Download Sequential deletion of C-terminal amino acids of the E1α component

© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 7 1041–1048
Sequential deletion of C-terminal amino acids of the
E1α component of the pyruvate dehydrogenase (PDH)
complex leads to reduced steady-state levels of
functional E1α2β2 tetramers: implications for patients
with PDH deficiency
Agnieszka Seyda, Gillian McEachern, Richard Haas1 and Brian H. Robinson+
Department of Biochemistry, University of Toronto, and Metabolism Research Programme, Research Institute,
The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada and 1Departments of
Neurosciences and Paediatrics, University of California—San Diego, San Diego, CA, USA
Received 5 November 1999; Revised and Accepted 1 February 2000
Human pyruvate dehydrogenase (PDH) complex
deficiency is an extremely heterogeneous disease
in its presentation and clinical course. We have
characterized novel mutations that affect the C-terminal
portion of the PDH-E1α-coding sequence. Although the
molecular defects underlying these mutations are
different, both effectively produce a stop codon
prematurely three amino acids from the C-terminus.
The clinical and biochemical consequences of these
mutations are unusual in that the affected individuals
are very long-term survivors with PDH complex
deficiency despite having low (<20%) activity in skin
fibroblasts. These findings prompted us to investigate
the C-terminus of E1α in greater detail. We constructed
and expressed a series of PDH-E1α deletion mutants in
a cell line with zero PDH complex activity due to a null
E1α allele. Sequential deletion of the C-terminus by one,
two, three and four amino acids resulted in PDH
complex activities of 100, 60, 36 and 14%, respectively,
compared with wild-type E1α expressed in PDH
complex-deficient cells. The immunodetectable protein
was decreased by the same amount as the activity,
suggesting that the stability and/or assembly of the
E1α2β2 heterotetramer might depend on the intactness
of the PDH-E1α C-terminus. In addition, we compared
the somatic and the testis-specific isoforms of E1α and
concluded that they are biochemically equivalent.
INTRODUCTION
The pyruvate dehydrogenase (PDH) complex is the link
between the glycolytic pathway and the tricarboxylic acid
cycle and catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA. The PDH complex is a multienzyme
system composed of multiple copies of three catalytic compo+To
nents, E1 (E1α and E1β), E2 and E3, and an additional component, protein X, which is thought to play a role as an E2–E3binding polypeptide (1–3). The activity of the PDH complex is
regulated by a PDH-specific kinase and a PDH-specific
phosphatase, which phosphorylate/dephosphorylate the E1α
component of the E1 subunit. PDH also utilizes four co-factors:
thiamine pyrophosphate (TPP), which binds non-covalently to
the E1 subcomponent; lipoic acid, which binds covalently to a
lysine residue located on the E2 subcomponent; FAD, which is
bound by E3; and NAD+, which is reduced by E3. TPP is probably bound by elements of both E1α and E1β subunits, so that
each α2β2 heterotetramer has two TPP molecules, one each
bridging E1α and E1β to form an active site (4).
PDH deficiency due to defective E1 is a well defined inborn
error of metabolism that is clinically very heterogeneous. It is
a major cause of primary lactic acidosis and Leigh disease in
both male and female children, often accompanied by cerebral
atrophy or microcephaly (1,5). In males, it may also present as
a milder carbohydrate-sensitive, intermittent ataxia syndrome
(1). Whereas there are two E1α genes, one encoded on the X
chromosome and one on chromosome 4, mutations in the
somatically expressed X-linked E1α gene are responsible for
producing the E1 deficiencies associated with PDH complex
defects (1,6–8). There is no expression of the chromosome 4
gene in tissues other than the testis (8). Suggestions that the
testis isoform could substitute for the X-linked coded E1α
protein if suitably expressed have been evaluated using a
bacterial expression system, with some success (9). The testis
E1α gene product will integrate to produce a fully functional
PDH complex able to undergo phosphorylation and
dephosphorylation, as does the complex made with the Xlinked E1α gene product (9). In this study, we have used human
skin fibroblasts with no endogenous E1 activity (the π0 cell
line), as a vehicle to express both the somatic and the human
testis-specific E1α isoforms and compare their activities.
In the past, there have been reports that some mutations
within the E1α-coding region that affect the C-terminus lead to
whom correspondence should be addressed. Tel: +1 416 813 5989; Fax: +1 416 813 8700; Email: [email protected]
1042 Human Molecular Genetics, 2000, Vol. 9, No. 7
a significant decrease in E1α- as well as E1β-immunoreactive
material (10–12). Two of these cases are female patients, and
the biochemical and protein expression data are hard to evaluate since one normally expressing E1α allele is present and
cell cultures are a mosaic (10). In the case of PDH deficiency
in a 10-year-old male with relatively mild symptoms of exercise intolerance and lactic acidemia, the E1α mutation resulted
in the addition of 33 amino acids with a residual PDH complex
activity of 27.4% (12). In these cases, little systematic investigation has been carried out to determine whether the decreased
amount of activity is due to the mutation itself, decreased
mRNA, decreased protein import into mitochondria or
increased mitochondrial degradation of imported mutant
protein.
Here we present results from three patients with mutations in
the C-terminal portion of the E1α-coding region, who are very
long-term survivors with PDH complex deficiency. We then
compare these results with those of a study in which four
C-terminal amino acids of the α subunit of E1 were deleted
sequentially and the resulting E1α variants were expressed in a
π0 cell line.
RESULTS
Case reports
Patients 1 and 2. These are twin brothers born in 1959 who
were normal until 2 years of age when they started to have
episodes of weakness, ataxia and hyperventilation. They were
labeled as having cerebral palsy in their early childhood. One
twin appears to be more adversely affected than the other,
though both have persevered with education at a school for the
disabled. Although they have learning difficulties, they are not
mentally retarded. A recent admission for one twin (G.F.) after
a collapse followed by dystonic movements revealed hypodensities in the internal capsule and basal ganglia on CT scanning. During this crisis, he had an elevated blood lactate but
this soon returned to normal. In cultured skin fibroblasts from
the patients, overall PDH complex activity was 12.2 and 12.4%
for the native complex (patients 1 and 2, respectively) and 13.8
and 14.6% for the dichloroacetate (DCA)-activated complex
(patients 1 and 2, respectively). In cultured lymphoblasts from
the patients, overall PDH complex activity was 25.7 and 22.8%
for the native complex (patients 1 and 2, respectively), and
32.5 and 25.7% for the DCA-activated complex (patients 1 and
2, respectively).
Patient 3. This patient, now 39 years old, first diagnosed as
PDH deficient by John Blass (13) many years ago, was seen
initially at 17 months for intermittent ataxia and dysarthria. He
continued to have 5–6 attacks per year until his teens, when the
frequency decreased to 1–2 episodes per year. Attacks are
usually precipitated by viral illness, stress or anxiety and are
associated with high blood pyruvate levels (0.4 mM). In addition to his intermittent symptoms, he has constant coarse
bilateral hand tremor, dysarthria and progressive Dupuytren’s
contractures in both hands. He also has persistent appendicular
and truncal ataxia which worsens during periods of metabolic
decompensation. Vertigo, particularly pronounced when
viewing rippling water, occurred in recent ataxic attacks. At
Figure 1. Western blot analysis of cultured skin fibroblasts (A) and lymphoblasts
(B). A 100 µg aliquot of total protein from mitochondria-rich fractions was
separated on a 12.5% SDS–polyacrylamide gel and transferred to nitrocellulose.
The filter was probed with an antibody against affinity-purified pig heart PDH
complex raised in rabbits. Lane 1, control (C); lane 2, patient 3; lane 3, patient 1;
lane 4, patient 2.
ages 34 and 37 years, he suffered acute psychotic episodes
requiring psychiatric in-patient care and antipsychotic medications, with paranoia, anxiety, memory loss and mutism. He has
also been severely depressed. Despite his episodic illness, the
patient attended university, obtaining a degree in computer
engineering. He works as a computer programmer. He has
been treated with intermittent acetazolamide, intermittent
prednisone and persistently with 300 mg/day of thiamine. In
cultured fibroblasts from the patient, overall PDH complex
activity was 14.7% for the native complex and 22.8% for the
DCA-activated complex.
Western blot analysis showed lowered PDH-E1α and -β
immunoreactivity for all three patients, with ∼15 and 30% of
levels of the control present in fibroblasts and lymphoblasts,
respectively (Fig. 1).
Characterization of the mutations
Patients 1 and 2 have an AAGT insertion at 1159 and patient 3
has a C1163T change. Both mutations effectively produce a
stop codon prematurely a short distance from the C-terminus
(Fig. 2).
Creation of the π0 cell line for E1α expression
We transformed a human skin fibroblast cell line derived from
a female patient with a mutation that nullified one of the PDHE1α alleles (see Materials and Methods). After transformation
and cloning of individual foci, two types of clone were identified: those that had normal PDH complex activity and those
Human Molecular Genetics, 2000, Vol. 9, No. 7 1043
Table 1. Total PDH complex activities recorded and averaged for PDH-E1α
variants transfected into the π0 cell line
E1α variant
PDH complex activity (nmol/min/mg)
Native
DCA activated
Control
0.98 ± 0.14 [10]
1.12 ± 0.16 [10]
100
E1α cDNA
0.47 ± 0.09 [5]
0.61 ± 0.06 [5]
55
Testis-specific
0.53 ± 0.10 [6]
0.59 ± 0.08 [6]
54
∆1
0.49 ± 0.11 [5]
0.62 ± 0.04 [5]
55
∆2
0.34 ± 0.04 [5]
0.37 ± 0.05 [5]
33
∆3
0.19 + 0.04 [4]
0.23 + 0.03 [4]
20
∆4
0.10 ± 0.03 [7]
0.09 ± 0.05 [7]
8
π0
0.0 [11]
0.0 [11]
0
% of controla
The numbers of determinations for each clone are given in square brackets.
approximation given to enhance the clarity of the table.
aAn
Expression of C-terminus deletion mutants of E1α
Figure 2. Sequencing of plasmids containing PCR products of PDH-E1α cDNA.
The arrow and brackets indicate the site of C1163A substitution and AAGT ins at
bp 1159, respectively. A control (C) and the affected males are shown.
Figure 3. Western blot analysis of the π0 cell line as well as the parental and control
cell lines. A 60 µg aliquot for the control and the parental cell line, and 100 µg for
the π0 cell line of total protein from mitochondria-rich fractions were separated on
a 12.5% SDS–polyacrylamide gel and transferred to nitrocellulose. The filter was
probed with an antibody against affinity-purified pig heart PDH complex raised in
rabbit. C, control cell line; P, parental cell line.
that had zero PDH complex activity, renamed π0. Both types as
well as the parental cell line (see Materials and Methods) were
probed with anti-total PDH complex antibody by western blotting. As shown in Figure 3, neither E1α nor E1β subunits were
expressed in the π0 cell line; however, the expression of the E2
component of the PDH complex remains unaltered. Clones
with normal PDH complex activities also had normal expression of the PDH complex subunits. The parental cell line,
however, showed a substantial decrease in both the E1α and
E1β protein levels. Densitometry analysis of protein peaks
reveals that the amount of each of the E1 subunits was ∼10% of
the control. This correlates with the 7.5 ± 2.3% residual
activity in the parental cell line.
To investigate the importance of the C-terminus of PDH-E1α
for the stability and/or assembly of the E1α2β2 heterotetramer,
we constructed a series of PDH-E1α deletion mutants in which
each mutant was sequentially shorter by one amino acid from
the C-terminus. cDNA constructs corresponding to these E1α
amino acid variants (∆1, ∆2, ∆3 and ∆4) as well as the testisspecific isoform were subcloned into a mammalian expression
vector and transfected into the π0 cell line. All PCR-generated
E1α cDNA variants were digested with KpnI–XhoI, and
subcloned into the mammalian expression vector pcDNA 3.1.
The individual clones were sequenced to confirm that there
were no PCR artifacts generated. The error-free clones were
used in the transfection experiments.
The π0 cell line was transfected with cDNAs encoding the
wild-type E1α, the testis-specific E1α as well as four different
C-terminus deletion mutants of E1α: ∆1, ∆2, ∆3 and ∆4. Stably
expressing clones were selected, subcloned and analyzed for
expression of the E1α variant proteins by the total PDH
complex activity assay (Table 1), as well as by the binding of
antibody directed against pig heart total PDH complex (Fig. 4).
Levels of exogenous E1α mRNA expression were examined by
quantitative PCR using phosphoglycerate kinase (PGK)
mRNA as an internal control (Fig. 5). This method was chosen
to differentiate between the endogenous and exogenous E1α
mRNA levels due to the fact that, although the π0 cell line
presents with a complete loss of enzyme activity and immunoreactive E1α protein, the transcription and message production
of the endogenous E1α remain unaltered.
Analysis of PDHC activity assays
Complete recovery of activity on post-transfectional wild-type
E1α cDNA expression in π0 cell lines was not achieved
(Table 1), and rates of only 55% compared with control were
recorded. Expression of the testis-specific E1α as well as the
∆1 variant resulted in a comparable restoration of activity:
∼55%. When rates for ∆2, ∆3 and ∆4 were recorded and averaged, they were 33, 20 and 8%, respectively (Table 1). There-
1044 Human Molecular Genetics, 2000, Vol. 9, No. 7
Figure 4. Western blot analysis of the exogenous expression of the wild-type and
variant PDH-E1α. An 80 µg aliquot of total protein from mitochondria-rich
fractions was separated on a 12.5% SDS–polyacrylamide gel and transferred to
nitrocellulose. The filter was probed with an antibody against affinity-purified pig
heart PDH complex raised in rabbit. The amounts of E1α were normalized by
densitometry analysis to the amounts of E2 (PDH core protein) and are expressed
underneath the figure as the ratio E1α:E2. The ratio E1α:E2 for the control SV40transformed cell line was taken as 1.
fore, a specific pattern emerged from this part of the study, in
which total PDH complex activity decreased as the C-terminal
residues were removed one by one from the E1α protein.
Figure 5. Quantitative RT–PCR specifically designed to pick up the exogenous but
not the endogenous E1α mRNA, performed on wild-type E1α (A), ∆1 variant (B)
and ∆4 variant (C). The experiment was performed on 1 µg of total RNA extracted
from a single confluent 100 mm plate. The ratio of PGK to E1α was determined by
densitometry and found to be ∼1:2. Similar results were obtained for the remaining
E1α variants. The number of PCR cycles performed is indicated on the top of each
panel.
Analysis of expression of variant E1α proteins
To compare the rates of total PDH complex activities with the
levels of E1α protein expression, western blotting was
performed using antibodies directed against total PDH
complex. The levels of E1α expression for each clone were
standardized to the corresponding levels of E2 (the core
protein) by densitometry analysis and expressed as a ratio
E1α:E2. The ratio E1α:E2 for the SV40-transformed control cell
line was taken as 1 (Fig. 4). Results of this experiment mirror
results obtained from the activity assays such that progressively lower amounts of immunoreactive E1α protein were
detected as the C-terminal E1α residues were removed. The
amount of E1α protein expressed for each mutant also correlates with the corresponding activity of the complex, perhaps
indicating that the specific activities of mutant complexes are
unaltered. The expressed levels of E1β progressively decrease
as well, implying that E1β is incapable of autonomous existence without E1α (1,14,15). The amount of protein detected for
the testis-specific and wild-type E1α were comparable, further
supporting the hypothesis that these isoforms are interchangeable within the PDH complex.
Analysis of mRNA levels for exogenous E1α variants
Results for the quantitative PCR performed on clones expressing
wild-type, ∆1 and ∆4 PDH-E1α are shown in Figure 5. Densitometric analysis of band intensities showed that when compared
with the internal control (PGK, upper band), the levels of
exogenous mRNA transcribed from the cytomegalovirus
(CMV) promoter of the transfected vectors were similar in each
cell line. Comparable results were obtained for the remaining
PDH-E1α clones (data not shown). Therefore, these findings
show that lower protein levels for E1α deletion mutants were not
caused by lower levels of mRNA transcription.
Import of E1α variants into the mitochondrial matrix
To investigate whether lower levels of immunoreactive E1α
deletion mutant proteins were due to deficient import into
Figure 6. In vitro import of human precursor PDH-E1α. Full-length precursor
wild-type and variant E1α proteins were synthesized using a TNT-coupled
reticulocyte lysate system (Promega) with [35S]methionine as radioactive label
(Amersham Pharmacia Biotech). Import into isolated rat heart mitochondria was
for 10 and 30 min, as indicated. (–) mitos, in vitro translated wild-type E1α
incubated without rat heart mitochondria; p-E1α, 49 kDa precursor; m-E1α,
42 kDa mature form.
mitochondria, all PDH-E1α variant proteins were synthesized
in vitro and added to freshly prepared rat heart mitochondria. A
mitochondrial matrix protein, MnSOD, was used to assess the
import competence of the system (data not shown). Results for
this experiment are shown in Figure 6. The 49 kDa precursor
proteins were imported into mitochondria isolated from rat
heart, being processed to the mature size 42 kDa protein. By
densitometric analysis, it was established that despite small
differences in overall amount adhering to mitochondria, by
comparing the amount of precursor E1α (p-E1α) with that of
mature E1α (m-E1), there was no difference in precursor
processing between wild-type E1α and any one of the investigated E1α deletion variants.
DISCUSSION
We have shown that three patients with a long-lived mild
phenotype of PDH complex deficiency display a strong correlation between residual PDH complex activity in fibroblasts
and lymphoblasts and the levels of the E1α and the E1β
subunits as seen by immunoblot. E1 subunit levels of 12–15%
Human Molecular Genetics, 2000, Vol. 9, No. 7 1045
seen in fibroblasts correlated with residual activities of the
PDH complex of ∼15%, and residual E1α levels of 25% seen in
lymphoblasts correlated with enzyme activities of 26–32% for
the PDH complex. These patients had E1α mutations that
effectively removed the three C-terminal amino acids
(SerValSer) from the protein, exposing a lysine as the Nterminal residue. For the majority of patients investigated with
PDHC-E1 deficiency, residual rates in fibroblasts and
lymphoblasts are the same, whereas rates in heart, kidney and
liver can vary in the same patient (1). Thus, the 2-fold difference in activity and protein seen in lymphoblasts versus fibroblasts was due to different residual levels of mutant E1 in the
complex presumably caused by differential rates of degradation. This conclusion that the missing C-terminal amino acids
led to increased degradation of a potentially functionally active
protein E1 component was investigated further by making
mutant cDNA constructs coding for subunits missing one, two,
three and four amino acids and expressing them in a π0 cell line
totally devoid of PDH complex activity. The residual activity
of the PDH complex again decreased with increasing number
of amino acids removed, correlating strongly with the amount
of immunotitratable E1α and E1β subunits in the complex,
suggesting no change in specific activity of E1 (Fig. 4). This
supports a mechanism in which truncations of E1α appear to
bestow instability either on the E1α2β2 tetramer or, alternatively, on the whole assembled complex. The fact that E2 levels
remain the same while E1α and E1β are depleted mitigates
against a mechanism involving the whole complex. Similarly,
differential degradation of E1α mRNA species and possible
failure of import of truncated E1α protein were ruled out by
examining mRNA levels and import, respectively (Figs 5 and
6). Missense mutations of E1α with no change in protein length
may also present in males as carbohydrate-sensitive ataxia (1).
In this case, the change in activity may be due to E1 protein
degradation or more usually due to a change in specific activity
of the E1 component (1,14–16).
The present study showed that it is possible to complement
the PDH enzyme defect caused by a faulty PDH-E1α gene by
transfecting the cells with the normal PDH-E1α cDNA.
Although normal levels of activity were never achieved, in
stably transfected populations significantly higher values were
recorded than had been reported previously by others for this
type of transfection (17). It is known that the E1α subunit alone
has no demonstrable enzymatic activity and requires assembly
with E1α in order to form an active α2β2 heterotetramer. Otherwise, the proteins degrade. Examples of the instability of individual subunits of heterotetramers have been seen in patients
with PDH deficiency (18,19), and branched-chain α-keto acid
dehydrogenase deficiency (20,21). Therefore, the overexpressed E1α proteins may degrade at a time when there is no
E1α protein available due to uncoordinated expression (22,23).
The influence of thiamine on patients with PDH complex
deficiency has been controversial, with many patients having
been treated from the time of diagnosis in infancy (1,24). TPP
is thought to be bound by elements of both E1α and E1β
subunits; each α2β2 heterotetramer has two TPP molecules,
one each bridging E1α and E1β to form an active site (4). The
patients here represent the two ends of the spectrum in that one
pair of sibs were totally untreated because of late diagnosis as
adults, whereas the other patients with the same mutation were
treated from an early age. It would seem that the treated
patients have fared better than the untreated patients. Patients
1, 2 and 3 belong to a group of relatively long-lived, mildly
affected males who have the presentation originally referred to
by us and others as carbohydrate-sensitive ataxia (25,26).
There are two further patients from the Maritime provinces of
Canada who have the 1159 AAGT ins mutation (19). These
unrelated males are both now 13 years of age, attend special
school and have bouts of ataxia, which are carbohydrate and
stress induced. So far, these patients have no detectable central
nervous system lesions and have been given thiamine supplements from an early age. Our conclusion, therefore, is that this
group of patients with apparently low residual fibroblast activities of PDH complex nevertheless have mild phenotypes
because the specific activity of E1 is not altered by C-terminal
truncation. Overall residual activity is governed by tissuespecific degradation mechanisms allowing the brain to have
the 30–40% normal activity necessary for normal function (1).
Despite its importance, the molecular mechanisms
governing the tight tissue-specific regulation of the testisspecific isoform of the PDH complex E1α subunit remain
poorly understood (27). Even more limited data have been
published on the biochemical aspects of this isoform. Results
obtained in this study show for the first time that the expression
of the testis-specific E1α isoform in a mammalian cell line
produces a fully functional PDH complex, supporting previous
in vitro findings (9). This shows that should a mechanism for
inducing somatic expression of the testis-specific E1α isoform
be found, correction of X-linked E1α defects might be feasible.
MATERIALS AND METHODS
Enzymology and immunological analysis
Cultured skin fibroblasts were grown from skin biopsies in
α-minimal essential medium (α-MEM) culture medium. The
activity of the PDH complex in the native and DCA-activated
state was determined in fibroblast extract by the method of
Sheu et al. (28). Western blotting was performed on mitochondrial extracts prepared by the method of Pitkanen et al. (29),
using a polyclonal antibody against porcine heart PDH
complex raised in rabbit (30).
RNA and cDNA preparation
Total RNA was extracted from cultured skin fibroblasts using
TRIzol reagent (Total RNA Isolation Reagent) from GibcoBRL (Burlington, Canada). First strand cDNA synthesis was
carried out using total cellular RNA (20 µg) and an E1αspecific oligonucleotide aG17′ (5′-TCTAGAATTCGTACAAACTGCATGCAATTAC-3′) with M-MLV reverse transcriptase (Gibco-BRL).
Amplification of DNA
DNA was amplified by PCR with cDNA as a template, 250 µM
of each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 10 µl
of 10× PCR buffer (Gibco-BRL), 1.5 mM MgCl2, oligonucleotide primer (1 µg each) and 2 U of Taq polymerase (GibcoBRL), in a total reaction volume of 100 µl. PCR amplification
(31) of the cDNAs extracted from primary cell lines was carried
out as described previously (19). Briefly, the total coding
sequence of the E1α subunit was amplified in two overlapping
1046 Human Molecular Genetics, 2000, Vol. 9, No. 7
fragments, subcloned into pSP65 T-A cloning vector (Clontech,
Palo Alto, CA) and sequenced using a series of α-specific
primers. DNA was prepared from cultured skin fibroblasts of
patients and control cell lines by a modified version of the
method of Miller et al. (32). After 30 cycles of amplification
under the conditions specified, the amplified fragments were
visualized on ethidium bromide-stained gels, extracted and
subcloned into pSP65. All DNA sequencing was performed by
the Sanger dideoxy chain termination method (33) on doublestranded templates using a T7 polymerase sequencing kit (Pharmacia, Quebec, Canada). To make E1α deletion mutants, 5 µl of
the cDNA reaction mixture was used for PCR using
E1α-specific forward primer Kpn-F (5′-TTTGGTACCTTGTGAGGAGTCGCCGCTGC-3′) and a series of reverse primers,
each containing an XhoI restriction site:
Xho-R (5′-TTTCTCGAGGAGAACACTGTCTGGTAGCC-3′)
for wild-type E1α cDNA;
E1α-∆1-R (5′-TTTCTCGAGTTAGACTGACTTAAACTTGATCCACTG-3′) for ∆1 cDNA;
E1α-∆2-R (5′-TTTCTCGAGTTARGACTTAAACTTGATCCACTGATTC-3′) for ∆2 cDNA;
E1α-∆3-R (5′-TTTCTCGAGCTTAAACTTGATCCACTGATTGGC-3′) for ∆3 cDNA;
E1α-∆4-R (5′-TTTCTCGAGAAACTTGATCCACTGATTGGCACC-3′) for ∆4 cDNA.
The last four primers were designed so that a series of E1α
cDNA clones was made that were sequentially shorter by three
nucleotides. On translation, they produce a series of E1α
proteins sequentially shorter from the C-terminus by one
amino acid.
The PCR conditions used were: 94°C for 1 min, 60°C for 1
min and 72°C for 1.5 min for 35 cycles. The PCR product was
then subcloned into pCR 2.1 (Invitrogen, Carlsbad, CA) using
a TA Cloning kit (Invitrogen).
Construction of the testis-specific variant of E1α
Since the testis-specific gene is intronless, its sequence was
obtained from genomic DNA by PCR. PCR primers were
designed with 5′ restriction sites for easy cloning: forward primer
Ts-F
(5′-TTTGGTACCTGCCATCTACAGCACTCCGT-3′)
and reverse primer Ts-R (5′-TTTCTCGAGCCTCCTTGAGTTGAGAACAC-3′). The PCR conditions used were: 94°C for 30 s,
58°C for 30 s and 72°C for 1 min 30 s. The testis-E1α was also
first subcloned into pCR 2.1 to find an error-free clone [in
comparison with the sequence published by Dahl (34)] and then
recloned into the KpnI–XhoI sites of pcDNA 3.1.
DNA sequence analysis
Plasmid clones containing PCR product cDNAs were
sequenced with the T7 Sequencing kit according to the manufacturer’s recommendations (Amersham Pharmacia Biotech).
The reaction mixture was separated on an acrylamide gel. The
entire coding region of the patient’s cDNA was sequenced
with the primers described (35).
Cloning of E1α variants into the expression vector
Wild-type and mutant clones were digested with KpnI–XhoI
and subcloned into the KpnI–XhoI site of pcDNA 3.1
(Invitrogen).
Construction of π0 (PDH complex-deficient) cell line
A human female skin fibroblast cell line with a 7 bp deletion at
base 931 (exon 10) in one of the E1α alleles was transformed
with SV40 large T antigen using a vector containing a large
T antigen DNA (courtesy of Dr John Dick, HSC, Toronto) by
transfection. Transfection was carried out using the Superfect
reagent (Qiagen, Mississuaga, Canada) according to the manufacturer’s specifications on cells 18–22 h after splitting at a
confluence of 60–80% in six-well plates (35 mm). A 2 µg
aliquot of column-purified (Qiagen) plasmid DNA was
combined with 8 µl of the Superfect reagent in 150 µl of serumfree α-MEM. Cells were split 24–36 h after transfection at a
ratio of one 35 mm plate to five 100 mm plates. After 10–14
days, transformed foci were cloned and assayed for PDH
complex activity. Clones that had zero PDH complex activity,
named π0, were chosen for further characterization. In order to
confirm the absence of the E1 protein, western blot analysis was
performed on mitochondria from the π0 cell line, the control cell
line and the parental cell line. Twice the amount of protein was
used for the π0 cell line than for the control or the parental cell
lines to ensure that the experiment was not performed at the antibody’s limit of immunoreactivity.
Expression of wild-type and mutant E1α variants in the π0
cell line
All transfections were performed as described above using the
π0 cell line and Qiagen column-purified DNA (either wildtype, mutant or vector alone), which was linearized with PvuI.
Two days post-transfection, cells were plated into 100-mm
plates and selection was applied at a concentration of 0.2 mg/
ml G418 for 14–20 days until visible colonies could be
detected. These colonies were cloned using cloning rings, and
PDH complex activity was determined.
Quantitative PCR
RNA for quantitative PCR was isolated from transfected human
skin fibroblasts using a total nucleic acid isolation kit (Qiagen). First
strand cDNA synthesis was performed as described above using
oligo(dT) as a primer. A negative control was designed so that first
strand synthesis was carried out without MLV reverse transcriptase
and used later in the PCR to ensure that no plasmid DNA contamination gave false-positive results. PCR was performed using a set of
target primers as well as a set of internal control primers targeted
against human PGK (both sets of primers were present in the same
PCR). Primer sequences were target primers: pcDNA 3.1F (5′TTAATACGACTCACTATAGGGAG-3′
dihydrolipoamide
acetyltransferase core) and pcDNA 3.1R (5′-AACTAGAAGGCACAGTCGAGG-3′); and internal control primers: PGK-For
(5′-CAGTTTGGAGCTCCTGGAAG-3′) and PGK-Rev (5′TGCAAATCCAGGGTGCAGTG-3′).
PCR was carried out as described above using the following
set of conditions: 94°C for 30 s, 59°C for 30 s, 72°C for 45 s in
a total volume of 50 µl. The following numbers of cycles were
performed: 18, 20, 22, 24, 26, 28 and 30. One PCR tube was set
up for each set of cycles. Samples were run on a 2% agarose
gel, in sequential order of the number of cycles, and the
amount of PCR product was quantitated by densitometry.
Human Molecular Genetics, 2000, Vol. 9, No. 7 1047
Cell culture
Untransfected SV40-transformed human skin fibroblasts were
grown in α-MEM supplemented with 15% fetal bovine serum
(FBS). Transfected SV40-transformed human skin fibroblasts
were grown in α-MEM supplemented with 15% FBS together
with 0.2 mg/ml G418. Increased thiamine concentration in the
medium was achieved whenever necessary by addition of
0.1 mg per 10 ml of medium.
Mitochondrial import studies
Mitochondria were isolated from rat hearts by the method of
Takahashi and Hood (36). Full-length cDNAs encoding wildtype and variant E1α as well as human MnSOD were subcloned
into pGEM 3Z. Proteins were synthesized with the TNTcoupled reticulocyte lysate system (Promega, Madison, WI)
using [35S]methionine as radioactive label (Amersham
Pharmacia Biotech). Mitochondria were incubated with lysate
containing the translated radiolabeled precursor proteins at 30°C
for various periods of time, as indicated below. Final import
reactions consisted of 40 µl (120 µg) of mitochondria, 5 µl of
reticulocyte lysate and 2 µl of translation reaction. Glutamate
was added as an additional respiratory substrate at a final
concentration of 33 mM. Following incubation, mitochondria
were recovered by centrifugation and washed twice with the
import buffer. Pellets were resuspended in 15 µl of import buffer
and 15 µl of 2× loading buffer [10% glycine (v/v), 80 mM SDS,
62.5 mM Tris–HCl pH 6.8, 5% 2-mercaptoethanol (v/v) and
10% Coomassie blue]. Samples were denatured and electrophoresed through a 12.5% SDS–polyacrylamide gel at 80 V. Gels
were fixed [30% methanol (v/v), 3% glycerol (v/v)], treated for
30 min with Amplify (Amersham Pharmacia Biotech), dried and
exposed to film (Bio-Max; Kodak, Rochester, NY) at –80°C.
Autoradiograms were quantified using densitometry.
ACKNOWLEDGEMENTS
We thank Dr Ives of St John’s, Newfoundland, for the cell
lines of patients 1 and 2, and MRC Canada for the financial
support of this research project.
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