(Received for publication, October 30, 1996, and in revised form, March 4, 1997)
From the Department of Pediatrics, ¶ Medical
Genetics and Neurology, University of Wisconsin Medical School, and
the Waisman Center, Madison, Wisconsin 53705
A mouse mutant with glutathionuria was discovered
by screening for amino acidurias in the progeny of
ethylnitrosourea-mutagenized mice. Total glutathione concentration was
increased in both blood and urine but decreased in liver homogenates
from affected mice. Glutathionuric mice exhibited lethargy, severe
growth failure, shortened life spans and infertility. -Glutamyl
transpeptidase activity was deficient in kidney homogenates of
glutathionuric mice. The glutathionuric phenotype in these mice is
inherited as an autosomal recessive trait. This mouse mutant will be a
useful animal model for the study of
-glutamyl transpeptidase
physiology and glutathione metabolism.
GSH is the most abundant cellular thiol and functions as the
principal reducing reagent in all cell types (1). A partial listing of
the antioxidative functions of GSH include: protection against
mitochondrial damage, protection against oxygen toxicity in the lung,
protection against lipid peroxidation, detoxification of electrophilic
compounds through conjugation, preservation of proper sulfide bonds in
proteins, a postulated function in anticarcinogenesis, and a role in
the immune system (2). GSH metabolism also provides a source of
cysteine for cells (3). -Glutamyl transpeptidase (
-GT; EC
2.3.2.2)1 catalyzes the initial step in the
degradation of GSH (4).
-GT is a key step in the
-glutamyl cycle
(5), a series of degradative and synthetic reactions that mediate
cellular GSH metabolism. Several reviews of
-GT physiology and
function have been published (4-6), but despite intensive
investigation, the exact role
-GT plays in GSH metabolism or its
putative contribution to renal amino acid transport have not been
definitively determined. Bound to secretory endothelial cell membranes
in several organs but predominantly in proximal renal tubule cells,
-GT participates in the transmembrane transport of GSH and in
interorgan GSH exchange (Fig. 1) (7). Meister (8)
proposed that
-GT also contributes to amino acid transport in the
proximal renal tubule through transpeptidation of GSH and subsequent
tubule cell uptake of
-glutamyl amino acids. In vivo
model systems that have lost
-GT activity are an exquisitely powerful tool for the study of
-GT function and its relationship to
GSH metabolism. Administration of chemical inhibitors of
-GT to
animals results in both glutathionuria and glutathionemia (9), but
chemically treated animal models are limited by several drawbacks including the temporary nature of inhibition and the difficulty of
long-term continuous inhibitor administration. Also, the degree and
specificity of enzyme inhibition in various tissues (particularly the
brain) of these chemically treated animals is unknown. Genetic
-GT
deficiency has been described in only five humans (6), and the effects
of various different disease states or environmental influences upon
-GT deficient individuals cannot be adequately evaluated given the
rarity of the disorder. A genetic animal model of total
-GT
deficiency overcomes the limitations of previously existing
experimental systems and provides a useful tool for the study of
-GT
function and GSH physiology.
N-Ethyl-N-nitrosourea (ENU) is the most potent
point mutagen known (10), yielding mutation rates up to 300 × 105 in mice, depending upon the specific locus tested.
ENU mutagenesis has been used by several laboratories to generate
mutant mouse strains that model specific human genetic diseases (11).
Since the report of phenylketonuria secondary to phenylalanine
hydroxylase deficiency in ENU-generated mouse mutants (12), our
laboratory has focused upon developing mouse models of other human
inborn errors of metabolism. To this end, we screen the progeny of
ENU-treated mice for metabolic abnormalities using a variety of urine
biochemical analyses. Using this protocol, we have previously isolated
a mouse strain with recessively inherited sarcosine dehydrogenase
deficiency (13). In this report, we describe a genetic mouse model of
autosomal recessively inherited
-GT deficiency developed by random
mutagenesis using ENU. These mice exhibit glutathionuria/emia, severe
growth failure, shortened life spans, and inability to reproduce. This strain, designated GGTenu1, provides a useful
experimental system for the study of
-GT physiology and GSH
metabolism.
All reagents were purchased from Sigma unless otherwise specified.
Mutagenesis and Metabolic Screening ProtocolMale C57Bl/6J mice were treated with 50 mg/kg body weight ENU weekly for a total of three doses according to the method of King et al. (14). Breeding and metabolic screening of potentially mutant progeny was carried out as described previously (13). Urine samples were collected on filter paper from all G3 progeny after weaning (21-28 days of age) following an overnight fast. Each urine sample was evaluated using a battery of qualitative chemical analyses including sodium cyanide-sodium nitroprusside reagent for the detection of disulfides and free sulfhydryls (15). Urine amino acids were examined with one-dimensional paper chromatography using butanol:acetic acid:water (12:3:5) solvent and detection with ninhydrin according to the method of Smith (16). Quantitative urine and plasma amino acid analysis and quantitative urinary organic acid analysis were performed in any mouse that had an abnormal result by amino acid paper chromatography. Quantitative urine and plasma amino acids were analyzed on a Beckman System 6300 automatic amino acid analyzer according to the methods of Slocum and Cummings (17). Quantitative urine organic acids were measured by trimethylsilyl derivatization followed by gas chromatography-mass spectrometry according to the method of Hoffmann et al. (18). Duplicate one-dimensional paper chromatograms were examined specifically for the presence of sulfur-containing amino acids using platinic iodide reagent (15) in any mouse that had an abnormal cyanide-nitroprusside screening test. Urine from the mouse mutant described in this report and the identity of the sulfur-containing compounds therein were further examined by two-dimensional high voltage thin layer chromatography with ninhydrin detection (15) in the laboratory of Dr. Vivian Shih, Massachusetts General Hospital, Boston, MA.
Tissue Glutathione ConcentrationTotal glutathione
concentration in urine, plasma, and tissues of control and
glutathionuric (GGTenu1) mice was determined
enzymatically using the method of Tietze (19). GSH reacts with the
sulfhydryl reagent 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB or
Ellman's reagent) to produce 2-nitro-5-thiobenzoic acid and its
glutathionyl adduct. Yeast glutathione reductase added to the reaction
mixture reduces GSSG in the presence of NADPH to form GSH which is then
free to react with DTNB. The rate of 2-nitro-5-thiobenzoic acid
production is measured by monitoring absorbance at 412 nm and is
proportional to the total GSH concentration in the solution. For the
assay, all reagents are prepared in 125 mM sodium
phosphate, pH 7.5, 6.3 mM sodium EDTA buffer. 700 µl of
0.3 mM NADPH, 100 µl of 6 mM DTNB, and 200 µl of sample are combined in a 1-ml cuvette and allowed to
equilibrate at 30 °C in a water bath for 5 min. The reaction was
initiated by adding 10 µl of 50 unit/ml yeast glutathione reductase,
and the increase in absorbance at 412 nm was recorded for 6 min at
30 °C. Total glutathione concentration in unknowns was calculated
from a standard curve of known GSSG concentrations varying from 0 to
0.02 mM but are reported as millimoles/liter GSH. The
absorbance at 412 nm does not appreciably change over 6 min without the
addition of glutathione reductase.
Urine samples for total glutathione measurement were collected from urine- soaked filter paper by NH4OH elution. Urine glutathione concentration was measured without further sample modification and was corrected for urine creatinine concentration. Blood was obtained by direct cardiac puncture and anticoagulated with 25 µl of 0.1 M EDTA. Plasma, collected by centrifugation, was deproteinized with 0.1 volume 35% w/v sulfosalicylic acid. Following centrifugation, total glutathione concentration was measured in the supernatant. Solid tissue samples were weighed and homogenized in 10 volumes of 10% w/v trichloroacetic acid with five up and down strokes of a Pro2000 tissue homogenizer (Pro Scientific, Inc.). Following centrifugation at 3000 × g, total glutathione concentration was measured in the supernatant and corrected for wet weight of the tissue sample.
Kidney-GT activities in whole kidney
homogenates of control (C57Bl/6J) and GGTenu1 mice
were determined by the method of Orlowski and Meister (20). 10% w/v
kidney homogenates were prepared in 0.1 M Tris-HCl, pH 9.0, with five strokes of a Pro2000 electric homogenizer while the samples
remained on ice. The samples were kept on ice or stored at
20 °C
until
-GT activity was measured. In this assay, the
-glutamyl
moiety of the artificial substrate,
-glutamyl-p-nitroanilide, is transferred to glycylglycine
by
-GT and the optically active product, p-nitroaniline,
is generated. 1.8 ml of 11.11 mM glycine-glycine + 2.78 mM
-glutamyl-p-nitroanilide in 0.1 M Tris-HCl, pH 9.0, is added to 10-500 µg of sample
protein in a 200-µl volume. The reaction is incubated at 37 °C for
10 min and then stopped with the addition of 50 µl of 50% w/v
trichloroacetic acid followed by 2 ml of 2 M acetic acid.
The absorbance of the sample at 410 nm is measured versus a
duplicate reaction to which trichloroacetic acid and acetic acid had
been added immediately to stop the reaction at time 0. The amount of
p-nitroaniline produced in the reaction was calculated using
the molar absorptivity of p-nitroaniline at 410 nm (
= 8.8 mM
1 cm
1) and
-GT
activity was expressed in terms of nanomoles of
p-nitroaniline produced/min/mg of protein. Protein
concentrations were determined by a modification of a bicinchoninic
acid method (Pierce) (21).
-GT was partially purified from wild type
Harlan Sprague Dawley rat, wild type C57Bl/6J mouse, and
GGTenu1 mouse kidney homogenates according to the
method of Hughey and Curthoys (22). Briefly, kidney microsomal
fractions were isolated by differential centrifugation and resuspended
in 0.1 M Tris buffer, pH 9.0, 1% Triton X-100. Alkaline
phosphatase (Sigma diagnostic kit) and
-GT activities were measured
on the microsomal fractions from each animal. Protein electrophoresis
was performed on SDS-10% polyacrylamide gels (23), and proteins were
transferred by electrophoretic elution (24) to polyvinylidene
difluoride membranes (Immobilon-P, Millipore).
-GT protein was
identified using polyclonal rabbit antisera (courtesy of Dr. Henry
Pitot) raised against purified rat
-GT. As a control, an integral
renal tubule membrane protein, the
1 subunit of Na,K-ATPase, was
detected on a duplicate immunoblot using polyclonal rabbit sera raised
against amino acids 152-340 of the rat Na,K-ATPase
1 subunit as
deduced from the cDNA sequence (Upstate Biotechnology, Lake Placid,
NY). On both blots, the primary antibodies were localized with alkaline
phosphatase-conjugated goat anti-rabbit IgG antibody (Sigma), and
alkaline phosphatase activity was detected using nitro blue
tetrazolium/bromochloroindolyl phosphate substrate (Pierce).
Male C57Bl/6J inbred mice were treated weekly with ENU by intraperitoneal injection for a total of three doses as described previously (14). After the mice had regained fertility, each was bred to C57Bl/6J females to generate five to ten G1 males. G1 (n = 31) males were bred with females to generate G2 offspring and then each of the G1 males were back-crossed with two or three of their G2 daughters to generate a total of 553 potentially homozygous mutant G3 mice. Urine samples from two male mice in a single sibship gave a positive test result with sodium cyanide-sodium nitroprusside reagent, indicating the presence of a disulfide or free sulfhydryl-containing compound in the urine. Qualitative urine amino acid analysis by one-dimensional paper chromatography and ninhydrin detection demonstrated a large red-purple spot with RF 0.13 and a smaller pink spot with RF 0.31 which are not present on the amino acid chromatograms of control mouse urine. The larger spot reacted with both the cyanide-nitroprusside and iodoplatinate reagents indicating the presence of sulfur in the compound. This large spot had the same RF and color on ninhydrin-staining as GSSG purchased from Sigma. The smaller pink spot corresponded to GSH, which was present at much lower concentrations than GSSG in the mutant mouse urine. Oxidized and reduced glutathione were also identified by two-dimensional high voltage electrophoresis of mutant mouse urine by Drs. Kimiyo Mogami and Vivian Shih, Massachusetts General Hospital, Boston, MA.
Quantitative urine amino acid analysis using the Beckman amino acid
analyzer (high pressure liquid chromatography) showed three abnormal
peaks on the chromatogram (Fig. 2). A single, sharp peak
with a retention time of 12.0 min co-chromatographed with both GSH and
-glutamylcysteine. A broad peak at 18-27 min corresponded to GSSG.
The third peak at 37 min was broad and smaller than the first two peaks
and has been tentatively identified as bis-
-glutamylcystine (
-glutamylcysteine disulfide), a substance which has also been detected in the urine of humans with
-GT deficiency (25). The method
of urine collection used in our screening protocol likely promoted
oxidation of GSH to GSSG (and of
-glutamylcysteine to bis-
-glutamylcystine), yielding the large amount of GSSG compared with GSH in most urine samples from the affected mice. Pretreatment of
mutant mouse urine with dithiothreitol resulted in complete disappearance of the broad peaks associated with GSSG and
bis-
-glutamylcystine and accentuation of the peak corresponding to
GSH and
-glutamylcysteine (data not shown).
Phenotype of the Affected Mice
At birth, glutathionuric mice
were indistinguishable from their unaffected litter mates. The total
number of offspring in litters which contained glutathionuric pups was
not significantly different from the size of control C57Bl/6J litters
(5-12 pups/litter) in our colony. By approximately 14 days of age,
glutathionuric pups appeared smaller than their litter mates, and at
weaning (3-4 weeks of age), the body weight of affected mice was
significantly lower (Fig. 3). Other than their size, the
mice at weaning appeared physically healthy and morphologically normal
(Fig. 4).
Severe growth failure persisted after weaning (Fig. 3). As the glutathionuric mice aged, their fur became rough and dull, but there was no change in coat color. The mice became less active, but were easily agitated. Many of the glutathionuric mice developed a severe thoracolumbar kyphosis. One severely kyphotic mouse developed paralysis of the hindlimbs, and two males suffered maceration of the penis due to priapism. Approximately 10% of the mice exhibit unilateral cataracts which are physically apparent by 2-3 months of age. A single mouse had bilateral cataracts. Unfortunately, neither male nor female glutathionuric mice have produced offspring. The line of glutathionuric mice has been maintained with considerable difficulty only by breeding unaffected siblings of glutathionuric mice which potentially are heterozygous for the glutathionuric trait.
Gross anatomic examination of several mice revealed no abnormalities except for small size of all organs in proportion to the small body size overall. In one mouse sacrificed at age 14 months, a large cystic mass completely replaced the right kidney. The right ureter, urinary bladder, and the left kidney and ureter appeared grossly normal. In another mouse sacrificed at age 15 months, a solitary solid tumor was found in the liver. Aortic perfusion with formalin-saline was performed on two male glutathionuric mice. Microscopic examination of most internal organs with hematoxylin-eosin staining was completely normal except for oligospermia apparent in the testes. Nissl staining of coronal sections from the lens of a single mouse confirmed the subcapsular location of a clinically apparent unilateral cataract. Histology of the contralateral lens in that mouse was normal. The brain and spinal cord of two mice were sectioned and examined with hematoxylin-eosin or Nissl staining. No anatomic abnormalities of the nervous system were detected. The hematocrit of four different glutathionuric mice was similar to that of control mice, ranging from 50-55%. The microscopic appearance of Wright-stained peripheral blood smears from glutathionuric mice was normal compared with blood smears from control C57Bl/6J mice. Neither male nor female glutathionuric mice have successfully produced offspring. The mean age of glutathionuric mice (n = 60) at death was 242 ± 15 days (±S.E.) with a range of 70-512 days, while the phenotypically normal siblings of glutathionuric mice (n = 88) lived an average of 339 ± 22 days (p < 0.001) with a range of 74-929 days.
Quantitative Amino Acid AnalysisIn addition to the large amount of glutathione detected, quantitative urine amino acid analysis revealed slight elevations of many amino acids including threonine, glycine, cystine, isoleucine, leucine, ornithine, and lysine compared with control mouse urine samples (Table I). This may indicate a mild generalized defect of amino acid reabsorption in the renal tubules of glutathionuric mice. Additionally, taurine, a sulfur-containing amino acid that is usually the most abundant amino acid in control mouse urine, was consistently low in urine from glutathionuric mice. Plasma cystathionine levels were slightly lower in mutant mice (3.1 ± 3.8 µmol/liter) than in controls (8.5 ± 1.8 µmol/liter), but no other significant differences in plasma amino acid concentrations were detected. Specifically, the plasma levels of other sulfur-containing amino acids including methionine and non-protein-bound cystine were normal in glutathionuric mice. Quantitative amino acid analysis was performed on trichloroacetic acid extracts of liver, kidney, and brain of glutathionuric and control mice. No significant differences in any amino acids including free cystine were detected (data not shown).
|
Using the method of Tietze (19), glutathione concentration was measured in urine and plasma, and trichloroacetic acid extracts of liver, kidney, and brain from glutathionuric and control mice (Table II). As expected, urine glutathione excretion was very elevated in the mutant mice. Plasma glutathione concentration was also elevated in the mutant mice. Total glutathione was also elevated in trichloroacetic acid extracts of kidney and brain from glutathionuric mice. However, total glutathione in liver extract from glutathionuric mice was significantly decreased compared with control mice.
|
-GT-specific activities were
measured in Tris-HCl extracts of whole kidney from glutathionuric and
control mice (Table III). Kidney
-GT activity of
glutathionuric mice was nearly 100-fold lower than
-GT activity
measured in control C57Bl/6J mice. Under reaction conditions similar to
those used to measure
-GT activity in kidney extracts from control
mice (25-50 µg of protein added to the reaction and 10 min of
incubation),
-GT activity was frequently undetectable in kidney
extracts from glutathionuric mice. Increasing the amount of
glutathionuric mouse kidney extract in the reaction to 500 µg of
protein allowed the measurement of a slight amount of
-GT activity.
Incubation of the reaction with glutathionuric mouse kidney extract at
37 °C for up to 4 h did not result in appreciably higher
p-nitroaniline production (data not shown). A 1:1 mixture of
kidney extracts from a glutathionuric mouse and from a control mouse
had measured
-GT activity equaling 50% of specific
-GT activity
measured in control extracts alone, indicating that kidney extract from
glutathionuric mice did not inhibit the
-GT reaction when active
enzyme was added. Kidney
-GT activity was also measured in four mice
that had produced glutathionuric offspring and were therefore carriers
of the glutathionuric trait. Mean kidney
-GT activity in the
carriers was 72% of that in control mice.
|
Kidney microsomal fractions from the Harlan
Sprague Dawley rat, C57Bl/6J mouse, and GGTenu1
mouse were analyzed for the presence of -GT protein by Western blot
analysis using polyclonal rabbit sera raised against purified rat
-GT protein. The specific activity of
-GT in each kidney microsomal fraction was 557 ± 10.1 nmol/min/mg of protein in wild type C57Bl/6J mouse, 64.6 ± 5.9 nmol/min/mg in
GGTenu1 mouse, and 2070 ± 365 nmol/min/mg in
the Harlan Sprague Dawley rat. The specific activity of alkaline
phosphatase, another renal tubule membrane-associated enzyme, in these
microsomal fractions was 788 ± 39.1 nmol/min/mg of protein in the
wild type C57Bl/6J mouse (ratio of
-GT to alkaline phosphatase
activity = 0.71), 839 ± 35.2 nmol/min/mg in the
GGTenu1 mouse (ratio = 0.071), and 703 ± 16.6 nmol/min/mg in the Harlan Sprague Dawley rat (ratio = 2.9).
Aliquots containing approximately 10 µg of total protein from each
microsomal fraction were loaded onto a 10% SDS-polyacrylamide gel.
Immunoblotting revealed two densely stained bands with apparent
molecular masses of approximately 55 and 35 kDa in the rat kidney
microsomal fraction (Fig. 5). This result agrees with
the immunoblot of purified rat
-GT obtained by Coloma and Pitot
(26). Two additional less intensely stained bands with apparent
molecular masses of 150 and 25 kDa were also detected in the rat. The
150-kDa band might be the
-GT precursor protein which contains both
the large and small subunits; the 25-kDa band could possibly be a
deglycosylated form of the small subunit (26). Alternatively, these
bands could be artefacts. Two polypeptides are detected in wild type
mouse kidney microsomal protein with apparent molecular masses of
approximately 55 and 25 kDa, but the smaller polypeptide in control
mouse stained less intensely than the larger subunit. This could be
caused by inefficient detection of mouse
-GT protein when using sera
raised against rat
-GT protein, or the smaller subunit in control
mouse could have been partially inadvertently degraded during the
isolation procedure. Only a small amount of a polypeptide with 150 kDa
apparent molecular mass is visible in the GGTenu1
lane. Neither mature
-GT subunit was detected in
GGTenu1 kidney in the trial depicted in Fig. 5, but
faint traces of each subunit (apparent molecular masses 55 and 35 kDa)
were occasionally detected in GGTenu1 kidney on
repeated immunoblots. This result is consistent with the small amount
of residual
-GT activity measured in GGTenu1
kidney homogenate.
Inheritance of Glutathionuria
Autosomal recessive inheritance
of glutathionuria in the mice was suspected from pedigree inspection.
No vertical transmission of glutathionuria was seen. Ninety-nine
glutathionuric mice were detected out of 323 total offspring (30.6%)
from 46 matings. Of these, 44 were male and 55 were female. In an
autosomal recessive model, 81 glutathionuric mice would have been
expected out of 323 total offspring (25%). This difference between the
expected and actual number of affected mice is statistically
significant (2 = 5.33, p < 0.05, one
degree of freedom) but becomes insignificant if one assumes that the
glutathionuric phenotype of only three mice had been assigned
incorrectly.
Because glutathionuric mice did not breed, the mutant strain has been maintained using two breeding schemes. First, unaffected female siblings of glutathionuric mice are bred to their carrier fathers. Second, unaffected male siblings of glutathionuric mice are bred to wild type C57Bl/6J females. The female offspring of this mating are backcrossed to their father and the next generation of offspring are then screened for glutathionuria. Using the latter breeding method, the glutathionuric trait (that is, the carrier state for glutathionuria) has been transmitted through 12 generations without any alteration in the glutathionuric phenotype of homozygous mice.
The glutathionuric mouse described here is an animal model of
genetic -GT deficiency. Glutathionuria was detected by paper chromatography in progeny of mice treated with ENU, a potent chemical point mutagen, and confirmed by high voltage two dimensional
electrophoresis, quantitative amino acid analysis and specific
spectrophotometric measurement of GSH concentration. Plasma glutathione
levels were also elevated in the glutathionuric mice. Given that
glutathionuria in association with
-GT deficiency has been described
in humans (27, 28) and in rats treated with chemical inhibitors of
-GT (9),
-GT deficiency is the most likely cause of
glutathionuria in the mutant mice.
-GT deficiency in kidney
homogenates from the glutathionuric mice was confirmed using a
spectrophotometric assay of
-GT activity. Western blot analysis of
Triton X-100 treated kidney microsomes from glutathionuric mice
revealed only a trace of
-GT cross reactive material compared with
control mouse kidney microsomes. Glutathionuria is inherited in an
autosomal recessive manner in this mouse strain. We conclude that this
mouse strain, which we designate GGTenu1, is a model
for genetic
-GT deficiency.
The progeny of ENU-treated mice may harbor random point mutations at
multiple genetic loci. We have not ruled out the possibility that
mutations at more than one genetic locus are required for the
GGTenu1 phenotype, but the autosomal recessive
inheritance pattern suggests that this phenotype is caused by mutation
at a single genetic locus. This claim is further supported by the fact
that glutathionuric offspring have been produced by twelfth generation
progeny of the original ENU-treated mouse. Mice in the 12th generation,
because of outbreeding to wild type C57Bl/6J mice, share only 10.6%
genetic identity (2 of 20 chromosomes) with first generation mice. In humans, a family of at least four -GT genes exists (29), mapping to
chromosome 22q (30). At least two of the human
-GT genes are
transcribed as the human kidney cDNA differs from the placental and
liver cDNAs (31). In rats (32) and mice (33), several different
-GT mRNAs with different 5
ends but identical coding regions
are transcribed from a single gene. In rat kidney, translation of
-GT mRNA results in a precursor polypeptide which is
subsequently cleaved into two glycoprotein subunits of
Mr ~29,000 and ~49,500 (34). Only traces of
-GT protein were detected by immunoblotting of kidney microsomal
fraction from glutathionuric GGTenu1 mice. These
data suggest that glutathionuric GGTenu1 mice are
homozygous for a mutation (probably at the
-GT locus) which
significantly interferes with either the production or stability of
active
-GT protein.
Recently, the technique of homologous recombination in embryonic
stem cells has been used to disrupt the murine -GT gene (35). Mice
that are homozygous for the
-GT deletion
(GGTml/GGTm1) have no
detectable
-GT activity in kidney, pancreas, small intestine, or
seminal vesicles. Plasma glutathione levels in
GGTm1/GGTm1 mice (175 µM) are very similar to our results in
GGTenu1 mice (213 µM), but urinary
glutathione excretion is six to seven times greater in
GGTm1/GGTm1 mice (15378 µM versus 2261 µM in
GGTenu1 mice). The growth pattern of
GGTm1/GGTm1 mice mirrors that of
GGTenu1 mice, but the life span of knock-out mice
(10% survival at 25 weeks of age) is shorter than in
GGTenu1 mice (50% survival at 25 weeks of age).
Other physical differences include the ubiquitous development of
bilateral cataracts and change in coat color from black to agouti in
GGTm1/GGTm1 mice. All these
differences may be explained by the presence of slight residual
-GT
activity in GGTenu1 mice compared with complete
-GT deficiency in GGTm1/GGTm1
mice. The phenotypic similarities between the two mouse strains further
supports our claim that GGTenu1 mice are a model for
genetic
-GT deficiency.
Urine GSH excretion from the GGTenu1 mice was almost
600 times greater than that from control mice (Table II). The mechanism
by which -GT deficiency causes glutathionuria has been previously proposed. Two-thirds of the glutathione in plasma is extracted by the
kidney (36). It is both filtered through the glomerulus and secreted by
the tubule cells into the proximal renal tubule (37). Renal proximal
tubule cells exhibit the highest
-GT activity of any tissue, and
this activity is mainly localized to the cell surface facing the tubule
lumen (38).
-GT catalyzes the initial step in the degradation of
renal tubular GSH by cleaving the
-glutamyl bond and transferring
the
-glutamyl moiety to a broad repertoire of acceptors including
many amino acids, dipeptides, water or GSH itself (Fig. 1). The
products of the
-GT reaction, namely cysteinylglycine and the
-glutamyl-acceptor complex, are transported into the renal tubule
cells and processed enzymatically to produce the amino acids cysteine,
glycine, and glutamate. Resynthesis of GSH from these three amino acids
completes the proposed
-glutamyl cycle (5). Without
-GT activity
in the brush border membrane, GSH cannot be recovered from glomerular
filtrate and is lost in urine. Glutathionuria in
GGTenu1 mice confirms the previous experience in
humans with genetic
-GT deficiency and in rodents with chemically
induced
-GT deficiency, demonstrating that
-GT activity is
required for reabsorption of GSH from the urine.
Plasma GSH concentration in the GGTenu1 mice was
increased 5-fold as compared with control mice (Table II). Chemical
inhibition of -GT activity in mice and rats also increases plasma
GSH levels (9, 39). In a
-GT-deficient person, plasma GSH levels
were increased to 4.2-7.3 µmol/liter above control values of 1-1.6 µmol/liter (27). A second patient also exhibited an elevated GSH
plasma level of 19.4 µmol/liter (28). Plasma GSH levels are
approximately 10-fold higher in
-GT-deficient mice than in
-GT-deficient humans, possibly suggesting greater residual
-GT activity in affected humans, but plasma GSH levels in control mice are
also substantially higher than in human controls (~25 versus ~1 µmol/liter, respectively). Species-specific
variation in normal GSH physiology among humans and mice most likely
accounts for the species-specific differences in GSH levels in normal
and
-GT-deficient individuals. The redundancy and tissue-specific expression of human
-GT genes could be responsible for the
phenotypic differences between
-GT-deficient mice and humans.
GSH levels were altered in other tissues of GGTenu1
mice. Total liver GSH content in GGTenu1 mice was
decreased 66% compared with control animals. In mice treated with the
-GT inhibitor
D-
-glutamyl-(o-carboxy)phenylhydrazide, total
liver GSH content was decreased by only 17%, probably because of
incomplete or temporary
-GT inhibition (9). An 80% decrease in
liver GSH was measured in
GGTm1/GGTm1 mice (35). One
possible explanation for decreased liver GSH content in
-GT
deficiency might be that failure of GSH recovery from urine leads to
systemic deficiency of GSH precursors and limited liver GSH synthesis.
Of the three amino acids which constitute GSH (glutamate, glycine, and
cysteine), only the supply of cysteine is likely to be a limiting
factor in GSH synthesis. An alternative explanation for liver GSH
deficiency is impaired recovery of GSH from bile due to
-GT
deficiency affecting biliary epithelium.
It has been proposed that renal brush border -GT catalyzes
transpeptidation with luminal amino acids, particularly cystine, and
that uptake of
-glutamyl-amino acids from the tubule lumen is an
important pathway for amino acid transport in the kidney (8). In
GGTenu1 mice, urinary threonine, glycine, cystine,
isoleucine, leucine, ornithine, and lysine concentrations were slightly
increased as compared with controls (Table I). Cystine is an excellent
-glutamyl acceptor (40), and a decrease in recovery of cystine from
the renal tubule due to
-GT deficiency could explain increased
urinary cystine excretion in GGTenu1 mice. Glycine
and lysine are also reasonable
-glutamyl acceptors, but other amino
acids which are active
-glutamyl acceptors, most notably glutamine,
are not elevated in urine from GGTenu1 mice. Amino
aciduria can be a manifestation of generalized proximal renal tubule
dysfunction (renal Fanconi syndrome), but histologic examination of
kidneys from GGTenu1 mice revealed no abnormalities,
and there is no evidence of renal losses of other metabolites such as
glucose. These data support but do not prove the theory that GSH and
-GT play a nonexclusive role in the reabsorption of amino acids from
the renal tubule. GGTenu1 mice will be a valuable
experimental model for the study of
-GT and its relationship to
amino acid transport in the kidney.
If -GT deficiency does impair amino acid reabsorption in the renal
tubule, disturbed amino acid flux could be responsible for the poor
linear growth and weight gain of GGTenu1 mice.
Systemic cysteine deficiency due to chronic urinary loss of GSH could
also contribute to poor growth. Levels of free cystine and other amino
acids are normal in plasma and tissue homogenates from
GGTenu1 mice, but these data may not accurately
reflect the status of intracellular amino acid pools or interorgan
amino acid flux. Decreased urinary excretion of taurine, an amino acid
derived from cysteine, in GGTenu1 mice may reflect a
relative deficiency of cysteine or other sulfur-containing amino acids.
GGTenu1 mice excreted 40-45 µmol of GSH/day based
upon an average daily creatinine excretion of 0.6 mg/creatinine/day in
normal mice (41). If a normal mouse consumes 5 g of mouse chow
(Teklad Mouse Breeder Diet 8626) per day, the average daily intake of
cysteine equals 130 µmol of cysteine per day. This cysteine intake
may not be adequate to replace urinary GSH losses and provide for
normal cysteine requirements. Plasma cystine deficiency was detected in
GGTm1/GGTm1 mice; the growth
velocity of GGTm1/GGTm1 mice was
almost completely corrected when the mice were given N-acetylcysteine supplements as a source of extra cysteine
(35). Studies to determine the effect of cysteine supplementation on the growth of the GGTenu1 mice are in progress. No
significant problems with appetite, linear growth, or weight gain were
reported in any of the glutathionuric humans.
GGTenu1 mice exhibit neurologic abnormalities
including decreased general activity but with agitation and tremor when
stimulated. Histological examination at the light microscopic level of
their brains did not reveal any abnormalities. Impaired GSH and amino acid transport in the brain could disrupt neurotransmitter or other
pathways. Three unrelated humans with -GT deficiency had mild mental
retardation and one of them had severe behavioral problems (27, 28,
42). Recently, two female siblings from Australia have been reported
with
-GT deficiency (43). The older sister was discovered at 6 weeks
of age to have glutathionuria through a total population screening
program examining urine amino acids by paper chromatography. Her
health, growth, and development have been normal except for an absence
seizure disorder which developed at 10 years of age. Her younger sister
has
-GT deficiency but no seizure disorder. Her mild mental
retardation has been attributed to Prader-Willi syndrome associated
with an interstitial deletion of chromosome 15q11-13. Although the
causal relationship between the abnormal neurologic signs and
-GT
deficiency remains uncertain in humans, our findings in the
GGTenu1 mice indicate that
-GT is required for
normal neurologic function.
In summary, GGTenu1 mice provide convincing evidence
that -GT function is required for normal growth and neurologic
function. Further studies to elucidate the mechanism by which
-GT
deficiency leads to the physical and neurologic abnormalities in
GGTenu1 mice will enable the metabolic and cellular
roles of
-GT to be better defined. The GGTenu1
mouse also provides a valuable experimental platform for the study of
-GT and GSH metabolism in response to infection, environmental toxins, carcinogenic agents, and other disease processes.
The authors gratefully acknowledge Prof. H. C. Pitot for useful discussions concerning -GT and GSH metabolism
and for the rabbit anti-rat
-GT antisera; J. D. McDonald, W. F. Dove, and A. Shedlovsky for guidance with ENU mutagenesis protocols; R. M. Pauli and F. L. Siegel for critical reading of the manuscript; E. Langer for preparation of tissue sections for histology; A. Messing for
review of gross and microscopic pathology; and C. Christen and S. Schneider for care, breeding, and testing of the animals.