins expressed in a null-background strain of yeast. (a) Soluble lysates from yeast expressing the indicated alleles of hGALE from low copy number (MM33) or high copy number (MM195) plasmids were subjected to Western blot analysis (see Materials and methods) with antisera specific for hGALE and the endogenous yeast protein cyclophilin A, which served as a loading control. (b) Soluble lysates from yeast expressing the indicated alleles of hGALE were assayed for epimerase activity as described in Materials and methods. Values represent the means ± SEM (n = 3) of activity normalised to the wild-type control. An asterisk (*) denotes GALE activity that is statistically significant from the negative control using a one-tailed t-test (p < 0.05).

Fig. 2

Growth and gal-1P accumulation in S. cerevisiae Δgal10 expressing wild-type or variant alleles of hGALE from centromeric plasmids. Yeast cells were grown at 30 °C in SGE-ura medium supplemented with (a) 0.002% (w/v) or (b) 0.01% (w/v) galactose. Growth was monitored by measurement of the optical density at 600 nm (OD₆₀₀). All strains grew normally in SGE-ura medium without galacose (not shown). (c) Intracellular gal-1P levels in JFy3835 yeast expressing wild-type hGALE (wt), no GALE (bb), or M284K-hGALE (M284K). Cultures grown in SGE-ura medium were supplemented with 0.01% galactose at t = 0; samples were harvested for analysis of intracellular gal-1P as described in Materials and Methods at t = 0 h, t = 12 h, and t = 24 h. Values plotted represent mean ± SEM, n = 3. The level of gal-1P detected in “bb” samples at 12 h and 24 h was statistically distinct from the corresponding values detected in wild-type or M284K samples by two-tailed Student t-test, p < 0.001. Note no gal-1P was detected at any time points in control cultures not supplemented with galactose (data not shown).

Fig. 3

Expression and purification of M284K-hGALE from E. coli. Expression levels in cells grown at 37 °C (a) and at 24 °C (b) were monitored by SDS-PAGE (10%), stained with Coomassie blue. Greater amounts of the protein could be produced when the cells were grown at 24 °C; however, even at this temperature some degradation is evident. In both (a) and (b) U, extract from cells immediately prior to induction; I, extract from cells at the end of the period of induction prior to harvesting; S, the supernatant remaining after sonication and centrifugation of the cells; F, the material passing through the column; E1 and E2, the first and second elutions respectively; M, molecular mass markers (kDa).

Fig. 4

The M284K variant has lower enzymatic activity than does wild-type hGALE. The steady state enzyme kinetics for (a) 5 nM wild-type and (b) 1000 nM M284K variant GALE are shown. Each point represents the mean of three independent determinations of the rate and the error bars represent the standard deviations of these means. In both cases, reactions were carried out at 37 °C and 24 °C in the presence of 1.2 μM coupling enzyme (UDP-glucose dehydrogenase) and 10 mM NAD⁺ at pH 8.8.

Fig. 5

Limited proteolysis of wild-type hGALE and the M284K variant showed greater degradation of the M284K variant compared to the wild-type protein. UDP-galactose partially protected the wild-type protein, but not the M284K variant from proteolysis. C, control, undigested protein (10 μM); P, protein digested with 600 nM trypsin; U, protein digested with 600 nM trypsin in the presence of 1 mM UDP-galactose. (b) Guanidine hydrochloride denaturation of the wild-type protein followed by ANS fluorescence. In the absence of UDP-galactose (left), a peak occurred at approximately 0.8 M GuHCl. This peak shifted to approximately 1.6 M GuHCl in the presence of 50 μM UDP-galactose (right). No such effects were seen with the M284K variant. The control data represent the fluorescence of ANS in increasing concentrations of GuHCl in the absence of protein. Each point represents the mean of three determinations and the error bars represent the standard deviations of these means.

Fig. 6

Met-284 is located towards the surface of the protein, away from the active site and NAD⁺ binding site. The left hand figure shows the location of this residue (circled) in both subunits of the hGALE homodimer. The right hand view shows a close-up of the residue in one subunit. The side chain of Met-284 (circled) points towards the centre of a bundle of three α-helices. Substitution of this residue for lysine will insert an unpaired positive charge into this bundle. The figures were made in PyMol (www.pymol.org) using PDB entry 1EK6 .