From the Department of Genetics and ¶ Graduate
Program in Genetics and Molecular Biology, Emory University, School of
Medicine, Atlanta, Georgia 30322
Received for publication, October 19, 2000, and in revised form, December 18, 2000
![]() |
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.
One of the fundamental questions with regard to classic galactosemia
concerns the identification of predictive factors that might be used to
distinguish those patients who will thrive long term from those who
will experience complications. Waggoner et al. (4) addressed
this issue a decade ago with an international retrospective
questionnaire study and found no clear correlation between long term
outcome and three of the most obvious candidate extrinsic factors: age
at diagnosis, presence of neonatal complications before treatment, or
strict dietary compliance. These data suggested that some intrinsic
factor(s) might serve a predominant role in defining outcome.
With regard to intrinsic factors, perhaps the most obvious is GALT
genotype. The number of candidate mutations identified in patient
alleles now exceeds 150, the majority of which are missense point
mutations (5). Indeed many if not most galactosemia patients studied
are compound heterozygotes, further complicating the picture. Although
most naturally occurring mutant alleles of hGALT have not been well
characterized with regard to function, high sensitivity biochemical
studies of large groups of ungenotyped patients have demonstrated clear
biochemical heterogeneity in the patient population (6, 7).
Furthermore, differences in the prevalence of specific hGALT genotypes
also have been observed in populations of patients with
versus without detectable GALT activity (8). These data
support the hypothesis that genotype may correlate with activity, which
in turn may influence metabolite levels and phenotypic outcome. Indeed,
ungenotyped patients with detectable GALT activity have been reported
to accumulate lower levels of gal-1-P and to experience a milder
clinical course and than do their counterparts without detectable GALT
activity (6, 7). Similarly, a number of studies report decreased levels of UDP-gal and altered ratios of UDP-glc/UDP-gal in samples from galactosemic patients compared with controls (2). Nonetheless, there
has been no direct test of the relationship of these parameters. Furthermore, although some retrospective outcome studies of patients with galactosemia have reported a statistically significant
relationship between genotype and outcome, others have not (9-12),
perhaps reflecting the complications of confounding variables and
limited sample sizes.
We report here the first quantitative and allele-specific test of the
hypothesis that there is a relationship between hGALT genotype,
activity, metabolite levels, and sensitivity to galactose in a
eukaryotic system. In particular, we have used yeast, extending from
initial observations by Douglas and Hawthorne (13), who reported that
GALT-deficient yeast, but not their wild-type counterparts, were
growth-arrested by the addition of small amounts of galactose to the
medium despite the presence of other metabolizable carbon sources. We
applied a previously described null-background yeast expression system
(14) to study 16 naturally occurring patient alleles of human GALT
(R67C, S135L, L139P, V151A, F171S, P183T, Q188R, R201H, R231H, R259W,
K285N, E291K, N314D, R333W, Y323D, and T350A). In particular, we
characterized each allele in terms of both abundance and activity of
the encoded human GALT protein. Three main groups of alleles were
identified: those with <1% wild-type activity, those with 1-5%
wild-type activity, and those with Plasmids--
All hGALT mutations were recreated by
site-directed mutagenesis of the otherwise wild-type sequence, as
described previously (15). The primers used to generate alleles R67C,
L139P, P183T, R201H, R231H, R259W, K285N, E291K, Y323D, and T350A
were hGR67CF (5'-GAAGACAGTGCCCTGCCATGACCCTCTC-3'), hGL139PF
(5'-GGATGTAACGCCGCCACTCATGTCG-3'), hGP183TF
(5'-GCTGTTCTAACACCCACCCCCACT-3'), hGR201HF
(5'-GATATTGCCCAGCATGAGGAGCGA-3'), hGR231H
(5'-TCAGGAAGGAACATCTGGTCCTAAC-3'), hGR259WF
(5'-GCTGCCCCGTTGGCATGTGCGGCGG-3'), hGK285NF
(5'-GCTCTTGACCAATTATGACAACCTC-3'), hGE291KF
(5'-GACAACCTCTTTAAGACGTCCTTTCC-3'), hGY323DF
(5'-CACGCTCATTACGACCCTCCGCTC-3'), hGT350AF
(5'-GAGGGACCTCGCCCCTGAGCAGGCT-3'), respectively. All resultant mutant
alleles were confirmed by dideoxy sequencing. Recreations of the
mutations S135L, V151A, F171S, Q188R, N314D, and R333W have been
described previously (14, 16-19).
For expression at low copy number, each allele was subcloned using the
enzymes EcoRI and SalI into the centromeric yeast
vector pMM22 (20). For expression at high copy number, alleles were subcloned into the 2-µm yeast vector pYEP-GAP (a generous gift of Dr.
Warren Kruger, Fox Chase Cancer Center). Both plasmids facilitate
expression of the introduced open reading frame from the constitutive
yeast GAP promoter.
Yeast Strains and Manipulations--
All yeast manipulations
were carried out according to standard techniques as described
previously (21). All YEP-GAP and MM22 plasmids were transformed into
yJFK1, a previously described haploid strain of Saccharomyces
cerevisiae deficient in GAL7, the endogenous yeast GALT
(14). Transformants were selected and maintained on the basis of
tryptophan prototrophy, conferred by the plasmid. Except where
otherwise noted, cells were cultured in media containing dextrose as
the sole carbon source to prevent any selective pressure for GALT activity.
Soluble cell lysates were prepared from 30-ml cultures grown at
30 °C to A600 = 1.5, essentially as
described previously (14, 22). Briefly, cell pellets were resuspended
in 500 µl of lysis buffer (20 mM HEPES, 1 mM
dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.3 mM
aprotinin, 1 mM pepstatin A, 2 mM antipain, 1 mM phosphoramidon, 0.2 µg/ml chymostatin, 8 mM E64, and 1 mM phenylmethylsulfonyl fluoride)
and transferred to 2-ml tubes. 100 µl of acid-washed glass beads
(0.5-mm diameter) were added to each tube, and the cells were disrupted
with 6 cycles of agitation (45 s on high followed by 45 s on ice)
using a multihead Vortex at 4 °C. Each disrupted cell suspension was
then transferred to a 1.5-ml tube and centrifuged in a
microcentrifuge on high speed for 10 min at 4 °C to pellet
insoluble material. Finally, each clarified supernatant was transferred
to a fresh tube and assessed for protein concentration using the
Bio-Rad protein assay reagent, as recommended by manufacturer, with
bovine serum albumin as a standard.
GALT Activity Assays--
GALT activity was determined in
soluble yeast lysates as described previously (14, 22). For lysates
prepared from yeast expressing low copy number plasmids (MM22
derivatives), between 1 and 20 µg of total protein, adjusted to
maintain linearity of the assay, was included in each reaction. 1 µg
of protein was used for assays of wild-type hGALT under these
conditions. For lysates prepared from yeast expressing high copy number
plasmids (YEP-GAP derivatives), between 0.3 and 180 µg of total
protein, adjusted to maintain linearity of the assay, was included in
each reaction. 0.3-0.5 µg of protein was used for assays of
wild-type hGALT under these conditions. All assays were performed in
triplicate (or greater) as indicated (Table I), representing extracts
prepared from independent colonies, and adjusted according to total
protein before being normalized to the appropriate wild-type values.
Western Blot Analyses--
Western blot analyses were performed
as described previously (18). SDS-polyacrylamide electrophoresis gels
to be blotted were loaded with 5 µg/lane of protein
representing yeast expressing low copy number plasmids (MM22
derivatives) and either 1 µg (wild-type) or 5 µg (mutants) of
protein/lane representing yeast expressing high copy number
plasmids (YEP-GAP derivatives). Both wild type and mutant forms of
human GALT were detected using a rabbit polyclonal antiserum raised
against hexahistidine-tagged hGALT at a dilution of 1:100,000. As a
control for loading, blots also were probed with an antiserum against
the endogenous yeast protein cyclophilin (at a dilution of 1:30,000)
(23). Signals were visualized using a horseradish peroxidase-conjugated
antiserum against rabbit Ig (Amersham Pharmacia Biotech, 1:5000
dilution) followed by reaction with the enhanced chemiluminescence
(ECL) system from Amersham Pharmacia Biotech, as recommended by the manufacturer.
Sensitivity of Yeast to Galactose--
Cultures inoculated from
colonies were grown initially in synthetic medium containing 2%
dextrose overnight to an A600 between 1 and 2. Cells were then diluted into 6 ml of fresh medium containing 2%
glycerol, 2% ethanol in place of dextrose at an
A600 = 0.1. These cultures were allowed to grow
to an A600 of about 1, at which point they were
again diluted in duplicate into 6 ml of fresh synthetic medium
containing 2% glycerol, 2% ethanol at an A600 = 0.1 to begin growth curves. Time points were taken at 0, 7, 22, and
31 h, at which point the A600 of each
culture was close to 0.4. At that point, galactose was added to a final
concentration of 0.05% to one culture from each pair, and all tubes
were returned to the rotator at 30 °C. Finally, measurements of
A600 were followed for each culture periodically
over the next 2-4 days, as indicated (Figs. 2-4).
Gal-1-P Measurements--
Samples were prepared from duplicate
30-ml cultures of cells grown as described above (before the addition
of galactose). At 31 h after inoculation, a 6-ml sample was
removed from each culture (zero time point), and galactose was added to
the remaining volume of one culture from each pair to a final
concentration of 0.05%. At 7, 15, and 63 h after the addition of
galactose, 6-ml samples from each culture were harvested, pelleted, and
frozen. Finally, cell pellets were lysed as described above, except
that protease inhibitors were not included in the lysis buffer. Protein concentrations were measured, as described above, using the Bio-Rad protein assay reagent as recommended by manufacturer, with bovine serum
albumin as a standard.
Next, each lysate was cleared of proteins by vigorous extraction with
methanol (500 µl of methanol/200 µl of aqueous sample) followed by
centrifugation to pellet the protein precipitate. Finally, each
clarified supernatant was transferred to a fresh 1.5-ml tube and dried
under vacuum. Pellets were resuspended in sterile, deionized water for
further analysis.
Gal-1-P levels were quantitated using a coupled spectrophotometric
assay described previously (17, 22) with slight modifications. In
particular, the assay buffer contained 100 mM
glycylglycine, pH 8.7, 6 mM dithiothreitol, 5 µM glucose-1,6-diphosphate, 5 mM MgCl2, 0.8 mM NADP, 0.1 µg of
phosphoglucomutase, and 0.06 µg of glucose-6-phosphate dehydrogenase
in a total reaction volume of 400 µl. All assays were carried out
using 300 ng of purified His6-tagged hGALT isolated from a
yeast expression system. A standard curve was generated using
UDP-glucose at a concentration of 0.6 mM with varying
concentrations of galactose 1-phosphate (0.075-1.2 mM). To
determine the levels of galactose 1-phosphate present in each test
sample, 50 µl of deproteinated extract (representing 20-416 µg of
original lysate protein) were used in place of a known quantity of
galactose 1-phosphate so that the amount of gal-1-P present in that
sample could be determined by interpolation from the standard curve.
Final values presented (Figs. 3 and 4) were normalized according to
total protein in each original extract.
To ensure that our measurements of gal-1-P were comparable with those
of other groups using the method of Bergmeyer (24), which utilizes
alkaline phosphatase and galactose dehydrogenase, we assayed a set of
test samples, some with high gal-1-P and others with low gal-1-P, by
both methods. In all cases comparable values were obtained from both
assays (data not shown).
UDP-gal and UDP-glc Measurements--
Samples were prepared from
cultures of yeast expressing either no GALT or wild-type human GALT
grown in the presence versus absence of galactose,
harvested, lysed, and cleared of proteins, as described above. UDP-gal
was measured against a standard curve established using a coupled
reaction with purified UDP-gal-4 epimerase (a kind gift of Drs. Jim
Thoden and Hazel Holden, University of Wisconsin, Madison, WI)
and UDP-glc dehydrogenase (Sigma), as described previously (25).
UDP-glc was measured directly in each sample in the absence of
epimerase using UDP-glc dehydrogenase (Sigma) and also quantitated by
comparison with a standard curve. Final values presented (Fig. 4) were
normalized according to total protein in each original extract.
Expression and Analysis of Patient-derived Alleles of Human GALT in
Yeast--
We have used site-directed mutagenesis of the wild-type
human GALT sequence to recreate each of 16 naturally occurring
mutations: R67C, S135L, L139P, V151A, F171S, P183T, Q188R, R201H,
R231H, R259W, K285N, E291K, N314D, Y323D, R333W, T350A. After
confirmation, each allele was introduced into both low copy number
(CEN, MM22) and high copy number (2 µm, YEP-GAP) yeast expression
plasmids containing the constitutive GAP promoter and
transformed into the previously described null background strain of
S. cerevisiae, yJFK1 (14). Plasmids encoding the wild-type
hGALT sequence and empty plasmids alone were also included in all
experiments as positive and negative controls, respectively. Unless
otherwise noted, all cultures were maintained in dextrose-containing
medium to prevent selective pressure based on encoded hGALT activity.
To confirm expression and to determine the relative abundance of each
substituted hGALT protein in yeast, soluble lysates prepared from cells
expressing each hGALT allele from a centromeric plasmid were subjected
to Western blot analysis with the rabbit polyclonal anti-hGALT
antiserum, EU70. As a control for loading of lanes, each filter was
also probed with a polyclonal antiserum that recognizes yeast
cyclophilin (23), an abundant endogenous yeast protein. As illustrated
in Fig. 1, wild-type hGALT was readily detectable in this system, and the negative control was clean. A faint
cross-reacting band that runs just above the position of hGALT was also
visible in all lanes, providing an additional internal control for
loading. Twelve of the 16 substituted hGALT proteins demonstrated
abundance comparable with that of the wild-type protein, whereas 4 (V151A, R231H, R259W, K285N) were below the threshold of detection. To
increase sensitivity of the assay, lysates were prepared from yeast
expressing each of these four alleles from high copy number plasmids.
Western blot analyses of these samples (Fig. 1, right
panel), again in parallel with wild-type hGALT, also expressed
from a high copy number plasmid, confirmed that each of the four mutant
proteins was expressed, albeit at markedly decreased levels (>20-fold)
relative to the wild-type protein.
Lysates representing each hGALT allele expressed from both low copy and
high copy number plasmids also were analyzed for GALT activity using a
standard in vitro assay, as described under "Experimental Procedures." As reported previously, lysates from yJFK1 expressing no
hGALT demonstrated no detectable GALT activity in these assays (Ref. 14
and data not shown). As illustrated in Table
I, the 16 mutant alleles tested displayed
a spectrum of activity levels ranging from below the threshold of
detection to nearly wild-type. Grouped roughly according to in
vitro activity, six (F171S, Q188R, R231H, R259W, K285N, R333W)
were below the threshold of detection, four (R67C, S135L, L139P, V151A)
displayed low but detectable activities (between 1 and 5% wild-type
levels), and six (P183T, R201H, E291K, N314D, Y323D, T350A) displayed
close to 10% or greater wild-type levels of activity.
Sensitivity of Yeast Expressing Mutant Alleles of Human GALT to
Galactose--
More than 30 years ago, Douglas and Hawthorne (13)
demonstrated that yeast deficient in gal7, the endogenous
yeast GALT, were sensitive to the presence of low levels of galactose
added to their culture medium despite the presence of other
metabolizable carbon sources such as glycerol and ethanol. We have
repeated these results with our strains and applied this system to
probe the relationship between hGALT activity and galactose toxicity in yeast.
In brief, yJFK1 expressing wild-type hGALT, no GALT, or each of the 16 substituted hGALT alleles described above were grown in duplicate
cultures of synthetic medium containing glycerol/ethanol as the carbon
source. At ~31 h after inoculation, when all cultures were growing
well (A600 ~ 0.4), galactose was added to
0.05% final concentration to one of each of the pairs of cultures. All
cultures were then returned to incubation at 30 °C with periodic
monitoring of A600 over the course of ~4 days.
As expected, all cultures grew indistinguishably in the absence of
galactose (data not shown). In the presence of galactose, however,
marked differences were apparent in the growth profiles of the
different strains (Fig. 2), and these
correlated well with the levels of in vitro GALT activity
associated with each strain.
In particular, those strains expressing Relationship between Galactose Sensitivity and Metabolite
Accumulation in Yeast--
As an initial step toward probing the
biochemical basis of galactose sensitivity in yeast, we repeated growth
studies of two cultures representing each of the three hGALT activity
groups described above (high, intermediate, and very low) and monitored intracellular gal-1-P levels at four time points after the addition of
galactose to each culture. The times plotted (Fig.
3) correspond to 0, 7, 15, and 63 h
after the addition of galactose. As illustrated in Figs. 3,
A and D, cells expressing high levels of GALT
activity (wild type or T350A) demonstrated no detectable accumulation
of gal-1-P at any time. Cells expressing intermediate (1-5% wild type) levels of GALT activity (S135L and L139P) demonstrated no detectable gal-1-P at 0 h but did show accumulation of gal-1-P at
the 7 and 15 h time points. By 63 h after the addition of
galactose, again no gal-1-P was detected in these cells. Finally,
cultures expressing no detectable GALT activity (null or Q188R)
demonstrated no detectable gal-1-P at 0 h but marked accumulation
of the metabolite at both the 7 and 15 h time points. By 63 h
after the addition of galactose, the gal-1-P levels in both cultures
had dropped precipitously, although both remained within the detectable
range. As expected, in duplicate samples of all cultures that received no galactose, no gal-1-P was detected at any time point tested (data
not shown).
As an additional test of potential metabolite imbalance, we grew
cultures of yeast expressing either no GALT or wild-type human GALT in
medium containing 2% glycerol, 2% ethanol to early log phase and then
added galactose to 0.05% to half of the cultures and harvested time
points, as described above. Measurements of gal-1-P, UDP-gal, and
UDP-glc were performed as described under "Experimental
Procedures." Consistent with clinical reports (2), we observed a
clear decrease in UDP-gal levels in samples prepared from
GALT-deficient yeast exposed to galactose but not in corresponding samples prepared from yeast expressing wild-type hGALT (Fig.
4, panels E and F).
In contrast, only minor changes in the levels of UDP-glc were observed
in these same samples (Fig. 4, panels G and H).
As expected, gal-1-P accumulated to high levels only in the
GALT-deficient cells exposed to galactose (Fig. 4, panels C
and D).
The results reported here are significant for two reasons. First,
although many patient-derived alleles of human GALT have been
identified (5), few have been well characterized in terms of degree or
nature of resulting enzyme impairment. Of the 16 mutant alleles studied
here, although 7 have been analyzed previously (S135L (19, 26-28),
V151A (19), F171S (16, 26), Q188R (14, 17, 29-32), R231H (33), N314D
(18, 34), and R333W (17, 29, 30)), nine have not (R67C, L139P, P183T,
R201H, R259W, K285N, E291K, Y323D, T350A). These results therefore
represent the first clear demonstration that these nine mutations are
functionally significant and not simply polymorphisms that occur in
linkage with some distinct but unknown causal mutation.
Second, the data reported here present the first clear evidence in a
genetically and biochemically amenable model system of a relationship
between hGALT genotype, encoded enzyme activity, in vivo
galactose toxicity, and aberrant metabolite accumulation. These data
lay a foundation for future studies into the biochemical basis of
galactose toxicity in yeast and perhaps other eukaryotes, including humans.
hGALT Expression, Abundance, and Activity--
Our data presented
here concerning the expression, abundance, and activity of
patient-derived alleles of hGALT demonstrate that, as recognized
previously in patients (6, 7), not all hGALT alleles derived from
patients with galactosemia are completely null. They represent a
spectrum, albeit generally at the low end of activity.
Three-dimensional homology modeling of the human GALT sequence onto the
Escherichia coli GALT crystal structure (35) predicts that
all four mutations associated with marked reduction in protein abundance (V151A, R231H, R259W, and K285N) impact residues near the
surface of the protein. None of these residues is within 13 Å of
either the active site, the metals, or the dimer interface. Although
compromised stability seems likely, the actual cause of the low
abundance for each of these substituted proteins remains unknown.
Similarly, in terms of activity, with few exceptions, all of the
mutations associated with very low or no detectable GALT activity
impact either residues near the active site (S135L, L139P, F171S,
Q188R) or near the dimer interface (R333W) or represent low abundance
proteins (V151A, R231H, R259W, and K285N). One notable exception is
R67C. Homology modeling predicts that the Arg-67 residue lies in a loop
that protrudes from the surface of the GALT protein. The underlying
explanation for why this substitution causes such marked catalytic
impairment, but no apparent loss in abundance, remains unclear. Yeast
expressing this same mutation also exhibit anomalous galactose
sensitivity relative to the degree of catalytic impairment observed
in vitro, as will be discussed below.
Comparison with Prior Studies--
Comparisons between data
presented here and those reported elsewhere reveal that, to the
resolution of these studies, the yeast system recapitulates accurately
what has been seen in humans carrying the corresponding hGALT
mutations. For example, F171S (8), Q188R (8, 14, 36), R231H (33), R259W
(37), K285N (38), and R333W (8) have all been associated with null or
close to null GALT activity in hemolysates and/or lymphoblasts of
patients homozygous or compounds heterozygous for these mutations. In
contrast, the mutations R67C (39, 40), S135L (8, 27), and V151A (19)
have all been associated with low but nonzero activity in patient
samples. The substitutions P183T (41) and T350A (37) have both been
associated with milder GALT impairment in patient hemolysates and/or
lymphoblasts. In addition, with regard to N314D, recent reports have
described a collection of both coding and noncoding sequence variations
that are found in cis with this substitution and that
distinguish the mildly under-expressed Duarte (D2), from the mildly
over-expressed Los Angeles (LA, D1) GALT alleles (42, 43), both of
which carry N314D (Ref. 41; reviewed in Ref. 5). These data reinforce
the hypothesis that N314D may be a polymorphism (34) that causes the
electrophoretic mobility shift associated with both the Duarte and LA
alleles (18) but not the abundance/activity differences observed
between them (reviewed in Ref. 5). Finally, although R201H, L139P, E291K, and Y323D have all been identified in samples derived from patients with galactosemia, no data have been reported assigning specific GALT activity levels to these substitutions (9, 44).
Comparisons between model system-derived data and relevant patient data
are clearly important. Unfortunately, the fact that many galactosemia
mutations have been identified in patients only in the compound
heterozygous state confounds accurate assignment of catalytic
impairment to each allele. Modeling studies in COS cells and in yeast
have previously attempted to address this caveat. For several of the
substitutions modeled in both systems, comparable results were obtained
(e.g. F171S (16, 26), R333W (17, 29, 30), and N314D (18,
34)). For other substitutions, however, disparate results were observed
(S135L (19, 26, 28), Q188R (14, 29), and R231H (33)). In each of these
cases, the data obtained in yeast more closely corresponded to those
derived from patient samples. The basis for the disparity between the
COS and yeast-derived results remains unclear; however, considering
that GALT functions as a dimer, one possibility may be interaction between the exogenous human protein and endogenous wild-type GALT subunits in the COS cells. The yeast system has been genetically modified to eliminate endogenous GALT protein (see "Experimental Procedures").
Galactose Sensitivity in Yeast--
Our results concerning an
inverse relationship between GALT activity in vitro and
galactose sensitivity in vivo are fully consistent with
prior studies of galactosemia patients (6) and demonstrate several
points. First, although we have presented the data (Fig. 2) in terms of
low, intermediate, and higher activity alleles, these are groupings of
convenience. The alleles themselves represent a spectrum, as does the
degree of galactose sensitivity observed, with an inverse correlation
between the two. Additional studies will be required to define more
accurately the exact nature of this relationship.
Second, with regard to the "intermediate" alleles demonstrating
between 1-5% wild-type activity (Fig. 2, middle panel),
the galactose sensitivity observed is transient. By about the 96-h time
point, the cultures begin to recover, resuming growth rates reminiscent
of the "higher activity" cultures. Two possible scenarios may
explain this apparent recovery, either (a) the environment has changed (presumably galactose in the cultures has been consumed over time and fallen below some threshold of toxicity) or
(b) the cells have changed (they have adapted). Studies are
currently under way to distinguish between these possibilities.
The third point demonstrated by the data in Fig. 2 is that there are
exceptions to the relationship between GALT activity measured in
vitro and sensitivity to galactose measured in vivo; R67C is a case in point. The explanation for why this allele exhibits intermediate levels of activity in vitro but behaves more
like a null allele in vivo will be a focus of future study.
The implication that GALT activity measured in vitro under
defined buffer conditions with excess substrates may not accurately
reflect function in vivo is not only possible but likely.
Other options, such as disrupted enzyme sequestration (45) or
macromolecular interactions normally required for function in
vivo, may also contribute to the discrepancy and will be explored.
Metabolite Accumulation and Galactose Sensitivity--
The results
presented in Figs. 3 and 4 illustrating a relationship between
galactose sensitivity and aberrant accumulation of gal-1-P and UDP-gal
in yeast are consistent with earlier patient studies (2, 6) and also
raise several important questions. First, are elevated levels of
gal-1-P or depleted levels of UDP-gal actually causal in mediating
galactose toxicity in either yeast or humans, or are these changes
merely correlated? Future studies will be required to distinguish
between these possibilities. It is important to note that studies of a
mouse GALT knock-out model have demonstrated, at least in that system,
that the biochemical absence of GALT does not always result in clinical
pathology despite accumulation of gal-1-P (46, 47). It is particularly
interesting to note, however, that GALT-deficient mice maintained on a
diet including galactose did not demonstrate any detectable depletion of UDP-gal (47) relative to their wild-type counterparts, whereas both
humans (2) and yeast (Fig. 4) do. Another important consideration involves aldose reductase, an enzyme that converts galactose to galactitol that is normally expressed in humans at significantly higher
levels than in mice. Ai et al. (48) recently reported studies of a mouse knock-out model for galactokinase deficiency galactosemia demonstrating that, despite the absence of galactokinase and abnormal accumulation of representative metabolites, these mice
failed to exhibit cataracts (the predominant feature of the human
disorder) until a human aldose reductase transgene was introduced into
the mouse genetic background. Clearly, a similar modification of the
GALT-deficient mouse may also impact outcome. It should be noted that
yeast do express aldose reductase (GRE3). Further studies
will be required to explain the underlying biochemical basis of the
apparent outcome differences observed between the mouse, human, and
yeast GALT-deficient systems.
A second question concerns the quantitative relationship between
gal-1-P levels and culture growth rates in yeast. It is important to
recognize that although the gal-1-P levels detected in yeast null for
GALT at the latest time point measured (Fig. 3, panel F) are
markedly lower than those detected earlier in the experiment, they are
nonzero. In contrast, yeast expressing intermediate activity alleles of
hGALT (panel E) had no detectable gal-1-P at the
corresponding time point. This small but significant difference may
account for the absence of clear growth in the GALT-null cultures
within the time frame of the experiment. It is also important to note, however, that as illustrated in panels B and E,
there was a lag of close to 48 h between loss of detectable
gal-1-P and recovery of growth in the cultures. Further studies will be
required to explore more fully the relationship between gal-1-P or
other metabolites and galactose toxicity in yeast.
A final question raised by the data presented here (Fig. 3) concerns
the observation that even in yeast expressing no detectable GALT
activity, although gal-1-P levels rise precipitously upon exposure to
galactose, they do come down again. Prior studies (31, 32) report
minute, but nonzero, levels of activity associated with Q188R-hGALT and
with its Q168R E. coli GALT counterpart. Although this
residual activity may be invoked to explain the decline of accumulated
gal-1-P over time in yeast expressing Q188R-hGALT, the same decline
also was observed in yeast completely devoid of GALT (panel
F). These data suggest that some GALT-independent pathway for the
metabolism of gal-1-P must exist in yeast. This observation is
consistent with the recent results of others (49-51), although the
biological significance of this alternative pathway under normal
(GALT-proficient) conditions remains to be clarified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
~10% wild-type activity.
Monitoring the impact of galactose exposure on strains of yeast
expressing each mutant allele, we observed that, with few exceptions,
yeast expressing the lowest activity hGALT proteins demonstrated the
most significant sensitivity to galactose and the most prolonged
accumulation of gal-1-P, an indicator of galactose metabolic imbalance.
Strains expressing the intermediate activity alleles of hGALT
demonstrated intermediate galactose sensitivity and transient gal-1-P
accumulation. Finally, those strains expressing the highest activity
alleles of hGALT demonstrated no sensitivity to galactose and no
detectable accumulation of gal-1-P. Furthermore, studies of UDP-gal and
UDP-glc accumulation in samples prepared from yeast expressing
wild-type human GALT versus no GALT showed a specific and
significant loss of UDP-gal, but not UDP-glc, only in the
GALT-deficient cells in response to the addition of galactose. These
results present the first clear evidence in a biochemically and
genetically amenable model system of a relationship between hGALT
genotype, encoded enzyme activity measured in vitro,
aberrant metabolite accumulation, and sensitivity to galactose measured
in vivo. As such, these data lay a foundation for future
studies into the underlying mechanism(s) of galactose toxicity in yeast
and perhaps other eukaryotes, including humans.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Western blot analyses of soluble whole cell
lysates from yeast expressing human GALT. 5 µg of total protein
derived from cells expressing each of the indicated hGALT alleles from
low copy number plasmids were loaded in each lane of the
gels (left and middle panels). 1 µg of total
protein (WT) or 5 µg (all others) derived from cells
expressing each of the indicated hGALT alleles from high copy number
plasmids were loaded in each lane of the gel depicted in the
panel on the right. The lower sections (cyc) of
both panels are presented as loading controls, as described under
"Experimental Procedures." CEN,
centromere.
Activity assays of soluble lysates from yeast expressing the indicated
alleles of human GALT
~10%
activity were determined using cells expressing the indicated hGALT
alleles from centromeric plasmids. In each case activity values
associated with the mutant hGALT proteins were normalized against
wild-type hGALT expressed from the same vector backbone.
View larger version (20K):
[in a new window]
Fig. 2.
Galactose sensitivity of gal7
yeast expressing patient alleles of human GALT. Yeast
expressing each of the indicated alleles of hGALT were cultured in
synthetic medium containing glycerol/ethanol, with galactose added to
0.05% final concentration at 31 h (arrows). Growth was
monitored for each culture by A600. Yeast
expressing higher activity alleles of hGALT are presented in the
left-most panel, yeast expressing intermediate activity
alleles (+ wild type (WT) as a control) are presented in the
middle panel, and yeast expressing the lowest activity
alleles (+WT as a control) are presented in the
right-most panel. All values plotted represent averages ± S.D. (n = 3). Parallel samples of all cultures
maintained in medium containing dextrose grew well (data not
shown).
~10% GALT activity
(left panel) grew well despite the addition of galactose to
their medium, although the two lowest GALT activity strains in this set, Y323D and T350A, did show a transient slow down in growth. In
contrast, cells expressing mutant hGALT alleles associated with between
1 and 5% wild-type activity demonstrated a marked slow down in growth
rate after the addition of galactose that lasted for several days (Fig.
2, center panel). All but one of these cultures (R67C)
eventually resumed essentially wild-type growth rates. Finally, cells
expressing either no GALT or inactive alleles of hGALT demonstrated a
profound and prolonged cessation of growth after the addition of
galactose. These cultures did not recover to normal growth rates within
the time frame of the experiment (Fig. 2, right panel).
View larger version (18K):
[in a new window]
Fig. 3.
Transient accumulation of gal-1-P in yeast
experiencing galactose toxicity. As in Fig. 2, yeast devoid of
endogenous gal7 but expressing the indicated patient alleles
of hGALT were cultured in synthetic medium containing glycerol/ethanol,
with galactose added to 0.05% final concentration at 31 h
(arrows). In addition to monitoring
A600 at the indicated times, samples of each
culture were harvested and analyzed for intracellular gal-1-P
accumulation. As illustrated, cells expressing the higher activity
alleles of hGALT demonstrated no measurable accumulation of gal-1-P,
whereas those cells expressing either intermediate and lower activity
alleles exhibited notable accumulations of gal-1-P.
View larger version (26K):
[in a new window]
Fig. 4.
Impact of galactose exposure on accumulation
of UDP-gal and UDP-glc in yeast. As in Fig. 3, yeast expressing
either no GALT or wild-type hGALT were cultured in synthetic medium
containing glycerol/ethanol, with galactose added to half of the
cultures at 0.05% final concentration at 40 h
(arrows). In addition to monitoring
A600 at the indicated times (panels A
and B), samples of each culture also were harvested and
analyzed for intracellular levels of gal-1-P (panels C and
D), UDP-gal (panels E and F), and
UDP-glc (panels G and H). In all
panels, samples representing cultures with galactose are
represented by open circles, and samples representing
cultures without galactose are represented by filled
circles. All values plotted represent average ± S.D.
(n = 3). In panel C, samples representing
yeast cultured in both the presence and absence of galactose are
plotted, although only one set is visible because the values at each
time point were coincident (all zero). Similarly, gal-1-P measurements
at the 40-h time point (panel D) representing yeast cultured
in both the presence and absence of galactose were both zero, so only
one is visible in the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Matt Rodeheffer and Kate Murphy for technical contributions in the early stages of this project and to Alice Watson for assistance with the molecular modeling work discussed here.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK46403 (to J. L. F.-K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work and should be considered co-first authors.
To whom correspondence should be addressed: Dept. of Genetics,
Emory University School of Medicine, Rm. 431C Dental Bldg., 1462 Clifton Rd., NE, Atlanta, GA 30322. Tel.: 404-727-3924; Fax: 404-727-3949; E-mail: jfridov@emory.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009583200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GALT, galactose-1-phosphate uridylyltransferase; hGALT, human GALT; gal-1-P, galactose 1-phosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Frey, P. A.
(1996)
FASEB J.
10,
461-470 |
2. | Holton, J. B., Walter, J. H., Tyfield, L. A. (2000) in Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, S. W., Valle, D., Eds., A., Childs, B., Kinzler, K. W., and Vogelstein, B., Assoc. eds) McGraw-Hill Inc., New York, pp. 1553-1587 |
3. | Segal, S., and Berry, G. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. , Beaudet, A. , Sly, W. , and Valle, D., eds) , pp. 967-1000, McGraw-Hill, Inc., New York |
4. | Waggoner, D. D., Buist, N. R. M., and Donnell, G. N. (1990) J. Inherited Metab. Dis. 13, 802-818[Medline] [Order article via Infotrieve] |
5. | Tyfield, L., Reichardt, J., Fridovich-Keil, J., Croke, D. T., Elsas, L. J., Strobl, W., Kozak, L., Coskun, T., Novelli, G., Okano, Y., Zekanowski, C., Shion, Y., and Boleda, M. D. (1999) Hum. Mutat. 13, 417-430[CrossRef][Medline] [Order article via Infotrieve] |
6. | Ng, W. G., Xu, Y.-K, Kaufman, F. R., Lee, J. E. S., and Donnell, G. N. (1991) Neonatal Screening in the Nineties in 8th International Neonatal Screening Symposium and Inaugural Meeting of the International Society for Neonatal Screening (Wilcken, B. , and Webster, D., eds) , pp. 181-188, Leura, AustraliaNov. 12-15, 1991 |
7. | Xu, Y.-K, Kaufman, F. R., Donnell, G. N., and Ng, W. G. (1995) Clin. Chim. Acta 235, 125-136[CrossRef][Medline] [Order article via Infotrieve] |
8. | Wang, B. B. T., Xu, Y.-K, Ng, W. G., and Wong, L.-J. C. (1998) Mol. Genet. Metab. 63, 263-269[CrossRef][Medline] [Order article via Infotrieve] |
9. | Elsas, L. J., Langley, S., Paulk, E. M., Hjelm, L. N., and Dembure, P. P. (1995) Eur. J. Pediatr. 154, (7 Suppl. 2), S21-S27[Medline] [Order article via Infotrieve] |
10. | Hirokawa, H., Okano, Y., Asada, M., Fujimoto, A., Suyama, I., and Isshiki, G. (1999) Eur. J. Hum. Genet. 7, 757-764[Medline] [Order article via Infotrieve] |
11. | Kaufman, F. R., Reichardt, J. K., Ng, W. G., Xu, Y. K., Manis, F. R., McBride-Chang, C., and Wolff, J. A. (1994) J. Pediatr. 125, 225-227[Medline] [Order article via Infotrieve] |
12. |
Shield, J. P.,
Wadsworth, E. J.,
MacDonald, A.,
Stephenson, A.,
Tyfield, L.,
Holton, J. B.,
and Marlow, N.
(2000)
Arch. Dis. Child.
83,
248-250 |
13. |
Douglas, H. C.,
and Hawthorne, D. C.
(1964)
Genetics
49,
837-844 |
14. | Fridovich-Keil, J. L., and Jinks-Robertson, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 398-402[Abstract] |
15. | McClary, J. A., Witney, F., and Geisselsoder, J. (1989) Biotechniques 7, 282-289[Medline] [Order article via Infotrieve] |
16. |
Crews, C.,
Wilkinson, K. D.,
Wells, L.,
Perkins, C.,
and Fridovich-Keil, J. L.
(2000)
J. Biol. Chem.
275,
22847-22853 |
17. |
Elsevier, J. P.,
Wells, L.,
Quimby, B. B.,
and Fridovich-Keil, J. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7166-7171 |
18. | Fridovich-Keil, J. L., Quimby, B. B., Wells, L., Mazur, L. A., and Elsevier, J. P. (1995) Biochem. Mol. Med. 56, 121-130[CrossRef][Medline] [Order article via Infotrieve] |
19. | Fridovich-Keil, J. L., Langley, S. D., Mazur, L. A., Lennon, J. C., Dembure, P. P., and Elsas, L. J. (1995) Am. J. Hum. Genet. 56, 640-646[Medline] [Order article via Infotrieve] |
20. |
Henderson, J. M.,
Wells, L.,
and Fridovich-Keil, J. L.
(2000)
J. Biol. Chem.
275,
30088-30091 |
21. | Guthrie, C., and Fink, G. (1991) Methods Enzymol. 194, 281-301[Medline] [Order article via Infotrieve]; 373-389 |
22. |
Quimby, B. B.,
Wells, L.,
Wilkinson, K. D.,
and Fridovich-Keil, J. L.
(1996)
J. Biol. Chem.
271,
26835-26842 |
23. |
Zydowsky, L. D.,
Ho, S. I.,
Baker, C. H.,
McIntyre, K.,
and Walsh, C. T.
(1992)
Protein Sci.
1,
961-969 |
24. | Bergmeyer, H. (ed) (1974) Methods of Enzymatic Analysis , pp. 1291-1295, Academic Press, Inc., New York |
25. | Wohlers, T. M., and Fridovich-Keil, J. L. (2000) J. Inherited Metab. Dis. 23, 713-729[CrossRef][Medline] [Order article via Infotrieve] |
26. | Reichardt, J. K. V., Levy, H. L., and Woo, S. L. (1992) Biochemistry 31, 5430-5433[Medline] [Order article via Infotrieve] |
27. | Lai, K., Langley, S. D., Singh, R. H., Dembure, P. P., Hjelm, L. N., and Elsas, L. J. (1996) J. Pediatr. 128, 89-95[Medline] [Order article via Infotrieve] |
28. | Wells, L., and Fridovich-Keil, J. L. (1997) J. Inherited. Metab. Dis. 20, 633-642[CrossRef][Medline] [Order article via Infotrieve] |
29. | Reichardt, J., Packman, S., and Woo, S. (1991) Am. J. Hum. Genet. 49, 860-867[Medline] [Order article via Infotrieve] |
30. |
Elsevier, J. P.,
and Fridovich-Keil, J. L.
(1996)
J. Biol. Chem.
271,
32002-32007 |
31. | Geeganage, S., and Frey, P. A. (1998) Biochemistry 37, 14500-14507[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Lai, K.,
Willis, A.,
and Elsas, L.
(1999)
J. Biol. Chem.
274,
6559-6566 |
33. | Ashino, J., Okano, Y., Suyama, I., Yamazaki, T., Yoshino, M., Furuyama, J.-I, Lin, H.-C, Reichardt, J. K. V., and Isshiki, G. (1995) Hum. Mutat. 6, 36-43[Medline] [Order article via Infotrieve] |
34. | Reichardt, J. K. V., and Woo, S. L. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2633-2637[Abstract] |
35. | Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569[CrossRef][Medline] [Order article via Infotrieve] |
36. | Leslie, N. D., Immerman, E. B., Flach, J. E., Florez, M., Fridovich-Keil, J. L., and Elsas, L. J. (1992) Genomics 14, 474-480[Medline] [Order article via Infotrieve] |
37. | Shin, Y. S., Gathof, B. S., Podskarbi, T., Sommer, M., Giugliani, R., and Gresser, U. (1996) Eur. J. Pediatr. 155, 393-397[CrossRef][Medline] [Order article via Infotrieve] |
38. | Zekanowski, C., Radomyska, B., and Bal, J. (1999) J. Inherited Metab. Dis. 22, 679-682[CrossRef][Medline] [Order article via Infotrieve] |
39. | Shin, Y. S., Zschocke, J., Das, A. M., and Podskarbi, T. (1999) J. Inherited Metab. Dis. 22, 327-329[CrossRef][Medline] [Order article via Infotrieve] |
40. | Sommer, M., Gathof, B. S., Podskarbi, T., Giugliani, R., Kleinlein, B., and Shin, Y. S. (1995) J. Inherited Metab. Dis. 18, 567-576[Medline] [Order article via Infotrieve] |
41. | Ninfali, P., Bresolin, N., Dallapiccola, B., and Novelli, G. (1996) J. Neurol 243, 102-103[Medline] [Order article via Infotrieve] |
42. | Andersen, M. W., Williams, V. P., Sparkes, M. C., and Sparkes, R. S. (1984) Hum. Genet. 65, 287-290[Medline] [Order article via Infotrieve] |
43. | Shin, Y. S., Koch, H. G., Kohler, M., Hoffmann, G., Patsoura, A., and Podskarbi, T. (1998) J. Inherited Metab. Dis. 21, 232-235[CrossRef][Medline] [Order article via Infotrieve] |
44. | Elsas, L. J., Langley, S., Steele, E., Evinger, J., Fridovich-Keil, J. L., Brown, A., Singh, R., Fernhoff, P., Hjelm, L. N., and Dembure, P. P. (1995) Am. J. Hum. Gen. 56, 630-639[Medline] [Order article via Infotrieve] |
45. | Christacos, N. C., Marson, M., Riehman, K., Wells, L., and Fridovich-Keil, J. L. (2000) Mol. Genet. Metab. 70, 272-280[CrossRef][Medline] [Order article via Infotrieve] |
46. | Leslie, N. D., Yager, K. L., McNamara, P. D., and Segal, S. (1996) Biochem. Mol. Med. 59, 7-12[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Ning, C.,
Reynolds, R.,
Chen, J.,
Yager, C.,
Berry, G. T.,
McNamara, P. D.,
Leslie, N.,
and Segal, S.
(2000)
Pediatr. Res.
48,
211-217 |
48. |
Ai, Y,
Zheng, Z.,
O'Brien-Jenkins, A.,
Bernard, D. J.,
Wynshaw-Boris, T.,
Ning, C.,
Reynolds, R.,
Segal, S.,
Huang, K. T.,
and Stambolian, D. T.
(2000)
Hum. Mol. Genet.
9,
1821-1827 |
49. | Mehta, D. V., Kabir, A., and Bhat, P. J. (1999) Biochim. Biophys. Acta 1454, 217-226[Medline] [Order article via Infotrieve] |
50. | Kabir, M. A., Khanday, F. A., Mehta, D. V., and Bhat, P. J. (2000) Mol. Gen. Genet. 262, 1113-1122[Medline] [Order article via Infotrieve] |
51. | Lai, K., and Elsas, L. (2000) Biochem. Biophys. Res. Commun. 271, 392-400[CrossRef][Medline] [Order article via Infotrieve] |