From the Department of Chemistry and Biochemistry and
the Molecular Biology Institute, UCLA,
Los Angeles, California 90095-1569, the § Gladstone
Institute of Cardiovascular Disease,
San Francisco, California 94141-9100, and the ¶ Department
of Medicine and the Cardiovascular Research Institute, University
of California, San Francisco, California 94143
Received for publication, February 1, 2001
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ABSTRACT |
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L-Isoaspartyl
(D-aspartyl) O-methyltransferase (PCMT1)
can initiate the conversion of damaged aspartyl and asparaginyl
residues to normal L-aspartyl residues. Mice lacking this
enzyme (Pcmt1 The spontaneous chemical modification of proteins by reaction with
oxygen, water, sugars, and other abundant metabolites is unavoidable.
The accumulation of such nonenzymatically altered proteins is
associated with normal aging as well as atherosclerosis, Alzheimer's
disease, and diabetes (1-3). Organisms have several strategies for
dealing with damaged proteins, including intracellular proteolysis
mediated by proteasome and lysosome action (4-6). Some types of
covalent damage, however, are simple enough to recognize and repair
directly (7). Enzymes such as prolyl cis-trans isomerase (8), methionine sulfoxide reductase (9), and disulfide isomerase (8)
can restore activity to proteins that have been chemically altered.
We are interested in a common type of spontaneous protein damage in
which L-aspartyl and L-asparaginyl residues
undergo an intramolecular reaction that converts them to
L-succinimidyl residues (10, 11). Nonenzymatic hydrolysis
of the succinimide ring readily occurs at either carbonyl to generate
both normal aspartyl residues and isoaspartyl residues, in which the
peptide backbone proceeds through the To minimize the accumulation of damaged aspartyl residues in cellular
proteins, all mammalian tissues possess an L-isoaspartyl (D-aspartyl) O-methyltransferase (EC 2.1.1.77;
designated PCMT11 in mice)
(14). This enzyme uses S-adenosyl-L-methionine
(AdoMet) to methylate L-isoaspartyl (and, less efficiently,
D-aspartyl) residues but not normal L-aspartyl
residues (15). Nonenzymatic deesterification of the methylated residues
returns them to the succinimide form much more rapidly than occurs in
the absence of methylation, resulting in the eventual conversion of
most of the damaged residues to the "repaired"
L-aspartyl form (16, 17).
The physiological importance of this pathway remained unclear until
Pcmt1 knockout (Pcmt1 These results have raised new questions. Does the repair
methyltransferase, although expressed in all tissues, have particular importance within the brain? Is it important in neurons or in nonneuronal cell types? If the seizures in the Pcmt1 Generation of Pcmt1 Transgenic Mice--
A rat neuron-specific
enolase (NSE) promoter was used to direct the expression of mouse
Pcmt1 cDNA in the brains of transgenic mice. The
methyltransferase coding sequence (including 119 base pairs of
5'-noncoding and 777 base pairs of 3'-noncoding sequence) was obtained
from a 1580-base pair murine testis cDNA clone (21) and was removed
from plasmid sequences with EcoRI. The proximal rat NSE
promoter was isolated from the plasmid NSE-APP695 (22) after digestion
with HindIII. After overhangs were filled with Klenow
polymerase, the mouse Pcmt1 cDNA and the rat NSE vector were ligated, and the NSE-Pcmt1 transgenic construct was
isolated by digestion with SalI. The transgene (2 ng/µl)
was microinjected into F2 C57BL/6 × SJL fertilized mouse eggs by
standard techniques (23). From 37 microinjected eggs, 33 pups were
obtained, and 4 harbored the Pcmt1 transgene. These
transgenic founders were identified by polymerase chain reaction with
primers corresponding to mouse Pcmt1 cDNA sequences
(5'-GCCAGCCACTCGGAGCTAATCC-3' from exon 1 and
5'-CCACTATTTCCAACCATCCGTGC-3' from exons 4 and 5). Southern blot
analysis of tail DNA confirmed the integration of the transgene (Fig.
1). Two of these mice, founders 27 and
29, were bred with C57BL/6 × 129/SvJae mice that were
heterozygous for a knockout mutation in the endogenous Pcmt1
gene (18). DNA samples from the tails of these mice were genotyped by
polymerase chain reaction, both to detect the transgene and to detect
the knockout mutation. Mice that were heterozygous for the knockout mutation and hemizygous for the transgene were selected for breeding. All mice were weaned at 21 days of age, housed in a barrier facility with a 12-h light/dark cycle, and fed a chow diet.
Preparation of Mouse Tissues--
Brain, heart, liver, kidney,
and testis were removed immediately from sacrificed animals and placed
in ice-cold buffer (5 ml/g wet weight) containing 250 mM
sucrose, 10 mM Tris-HCl, 1 mM disodium EDTA, pH
7.4, and the protease inhibitor phenylmethylsulfonyl fluoride (25 µM). The tissues were disrupted in a glass homogenization tube with a Teflon pestle rotating at 310 rpm for four 10-s intervals. The homogenates were placed in 1.5-ml tubes and centrifuged at 20,800 × g for 10 min. The resulting supernatant
fractions contained both cytosolic proteins and microsomes and were
kept frozen until used.
Whole blood (100-200 µl) was taken from the tail or heart and
combined with 200 µl of 2 mg/ml disodium EDTA, 10 mM
sodium phosphate, 137.9 mM sodium chloride, pH 7.4. Erythrocytes were collected by centrifugation at 4000 × g for 4 min and washed four times with 1 ml of the above
buffer. Pelleted erythrocytes were lysed in 5 volumes of 5 mM sodium phosphate, 1 mM disodium EDTA, pH
7.4, and 25 µM phenylmethylsulfonyl fluoride. The lysates
were centrifuged in 1.5-ml conical tubes at 20, 800 × g for 10 min to remove the membrane ghosts, and the
supernatant fractions were stored at Protein Assay--
A modified Lowry assay was used to determine
protein concentrations in the extracts. Assays were done in duplicate
with bovine serum albumin as a standard (18).
Assay of L-Isoaspartyl (D-Aspartyl)
O-Methyltransferase Activity--
Methyltransferase activity was
assayed by its ability to transfer the methyl group from
S-adenosyl-L-methionine to ovalbumin. The
supernatant fraction from homogenized tissues (10-60 µg of brain,
heart, or testis protein; 0.6-0.8 mg of erythrocyte protein) was
incubated with 0.8 mg of ovalbumin (Sigma, Grade V) in 0.2 M BisTris-HCl, pH 6.0, containing 10 µM
[14C]AdoMet (53 mCi/mmol; Amersham Pharmacia Biotech) in
a final volume of 40 µl at 37 °C for 15 min. NaOH (80 µl of a
200 mM solution) was added to stop the reaction and to
hydrolyze the [14C]methyl esters formed on ovalbumin to
[14C]methanol. The reaction mixture was immediately
spotted onto an 8 × 2-cm piece of thick filter paper and
incubated above 5 ml of Safety-Solve scintillation fluid
(Research Products International) in the neck of a sealed 20-ml
scintillation vial at room temperature for 2 h to allow
[14C]methanol to diffuse into the scintillation fluid.
The filter paper was removed, and the scintillation fluid was counted
for radioactivity. Incubations containing
S-adenosyl-L-[methyl-14C]methionine,
ovalbumin, and buffer constituted the "blank" for the assay; the
radioactivity in these tubes (typically <5% of the nonblank samples)
was subtracted from total counts in the determination of enzyme activity.
Quantitation of Damaged Aspartyl Residues--
Cellular proteins
were incubated at 37 °C for 2 h with 0.8 µg of recombinant
human L-isoaspartyl methyltransferase (specific activity,
10,000 pmol of methyl esters formed on ovalbumin at 37 °C/min/mg
protein) (24) in 0.2 M BisTris-HCl, pH 6.0, and 10 µM [14C]AdoMet in a final volume of 40 µl. After base hydrolysis, [14C]methanol production was
measured as described above to quantitate L-isoaspartyl and
D-aspartyl methyl-accepting sites in cellular proteins.
Incubations containing [14C]AdoMet, recombinant
methyltransferase, and buffer constituted the blank for the
assay; the radioactivity in these tubes was subtracted from each
sample's total counts. Each sample was assayed in duplicate or
triplicate, and the average value is reported.
Quantitation of Endogenously Methylated Damaged Aspartyl
Residues--
Damaged residues that are already methylated within
cells by the endogenous methyltransferase and
S-adenosyl-L-methionine are not measured in the
assay described above but can be quantified after mild base treatment.
Protein (8.3-9.6 mg) from homogenized Pcmt1 Urine Collection and Analysis--
Urine, freshly voided on
Parafilm, was collected with a pipette and stored frozen until used.
Creatinine in the urine was measured by a modified form of the
procedure of Bosnes and Taussky (25). An aliquot of each urine sample
(0.3-1 µl) or standard creatinine (0-25 µg) was diluted to 50 µl with water in duplicate tubes. Picric acid was added (25 µl of a
40 mM solution), and the tubes were capped and incubated in
a boiling water bath for 45 min. After cooling to room temperature, 25 µl of 0.75 M NaOH was added. Within 15 min, 90 µl of
each sample was transferred to a flat-welled microtiter plate (Costar),
and absorbance was measured at 525 nm with a Beckman DU-600 plate
reader. Damaged aspartyl residues in the urine were assayed with
recombinant human methyltransferase as described above.
Amino Acid Analysis--
Free amino acids and
isoaspartyl-containing dipeptides in urine were derivatized with
o-phthalaldehyde and 2-mercaptoethanol, separated by
reverse-phase high pressure liquid chromatography, and quantitated by
fluorescence as described previously (26). A precipitate that formed
upon mixing of the urine and derivatization reagent was removed by
centrifugation at 20,800 × g for 3 min prior to
injection. Urine that had been dried in 6 × 50-mm glass tubes was
hydrolyzed in vaporized hydrochloric acid in vacuo at 108 °C for 3 h with a PicoTag Work Station (Waters); amino
acids in the hydrolysates were quantitated as described above. The
fluorescence color constants for these derivatives were determined with
amino acid and dipeptide standards.
Expression of a Pcmt1 Transgene in Neurons Prolongs the Lives of
Mice Lacking Endogenous Pcmt1--
We generated two Pcmt1
transgenic mouse lines, lines 27 and 29, in which the murine
Pcmt1 methyltransferase cDNA was placed under the
control of a neuron-specific promoter. We then compared the survival
of "transgenic Pcmt1
Line 27 and most of the line 29 mice possessing one or two copies of
the endogenous Pcmt1 gene were indistinguishable from comparable nontransgenic mice in size, weight, and behavior, although 15 of 94 line 29 mice ran rapidly in circles. Line 27 and line 29 mice
appeared to have normal physiological functions and had unremarkable
tissue histology. The transgenic Pcmt1 Localization of Methyltransferase Activity in Tissues of Line 27 and Line 29 Transgenic Mice--
The Pcmt1 transgene
controlled by a neuron-specific promoter appeared to rescue, at least
partially, the early death phenotype seen in mice lacking the
endogenous methyltransferase. We next investigated where in the mouse,
and at what level, the transgene was being expressed by assaying
methyltransferase activity in various mouse tissues. As expected,
transgenic mice possessing one or two copies of the endogenous
Pcmt1 gene expressed the methyltransferase in all tissues
assayed, including brain, heart, testes, erythrocytes, liver, and
kidney, at levels similar to those observed in nontransgenic mice
(Table I) (18). In contrast, transgenic
Pcmt1 Accumulation of Damaged Aspartyl Residues in Brain Tissue--
The
finding that transgenic mice expressing methyltransferase solely in
neurons lived longer than nontransgenic knockout mice led us to compare
the accumulation of damaged aspartyl residues in the brains of these
animals. Recombinant human methyltransferase was used to label these
residues in cytosolic/microsomal proteins with
[14C]methyl groups from [14C]AdoMet
in vitro. Examining 39 transgenic and 13 nontransgenic Pcmt1
Examination of the levels of aspartyl damage in the brain with respect
to age revealed several interesting points. First, these levels
increased with age in young animals; 40-day-old and older mice had
significantly more damaged residues per mg of protein than did the
13-21-day-old mice (p = 0.001 for
Pcmt1
In control experiments, we asked whether the low level of damaged
residues measured in the assays of Pcmt1+/ Limited Accumulation of Damaged Aspartyl Residues in Heart, Testes,
and Erythrocytes--
The longer life span of the transgenic
Pcmt1
The rates at which damaged residues in cytosolic proteins accumulated
in young Pcmt1 Damaged Aspartyl Residues in Urine--
The absence of increasing
accumulation of damaged aspartyl residues in the cytosolic proteins of
adult mice can result from repair of the damaged residues or from
turnover of the proteins. Few peptidases cleave isoaspartyl bonds, but
proteolysis of the surrounding residues creates isoaspartyl-containing
dipeptides and tripeptides that can be excreted in the urine
(33-36). Thus, if mice lacking endogenous PCMT1 do not have another
repair pathway, they might excrete the damaged residues that are
normally repaired in the cells of wild-type mice. To test this
hypothesis, we examined urine from Pcmt1
First, we directly quantitated urinary dipeptide levels after
derivatization with o-phthalaldehyde and
Second, peptides with an N-terminal isoaspartyl residue might
accumulate because the isopeptide bond is resistant to aminopeptidase activity. To determine whether more of these peptides accumulated in
Pcmt1
Finally, we used the recombinant
L-isoaspartyl/D-aspartyl methyltransferase as
an analytical probe for L-isoaspartyl residues in peptides
large enough to be methylated efficiently (tetrapeptides and larger)
(15). Methylatable residues were found in all of the urine samples
assayed, and the level of damage increased with the age of the mouse
(Fig. 7). We observed no significant
difference in the number of methylatable sites, relative to the amount
of creatinine, in urine from Pcmt1 We have been examining mice lacking PCMT1 to determine whether
ineffective repair of damaged aspartyl and asparaginyl residues contributes to disease or to the deleterious effects of aging. We
report here that damaged aspartyl residues in Pcmt1 The high level of PCMT1 activity in wild-type brain, the fact that
damaged residues rapidly accumulate in the brain in the absence of the
methyltransferase, and the seizures in the Pcmt1 To answer these questions, we performed a "brain rescue" experiment
by creating mice with a Pcmt1 transgene under the control of
a neuron-specific promoter in a genetic background lacking the
endogenous Pcmt1 gene. Here, we obtained mice expressing the methyltransferase only in the brain. Although the expression of the
transgene-derived methyltransferase activity in the brains of these
mice was only 6.5-13% of the level in wild-type mice, the transgenic
mice lived much longer and accumulated only half the damaged residues
found in nontransgenic knockout animals. The success of this rescue
experiment further supported the importance of the methyltransferase in
the brain.
We reported previously (18) that mice lacking the repair
methyltransferase are more sensitive to the seizure-inducing drug metrazol than are wild-type mice and that the anti-seizure drugs valproic acid and clonazepam prolonged the life span of these animals
(20). It is likely that, by decreasing the accumulation of damaged
aspartyl residues, the transgene-derived methyltransferase raised the
seizure threshold of the rescued mice to an intermediate level between
the thresholds of nontransgenic Pcmt1 Because many of the transgenic Pcmt1 Isoaspartyl linkages themselves are generally not cleaved by mammalian
proteases (33, 34). We have thus investigated the possibility that
proteolysis of proteins containing damaged aspartyl residues in
PCMT1-deficient mice may be reflected in the increased urinary output
of peptides containing L-isoaspartyl residues. In fact,
damaged aspartyl residues in proteins fed to rats are excreted as
isoaspartyl-containing dipeptides in the urine (40), and it has been
proposed that at least some endogenous isoaspartyl residues are dealt
with in a similar manner (41). Furthermore, an
L-isoaspartyl residue that arises in collagen has been
found in human urine as part of an eight-residue peptide (42). We found
that urine from Pcmt1 Is the elevated level of altered aspartyl residues in the urine from
Pcmt1 Interestingly, the ability of the proteolytic pathway to stem the
accumulation of damaged aspartyl residues is apparently insufficient to
prevent seizures in animals lacking the repair methyltransferase. Thus,
the repair methyltransferase is needed (at least in the brain) to lower
the level of damaged residues beyond what the degradation mechanisms
can accomplish. Furthermore, it is possible that proteolysis of
proteins that contain covalent modifications important to learning and
memory could have undesirable effects in that their replacement
proteins would not be appropriately modified (43).
Unlike the brain, other tissues appear to be able to function
relatively normally with higher levels of damaged aspartyl residues. The importance of the methyltransferase in maintaining a lower steady
state level of damaged aspartyl residues in these tissues remains to be
determined. It is certainly possible that mice reared under other
conditions (e.g. outside of a vivarium) would be more susceptible to pathologies in the absence of the repair methyltransferase.
/
mice) have elevated levels of damaged
residues and die at a mean age of 42 days from massive tonic-clonic
seizures. To extend the lives of the knockout mice so that the long
term effects of damaged residue accumulation could be investigated, we
produced transgenic mice with a mouse Pcmt1 cDNA under
the control of a neuron-specific promoter. Pcmt1 transgenic
mice that were homozygous for the endogenous Pcmt1 knockout
mutation ("transgenic Pcmt1
/
mice") had brain PCMT1
activity levels that were 6.5-13% those of wild-type mice but had
little or no activity in other tissues. The transgenic Pcmt1
/
mice lived, on average, 5-fold longer than
nontransgenic Pcmt1
/
mice and accumulated only half as
many damaged aspartyl residues in their brain proteins. The
concentration of damaged residues in heart, testis, and brain proteins
in transgenic Pcmt1
/
mice initially increased with age
but unexpectedly reached a plateau by 100 days of age. Urine from
Pcmt1
/
mice contained increased amounts of peptides
with damaged aspartyl residues, apparently enough to account for
proteins that were not repaired intracellularly. In the absence of
PCMT1, proteolysis may limit the intracellular accumulation of damaged
proteins but less efficiently than in wild-type mice having
PCMT1-mediated repair.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbonyl rather than the
-carbonyl moiety (12). The succinimide also racemizes more rapidly
than do the open chain forms, and hydrolysis of the
D-succinimide produces D-aspartyl and
D-isoaspartyl residues (12). Local protein structure causes
some L-aspartyl and L-asparaginyl residues to
be especially prone to succinimide formation, and the presence of
damaged aspartyl residues at these sites can significantly alter the
structure, function, and immunogenicity of the protein (10, 13).
/
) mice were created and
found to display a distinctive phenotype (18, 19).
Pcmt1
/
mice have 2-6-fold higher levels of damaged
aspartyl residues in their brain, heart, liver, and erythrocytes than
are observed in wild-type tissues (18). Furthermore,
Pcmt1
/
mice are smaller than their Pcmt1+/
and Pcmt1+/+ littermates, undergo severe tonic-clonic seizures, and die at an average age of 42 days (18, 19).
Electroencephalographic analysis shows that these mice suffer abnormal
brain activity about 50% of the time, not just during the tonic-clonic
seizures (20). Administration of the anti-seizure drug valproic acid enabled Pcmt1
/
mice to attain the same size and weight
as their wild-type littermates, suggesting that the absence of the
methyltransferase did not interfere directly with food intake or
metabolism (20). The combination of valproic acid and clonazepam
prolonged mean survival but only by 36 days (20).
/
mice were prevented, would other organs function abnormally as damaged
aspartyl residues accumulated? In the current study, we have approached these questions by producing transgenic mice that express Pcmt1 solely
within neurons.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of transgenic mice from two
different Pcmt1 transgenic mouse lines. Mouse
genomic DNA was digested with EcoRI and examined by Southern
blotting with a neuron-specific enolase promoter probe. Nontransgenic
mice had a single band corresponding to the endogenous enolase gene.
Transgenic mice were identified by the presence of additional bands
(two larger bands in the case of line 27 and one larger band
in the case of line 29).
20 °C.
/
and
Pcmt1+/+ brains was incubated in 20 µl of 75 mM potassium borate, pH 10.2. After times ranging from
5 s to 360 min, 10 µl of 500 mM BisTris-HCl, pH 5.7, was added to lower the pH to about 6. Then, recombinant human
methyltransferase (5 pmol/min) and S-adenosyl-L-[methyl-14C]methionine
(10 µM final concentration) in 10 µl of 150 mM BisTris-HCl, pH 6.0, was added, and these reaction
mixtures were incubated at 37 °C for 135 min. The reaction was
stopped by freezing on dry ice, and then the base-labile methyl esters
were quantitated as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice" with that of
"nontransgenic Pcmt1
/
mice." Whereas nontransgenic
Pcmt1
/
(n = 129) mice died at a median
age of 44 days (with only one mouse living beyond 150 days), the
transgenic Pcmt1
/
mice lived much longer (Fig. 2). Of 11 line 27 transgenic
Pcmt1
/
mice examined in this study, 6 died between 30 and 90 days of age, but 4 lived from 549 to 757 days. Line 29 transgenic Pcmt1
/
mice (n = 19) died at
a median age of 213 days (Fig. 2), and 3 lived more than 400 days. The
nontransgenic Pcmt1
/
mice began to die at about 21 days. In contrast, none of the line 29 transgenic Pcmt1
/
mice
died at less than 52 days of age (Fig. 2).
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Fig. 2.
Enhanced survival of Pcmt1 /
mice expressing a Pcmt1 transgene driven by a
neuron-specific promoter. Shown are data for Pcmt1
/
mice (n = 129) lacking the transgene (solid
line) and for line 29 mice in the Pcmt1
/
background
(n = 19; dashed line) or in the
Pcmt1+/
or Pcmt1+/+ backgrounds
(n = 36; dashed-dotted line). These data
represent mice that died spontaneously or are still alive.
/
mice, however, differed from the nontransgenic Pcmt1
/
animals in
several ways. First, although nontransgenic Pcmt1
/
mice
weighed significantly less than age- and sex-matched
Pcmt1+/
and Pcmt1+/+ littermates (18), the
weights of transgenic Pcmt1
/
mice were identical to
their Pcmt1+/
and Pcmt1+/+ littermates (data
not shown). Second, due to their low grade seizure activity
(e.g. facial grooming and myoclonic jerks), the
nontransgenic Pcmt1
/
mice could often be distinguished
by observation from wild-type and heterozygous littermates. In
contrast, these abnormalities were not observed in the transgenic
Pcmt1
/
mice.2
Finally, nontransgenic Pcmt1
/
mice of either sex never
produced litters, even when housed with Pcmt1+/+ animals and
given the anti-seizure drugs valproic acid and clonazepam, and only a
single mating was observed (20). Two line 27 Pcmt1
/
animals, however, produced two small litters without the administration
of any drug treatments, and 17 pairings involving male and/or female
line 29 Pcmt1
/
mice have produced three litters (from
two different Pcmt1
/
mothers).
/
mice expressed methyltransferase activity in the
brain but not in the other tissues (Table I), and Western blot analysis
detected PCMT1 protein only in brain homogenates (data not shown),
suggesting that the neuron-specific enolase promoter was properly
directing expression to neurons. However, the activity of this
transgene-derived methyltransferase in the brain was relatively low;
line 27 Pcmt1
/
brains had only 6.5% and line 29 Pcmt1
/
brains only 13% of the PCMT1 activity observed
in wild-type brains. The amount of activity in brains from young (50 days) and older (370 days) transgenic Pcmt1
/
animals was
not significantly different (data not shown). Since about half of the
cells in the brain are not neurons (27), some reduction of
methyltransferase activity was expected in these brains. The low
activity observed here, however, suggested that even in neurons the
expression of the transgene was weaker than that of endogenous
Pcmt1 in wild-type neurons. Relatively low levels of
neuronal expression from the neuron-specific enolase promoter have been
reported by other investigators (28, 29).
L-Isoaspartyl (D-aspartyl) O-methyltransferase
activity in mice possessing the Pcmt1 transgene with a neuron-specific
enolase promoter
/
mice, we found about 50% fewer damaged aspartyl
residues when the transgene was present, indicating that the
transgene-derived enzyme was repairing damaged neuronal proteins.
However, the transgenic Pcmt1
/
brains still had about
4.5-fold more damaged residues than did Pcmt1+/
and
Pcmt1+/+ brains (Fig. 3).
These damaged residues could be accumulating both in neurons, due to
the relatively low methyltransferase activity, and in glia, which
should not express the transgene at all.
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Fig. 3.
Accumulation of damaged aspartyl residues in
brain cytosolic/microsomal polypeptides. Recombinant human
L-isoaspartyl (D-aspartyl)
O-methyltransferase was used to quantitate damaged aspartyl
residues in polypeptides that remain in the supernatant following a
20,800 × g centrifugation of whole brain homogenates
as described under "Experimental Procedures." Open circles,
Pcmt1 /
(n = 14); gray circles,
transgenic line 27 Pcmt1
/
(n = 21);
black circles, transgenic line 29 Pcmt1
/
(n = 18); open
crosses, Pcmt1+/
(n = 14);
black crosses, transgenic line 29 Pcmt1+/
(n = 5); open triangles, Pcmt1+/+
(n = 17); open diamonds, Pcmt1+/
or
Pcmt1+/+ (n = 6); gray triangles,
transgenic line 27 Pcmt1+/+ (n = 2); and black triangles, transgenic line 29 Pcmt1+/+ (n = 3).
/
mice; p = 10-8 for
Pcmt1+/
and Pcmt1+/+ mice; Fig. 3). Second, the
13-14-day-old Pcmt1
/
animals already possessed about
8-fold more damaged aspartyl residues per mg protein than did
age-matched Pcmt1+/
and Pcmt1+/+ animals (Fig.
3). The difference in the amount of damage remained 8-11-fold as these
mice aged to 91 days, demonstrating that both nursing and weaned
Pcmt1
/
mice accumulate these residues. Finally, there
was no significant increase in the level of damaged aspartyl residues
in transgenic Pcmt1
/
mice after about 100 days of age (Fig. 3). In addition, although line 29 Pcmt1
/
mice
averaged twice as much brain PCMT1 activity as did comparable line 27 mice, the plateau level of damaged aspartyl residues in these two lines was not significantly different. The quantity of damaged residues in
Pcmt1+/
and Pcmt1+/+ mice also attained an
apparent steady state, although at a much lower level (Fig. 3). Because
damaged residues were arising continuously in the cellular proteins,
this stable level of damage probably represents a steady state between new damage, methyltransferase-linked repair, protein turnover, and
perhaps unknown factors.
and
Pcmt1+/+ mice resulted in part from the fact that some were
already methylated by the endogenous enzyme. We therefore incubated
brain cytosolic proteins under basic conditions where
L-isoaspartyl
-methyl esters should hydrolyze within a
few minutes to generate L-isoaspartyl residues with about
an 80% yield (30, 31). Analysis of these samples with recombinant
methyltransferase (data not shown) indicated that the true number of
L-isoaspartyl residues in the Pcmt1+/
and
Pcmt1+/+ proteins could be as much as 2.4-fold higher than the data shown in Fig. 3.3
Coupled with the 8-11-fold higher levels actually observed, this result indicates that the level of damaged aspartyl residues in Pcmt1
/
brain proteins was at least 4-fold higher than in
heterozygous and wild-type brain proteins.
/
mice enabled us to investigate whether
accumulation of damaged aspartyl residues continued in tissues
completely lacking methyltransferase-mediated repair as animals aged
beyond the limit set by the early deaths of the nontransgenic knockout
mice. In addition to heart (Fig. 4) and
testis (Fig. 5), we also examined
erythrocyte cytosol as a control (Fig.
6). Because erythrocytes are normally
removed from the circulation after about 40 days (32), their average age in adult mice is constant, and older proteins cannot accumulate. Unexpectedly, the accumulation of damaged aspartyl residues in all of
these tissues was quite similar. As in brain, the number of
methylatable residues per mg protein increased only in animals younger
than about 60 days and then leveled off as the mice got older (Figs.
4-6). The apparent plateau levels of damaged aspartyl residues in
heart, testes, and erythrocytes from transgenic Pcmt1
/
mice averaged 4.5-, 4.7-, and 5.2-fold higher, respectively, than the
average levels in tissues from mice expressing the endogenous methyltransferase, very similar to the 4.5-fold difference observed in
brain. However, the absolute plateau level of damage in each tissue was
significantly different, with brain having the highest and erythrocytes
having the lowest levels in both the presence and in the absence of
endogenous PCMT1 (Table II). As expected, the transgenic PCMT1 had no effect in tissues where it is not expressed.
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Fig. 4.
Accumulation of damaged aspartyl residues in
heart cytosolic/microsomal polypeptides. Levels of damage were
assayed as described in Fig. 3. Open circles,
Pcmt1 /
(n = 12); gray circles,
transgenic line 27 Pcmt1
/
(n = 12); black circles, transgenic line 29 Pcmt1
/
(n = 8); open crosses,
Pcmt1+/
(n = 6); gray cross,
transgenic line 27 Pcmt1+/
(n = 1); open triangles, Pcmt1+/+ (n = 9); and black triangle, transgenic line 29 Pcmt1+/+ (n = 1).
View larger version (23K):
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Fig. 5.
Accumulation of damaged aspartyl residues in
testis cell cytosolic/microsomal polypeptides. Levels of damage
were assayed as described in Fig. 3. Open circles,
Pcmt1 /
(n = 7); gray circles,
transgenic line 27 Pcmt1
/
(n = 12); black circles, transgenic line 29 Pcmt1
/
(n = 8); open crosses,
Pcmt1+/
(n = 3); gray crosses,
transgenic line 27 Pcmt1+/
(n = 2); black crosses, transgenic line 29 Pcmt1+/
(n = 4); open
triangles, Pcmt1+/+ (n = 5); gray
triangle, transgenic line 27 Pcmt1+/+
(n = 1); and black triangles, transgenic
line 29 Pcmt1+/+ (n = 3).
View larger version (24K):
[in a new window]
Fig. 6.
Accumulation of damaged aspartyl residues in
erythrocyte cell cytosolic polypeptides. Levels of damage were
assayed as described in Fig. 3. Open circles,
Pcmt1 /
(n = 8); gray circles,
transgenic line 27 Pcmt1
/
(n = 20); black circles, transgenic line 29 Pcmt1
/
(n = 16); open
crosses, Pcmt1+/
, (n = 8); gray
crosses, transgenic line 27 Pcmt1+/
(n = 2); black crosses, transgenic
line 29 Pcmt1+/
(n = 5);
open triangles, Pcmt1+/+ (n = 9);
gray triangle, transgenic line 27 Pcmt1+/+
(n = 1); and black triangles, transgenic
line 29 Pcmt1+/+ (n = 3).
Levels of damaged aspartyl residues in tissues of adult mice over 100 days in age
/
mice have been calculated from the data
in Figs. 3-6. These rates differ greatly between tissues,
ranging from 51 pmol of methylatable residues/mg of protein/day in
brains to 1.8 pmol of methylatable residues/mg of protein/day in
erythrocytes (Table III). Because the
rate at which aspartyl and asparaginyl residues arise in proteins
should not decrease during the life of a mouse, we can use these rates
to estimate the total number of damaged residues arising in adult mice.
Assuming that a mouse is 15% protein by weight and that half of this
protein is intracellular, a 20-g mouse would have 1.5 g of
intracellular protein. If the average rate of damaged residue formation
throughout the mouse is between 5 and 20 pmol/mg/day, as predicted from
the values in Table III, there should be 7.5-30 nmol of newly damaged
residues arising each day within the cells of an adult mouse.
Rates of accumulation of damaged aspartyl residues in Pcmt1/
mouse
tissues
/
mouse tissues over the first 21 days after birth
were calculated from the data in Figs. 3-6. It was assumed for these
calculations that there are no damaged residues in newborn mice.
/
and
Pcmt1+/+ mice in several ways.
-mercaptoethanol
and separation by reverse-phase high pressure liquid chromatography. We
did not detect significantly higher levels of isoaspartyl-containing dipeptides in urine from Pcmt1 knockout mice (data not
shown). For example, isoaspartylglycine ("
-aspartylglycine"),
the most abundant of these dipeptides in human urine (35), was present at 28.2 pmol/µg creatinine in urine from a 91-day-old
Pcmt1
/
mouse and at 27.5 pmol/µg creatinine in urine
from a 40-day-old wild-type mouse. These results suggest that much of
this dipeptide might originate in dietary, extracellular, or
unrepairable intracellular proteins rather than from the lack of repair
in Pcmt1
/
cells.
/
than in Pcmt1+/+ mice, we compared the
urinary levels of free amino acids and of amino acids released by acid
hydrolysis, which cleaves both aspartyl and isoaspartyl bonds. Although
wild-type urine contained 1.5-fold more total amino acid residues (free and peptide bound) than did knockout mouse urine, both urine samples contained 8-9-fold more peptide-bound residues than free residues (data not shown). This indicates that the knockout mice were not excreting elevated levels of peptides or proteins relative to wild-type mice.
/
animals and from
Pcmt1
/+ and Pcmt1+/+ controls in the 13-14-
and 20-21-day-old groups (Fig. 7). However, urine from older
Pcmt1
/
mice contained almost 2-fold more methylatable
sites than urine from control animals (Fig. 7). Assuming that
435-783-day-old mice weighing 20 g excrete 0.67 mg of creatinine
per day (37), then Pcmt1
/
mice excrete about 9.5 nmol
and age-matched control animals about 5.1 nmol of methylatable L-isoaspartyl residues per day. Thus, at least 4.4 nmol of
damaged residues that are repaired in wild-type mice are excreted each day by knockout mice that are not accumulating additional damaged residues within their cells. This daily excretion of damaged aspartyl residues in the urine represents 15-67% of the total daily production of intracellular aspartyl damage estimated above. This result indicates
that proteolysis leading to urinary excretion is a significant factor
in limiting the accumulation of damaged proteins in the absence, and
perhaps as well in the presence, of the repair methyltransferase.
View larger version (27K):
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Fig. 7.
Damaged aspartyl residues in mouse urine that
are recognized in vitro by the
L-isoaspartyl (D-aspartyl)
O-methyltransferase. Each urine specimen was
collected in a single voiding and was assayed without further
treatment. Methylatable damaged residues were quantitated with
recombinant human methyltransferase as described in Fig. 3. Open
bars, urine from nontransgenic Pcmt1 /
mice;
shaded bars, urine from nontransgenic Pcmt1+/
and Pcmt1+/+ mice. The difference between the values for
these groups is statistically significant for the 43-103- and
435-783-day-old mice (p = 0.006 and 0.020, respectively) but not for the 13-14- or 20-21-day-old mice
(p = 0.161 and 0.263, respectively).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
brain proteins from 13 to 14-day-old mice were already eight times more abundant than those in aged-matched wild-type mice and accumulated at a
rate of about 52 pmol/mg of protein/day until 20-21 days. Damaged
residues continued to accumulate but at a lower rate, averaging 9 pmol/mg protein/day between days 21 and 55. The rate of accumulation
appeared to decrease further in the brains of older
Pcmt1
/
mice, but few of these mice live beyond this age. A similar pattern of damage accumulation has been recently observed by
Shimizu et al. (38) in independently derived
Pcmt1
/
mice. In contrast to the Pcmt1
/
mice, however, Pcmt1+/+ and Pcmt1+/
mice kept
the level of damaged residues very low for at least 2 years. Although
they had only about half of the methyltransferase activity,
Pcmt1+/
mice survived as long as Pcmt1+/+ mice.
/
mice
suggested that this enzyme plays a critical role in normal brain
function. PCMT1, however, is expressed in all mammalian tissues and
presumably is involved in repair throughout the body. We were therefore
also interested in examining what would happen if tissues lacking this
repair were allowed to accumulate higher levels of damage over several years.
/
and wild-type mice. Immunohistochemical studies revealed that the transgene-derived methyltransferase was expressed largely in neurons, in both line 27 and
line 29. However, the pattern of neuronal expression was distinct in
the two lines.4 These
findings indicate that the expression of Pcmt1 in neurons (rather than glia) is paramount in the prevention of the fatal seizure
disorder. At this point, however, we do not know whether the
methyltransferase is critical for the function of all neurons or only
for certain types of neurons.
/
mice lived for
several hundred days, we were able to examine the long term
accumulation of damaged residues in peripheral organs, where the
methyltransferase is not expressed at all. We found no apparent defects
in these mice, and initial pathological studies were unremarkable. We
found that damaged aspartyl residues accumulated in an
age-dependent manner only in relatively young mice and
attained plateau levels by 100 days of age. At first approximation, the
rate at which damaged residues arise in proteins remains constant with
time; thus, the plateau levels of damage in tissues of older mice must result from processes of repair and/or turnover of these residues. We
have been especially interested in the situation in mouse tissues that
lack the repair methyltransferase, where the levels of damage are much
greater but which still approach plateau values with age. We suggest
that enhanced proteolytic degradation may limit the accumulation of
proteins containing damaged aspartyl residues, especially in
methyltransferase-deficient tissues. Clear evidence has been presented
for the selective proteolytic degradation of spontaneously damaged
calmodulin in both HeLa cells (6) and in Xenopus oocytes
(39).
/
mice contains more peptides with damaged aspartyl residues than urine from Pcmt1+/+ mouse
urine. Because PCMT1 repairs intracellular proteins (14), our results provide the first evidence that damaged aspartyl residues that arise
within cells can be excreted in the urine.
/
mice enough to account for the observed steady state level of damaged proteins in tissues despite continuing spontaneous generation of isoaspartyl residues? From the rates of
damage accumulation measured in tissues of young Pcmt1
/
mice, we estimated that 7.5-30 nmol of newly damaged aspartyl residues are generated each day. The daily excretion of damaged residues in the
urine of Pcmt1
/
mice was 4.4 nmol more than that in
wild-type mice, demonstrating that a significant fraction of the damage can be metabolized by proteolysis. Additional isoaspartyl peptides may
be present in the urine that are not readily recognized by the
methyltransferase (15), although we did not detect increased levels of
generally poorly recognized dipeptides and N-terminal L-isoaspartyl peptides.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AG15451, HL41633 (to S. G. Y.), GM26020, and AG18000 (to S. C.), and by a grant from the University of California Tobacco-related Disease Research Program (to S. G. Y.).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.
To whom correspondence should be addressed: 640 Paul D. Boyer
Hall, 611 Charles E. Young Drive East, Los Angeles, CA 90095-1570. Tel.: 310-825-8754, Fax.: 310-825-1968; E-mail:
clarke@mbi.ucla.edu.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M100987200
2
Nonfatal running/jumping seizures have been
observed in three transgenic Pcmt1+/ and
Pcmt1+/+ mice but never in nontransgenic animals.
3
When damaged aspartyl residues in brain proteins
from Pcmt1/
mice were quantitated as a function of time
of base treatment, a linear increase of 1.8 pmol of damaged residues/mg
of protein/min was obtained. This increase resulted from the creation
of new damaged residues in the proteins. In contrast, quantitation of damaged residues in brain proteins from Pcmt1+/+ mice gave a
biphasic increase with time of base treatment. For the first 5 min, the slope of the line was 33.4 pmol of damaged residues/mg of protein/min (largely reflecting new sites generated by hydrolysis of endogenous methyl esters), but for the next 355 min the slope was 1.8 pmol of
damaged residues/mg of protein/min, reflecting base-catalyzed generation of new damaged sites as for the Pcmt1
/
proteins. It is thus possible to correct the data to quantitate
endogenous methylated sites. We found that inclusion of endogenous
methyl esters increases by 2.1-fold the damage estimate in extracts
from wild-type brain. Because deesterification of a methylated
L-isoaspartyl residue produces an unmethylated
L-isoaspartyl residue only about 80% of the time, the true
number of damaged residues in wild-type brain proteins can be as much
as 2.4-fold higher than the number of residues detected in the absence
of base treatment. Even with this increase, however, there are still
about 4-fold more damaged residues in Pcmt1
/
brain
proteins than in wild-type brain proteins.
4 C. Farrar, E. Kim, S. Young, S. Clarke, and C. Houser, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PCMT1, L-isoaspartyl (D-aspartyl) O-methyltransferase; AdoMet, S-adenosyl-L-methionine; [14C]AdoMet, S-adenosyl[methyl-14C]-L-methionine; NSE, neuron-specific enolase; BisTris, 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol.
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