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Molecular basis of maple syrup urine disease and stable correction by retroviral
gene transfer

Article in Journal of Nutrition · July 1995

DOI: 10.1093/jn/125.suppl_6.1766S · Source: PubMed

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 Symposium: Alpha-Keto Acid Dehydrogenase Complexes:
nutrient Control, Gene Regulation and Genetic Defects
Molecular Basis of Maple Syrup urine Disease and Stable
Correction by Retro viral Gene Transfer12
DAVID T. CHUAHG,3 JAMES R. DAVIE,4 R. MAX IVW/Y, JACINTA
HIROHISA KOYATA5 AND RODY P. COX
Departments of Biochemistry and Internal Medicine,
Dallas, TX 75235
ABSTRACT Maple syrup urine disease (MSÃœD)or
branched-chain ketoaciduria is caused by a deficiency
of the branched-chain a-keto acid dehydrogenase
(BCKAD) complex. This results in the accumulation of
the branched-chain amino acids (BCAA) and branched-
chain a-keto acids (BCKA), which often produce severe
neurological damage and mental retardation. The pres
ent studies focus on mutations in the Eia gene of the
BCKAD complex and their effects on the assembly of
the El decarboxylase component of the enzyme com
plex. We have developed an efficient histidine-tagged
bacterial expression system that allows the folding and
assembly of Eia and El/9 subunits into the El heter-
otetramer (a^ßz)in the presence of overexpressed
chaperonins GroEL and GroES. The results of pulse-
chase experiments with this bacterial expression sys
tem showed that a majority of the 15 known Eia mu
tations, including the prevalent Y393N of Mennonite
MSUD patients, decrease the rate of association of nor
mal El/3 with mutant Eia. This results in limited or
no assembly of mutant El. It is concluded that the
carboxy-terminal region of the Eia subunit encoded by
exons 7-9 is important for subunit interaction. To stably
correct MSCID, we have developed a retroviral vector
that contains a normal Eia precursor complementary
DMA. Transduction of cultured lymphoblasts from a
Mennonite MSUD patient with this recombinant retro-
viral vector completely restored the rate of decarbox-
ylation of BCKA. The normal decarboxylation activity
in transduced MSCID cells remained stable without an
tibiotic selection during the 14-week study. The results
provide a paradigm for the development of somatic
gene therapy for MSUD. J. Nutr. 125: 1766S-1772S,
1995.
INDEXING KEY WORDS:
•maple syrup urine disease •branched-
chain a-keto acid dehydrogenase
•protein assembly defect •retrouiral
gene transfer
0022-3166/95 S3.00 ©1995 American Institute of Nutrition.
1766S
L. CHUANG,
University of Texas Southwestern Medical Center,
The mammalian branched-chain a-keto acid dehy
drogenase (BCKAD) complex belongs to the family of
highly conserved mitochondrial a-keto acid dehydro
Downloaded from jn.nutrition.org by guest on July 13, 2011
genase complexes (Yeaman 1989). The BCKAD com
plex consists of three catalytic components, i.e., a de
carboxylase or El consisting of two a and two ß sub-
units, a transacylase comprising 24 lipoic acid-bearing
subunits and a dehydrogenase, which is a homodimeric
flavoprotein. The enzyme complex also contains two
regulatory enzymes, i.e., a specific kinase and a specific
phosphatase that regulate E1 activity through a phos-
phorylation (inactive)/dephosphorylation (active) cy
cle. There are six genetic loci that encode the mam
malian BCKAD complex. A mutation in any one of
these loci can cause a dysfunction of the complex, re
sulting in maple syrup urine disease (MSUD) (Chuang
and Shih 1995).
The organization of the BCKAD complex is such
that E2 forms a structural core, to which other enzyme
components attach through ionic interactions (Yeaman
1 Presented as part of the symposium "Alpha-Keto Acid Dehy
drogenase Complexes: Nutrient Control, Gene Regulation and Ge
netic Defects" given at the Experimental Biology '94 meeting, An
aheim, CA, on April 27, 1994. This symposium was sponsored by
the American Institute of Nutrition. Guest editors for this sym
posium were Mulchand S. Patel, State University of New York at
Buffalo, Buffalo, NY and Robert A. Harris, Indiana University School
of Medicine, Indianapolis, IN.
2 Supported by Grants DK-26758 and DK-37373 from the Na
tional Institutes of Health and Grant 1-1149 from the March of
Dimes Birth Defects Foundation.
3 To whom correspondence should be addressed: Department of
Biochemistry, University of Texas Southwestern Medical Center,
5323 Harry HiñesBoulevard, Dallas, TX 75235-9038.
4 Medical Scientist Trainee supported by Grant 5-P32 GM-08014
from the National Institutes of Health and by the Perot Family
Foundation.
5 Present address: Department of Biochemistry, Toyama Medical
and Pharmaceutical University School of Medicine, Toyama, Japan.
 MAPLE SYRUP URINE
1989). The heterotetramer (a2ß2} of El binds to the
E1/E3 binding domain of the E2 chain through inter
actions with the El/3 subunit. The human El«subunit
contains the two phosphorylation sites (serine 292 and
serine 302). Both Eia and EIß appear to contribute to
the binding pocket for cofactor thiamine pyrophos-
phate (TPP). The crystal structure of the 24-meric E2
inner core (Mattevi et al. 1992) and E3 homodimers
of the related pyruvate dehydrogenase complex from
Azotobacter vinelandii has been recently solved. A
model was proposed that depicted that the peripheral
El and E3 components are distributed on the surface
of the cubic E2 inner core (Mattevi et al. 1992). A major
interest in our laboratory has been the effect of MSUD
mutations on the folding and assembly of El hetero-
tetramers, which will constitute the first part of this
discussion.
Clinical and genetic heterogeneity in MSUD
MSUD is clinically heterogeneous with five recog
nized distinctive clinical phenotypes, i.e., classic, in
termediate, intermittent, thiamine-responsive and E3-
deficient (Chuang and Shih 1995). Seventy-five percent
of the patients are of the classic type with low residual
enzyme activity. Clinical manifestations include early
onset of severe ketoacidosis, seizures and mental re
tardation. Many patients die during metabolic decom
pensation precipitated by infection or stress. The in
termediate form is milder with progressive mental re
tardation. The intermittent type has late onset of
ketoacidosis associated with stress, but many have
normal intelligence. The thiamine-responsive patients
resemble the intermediate phenotype but show a dra
matic response to pharmacological doses of thiamine.
The mechanism of the thiamine response is not clear.
E3 deficiency is associated with combined a-keto acid
dehydrogenase deficiency because E3 is a common
component of the pyruvate, a-ketoglutarate and
BCKAD complexes. Death occurs during infancy as
the result of lactic and keto-acidoses.
MSUD also is genetically heterogeneous as shown
by mutations in the different loci, which are termed
molecular phenotypes as shown in Table 1. Type IA
refers to mutations in Eia, Type IB in E1/3,Type II in
E2 and Type III in E3. Types IV and V are reserved
for mutations in the kinase and the phosphatase. Most
mutations identified thus far belong to Type IA or El a
and Type II or E2. No mutations are known to occur
in Types IV and V. There is little correlation between
the clinical and molecular phenotypes with the excep
tion of Type III. For example, the classic clinical phe
notype can be caused by mutations in Types IA, IB,
and II.
Mutations in Type IA MSUD
There are 15 mutations in the human Eia gene
(Type IA MSUD), which will be discussed here. Two
DISEASE 1767S
TABLE 1
Genetic heterogeneity in MSUD
ofmutationsidentified1
Molecularphenotype*IAIBIIIIIIVVAffectedlocusEiailߣ2E3KinasePhosphataseClin
intermediateClassicClassic,
thiamine-responsive
E3-deficientUnknownUnknownNo.
* Refers to the various loci of the BCKAD complex affected in
MSUD.
of these are frame-shift mutations caused by a single
C insertion (exon 2) and an 8-bp deletion (exon 7). The
Downloaded from jn.nutrition.org by guest on July 13, 2011
remaining 13 missense mutations can be roughly di
vided into two clusters; one is associated with the TPP
binding, and the others are near the carboxyl terminus
and are related to the subunit interaction. The latter
is exemplified by the Y393N (Tyr-393 to Asn) muta
tion. This mutation was first described by Zhang et
al. (1989) in a compound hétérozygote of Italian de
scent. Later, it was shown to be homozygous in classic
Mennonite patients (Fisher et al. 1991a, Matsuda et
al. 1990). The mutation was a T to A transversion in
exon 9 and is important because of its high frequency
associated with the inbred Mennonite population.
To establish that the Y393N mutation is the cause
of MSUD, our laboratory carried out transfection
studies with this mutation (Fisher et al. 199Ib). We
utilized an EBO vector that contains an Ori P origin
of replication and a sequence for Epstein-Barr nuclear
antigen-1 (EBNA-1). These features allowed the rep
lication of the vector in EB-transformed lymphoblasts.
A hygromycin phosphotransferase-selective marker
conferred antibiotic resistance. The Eia deficient
(Type IA MSUD) lymphoblasts (Lo) were transfected
with the EBO vector carrying either normal or Y393N
Eia precursor complementary DNA (cDNAs) and se
lected for growth in hygromycin. Both untransfected
and transfected cells were assayed for the ability to
decarboxylate a-keto[l-14C]isovalerate by the intact
cell assay. Figure 1 shows that the untransfected Lo
host has low residual activity compared with untrans
fected normal cells. Transfection with the vector
without insert did not increase the residual activity.
Transfection of Lo cells with the vector carrying nor
mal Eia restored the decarboxylation rate to that of
normal cells. In contrast, when the Lo cells were trans
fected with the vector carrying the Y393N mutation,
no activity was detected. These results provide direct
evidence that the Y393N mutation is responsible for
1768S SUPPLEMENT
l I
FIGURE 1 Decarboxylation rates of intact normal and
El a-deficient lymphoblasts (Lo)transfected with EBOvectors
containing normal and mutant Y393N Eia cDNA. The de-
carboxylation rate is expressed as nmol CO2/min/mg protein.
The vector alone without insert serves as a control. The ac
tivities of untransfected normal and Lo cells are in the two
left bars. Results are expressed as a means ±SEM(n = 6). The
rates of decarboxylation of [1-UC] pyruvate were normal in
both untransfected and transfected cells (data not shown).
Reproduced from Fisher et al. (l99Ib) with permission.
the MSUD phenotype in homozygous Mennonite pa
tients. The lysates of untransfected and transfected
lymphoblasts were analyzed by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis and Western
blotting using either anti-El a or anti-El ß antibodies
as a probe (Fig. 2). Both recombinant normal and
Y393N Eia subunits were expressed in Lo cells to a
level comparable with untransfected normal cells (top).
However, a fraction of the recombinant El«appeared
to be partially degraded. The EIßsubunits were not
detectable in untransfected Lo cells with Type IA mu
tations. Introduction of a normal El«cDNA into Lo
cells restored the E1/3to a level significantly less than
untransfected normal lymphoblasts. This result was
surprising in view of the normal levels of decarbox-
ylase activity in transfected Lo cells. This suggested
that there may be an excess of unbound El in normal
cells. On the other hand, in cells transfected with the
Y393N Eia, there was no restoration of the Elßsub-
unit. This strongly suggested that Y393N Eia failed
to assemble with EIß or forms an unstable a2ßi com
plex with degradation of the EIß subunit.
Expression and assembly of the human El
component in Escherichia coli
To elucidate the putative El assembly defect, we
developed a bacterial expression system in which Eia
and E1/8subunits were coexpressed on the same plas-
mid (Davie et al. 1992). The rationale was that inde
pendent expression and mixing of Eia and EIßsub-
units in vitro did not result in assembly of a functional
a2/32heterotetramer. In the coexpression vector, the
amino-terminal end of mature Eia cDNA was fused
to a mature bacterial maltose-binding protein (MBP)
sequence through a specific linker recognized by en-
doprotease factor Xa. The coexpression vector was
transformed into £.coli. The assembled fusion protein
MBP-E1 was purified from the bacterial lysate by
amylose resin affinity chromatography, followed by
elution with a maltose solution. The purified MBP-E1
was digested with factor Xa to remove the MBP ligand.
The digest was separated by a sizing chromatography
on a Sephacryl S-300, and the column fractions were
analyzed by Western blotting and El activity assay.
The results show that a fraction of nonfused Eia and
a trace of E10 were aggregated and eluted in the void
volume. A significant amount of assembled El eluted
Downloaded from jn.nutrition.org by guest on July 13, 2011
as a 160-kDa species, which is consistent with an a2ß2
composition. The activity profile coincided with that
of assembled El. When the same experiments were
carried out with MBP-Ela containing the Y393N mu
tation, there was no assembly of a functional El with
the expected size of 160 kDa, as indicated by the
Western blot profile of column fractions. Incompletely
digested mutant MBP-Ela, Y393N-Ela and Elßco-
eluted as large aggregates in the void volume. These
E1a
-E1ß
FIGURE 2 The protein assembly defect associated with
the Y393N mutation in the Eia subunit. Western blot anal
ysis was carried out using lysates from normal and Eia de
ficient (Lo)cells untransfected or transfected with EBO vec
tors containing the normal or Y393N form of Eia cDNA.
Each lane contains 200 tig of protein. After SDS-polyacryl-
amide gel electrophoresis, proteins were electrotransferred
to polyvinylidene difluoride membranes. The filters were
probed with either Eia (top) or E1/3(bottom) antibodies ra-
diolabeled by coupling with U5I-protein A. Reproduced from
Fisher et al. (1991b) with permission.
 MAPLE SYRUP URINE
results supported the thesis that the Y393N mutation
impedes the El assembly, resulting in dysfunction of
the BCKAD complex.
The study thus far established the protein assembly
defect in Mennonite MSUD patients. However, the
bacterial expression system was inefficient in that the
majority of normal recombinant El subunits occurred
in inclusion bodies as aggregated polypeptides. Be
cause the BCKAD complex is a mitochondrial mac-
romolecule, we suspected that molecular chaperones
might be required to assist in folding and assembly.
Molecular chaperones are a class of oligomeric proteins
that facilitate folding of other proteins by suppressing
aggregation or inhibiting kinetic trapping in the fold
ing pathway but are themselves not part of the final
structure (Ellis and van der Vies 1991). Thechaperone
proteins are highly conserved from bacteria to humans.
In E. coli there are homologs of mitochondrial hsp60
and hsplO called GroEL and GroES, respectively.
GroEL and GroES collaborate in facilitating the head
assembly of bacteriophages T4 and T5 (Ellis and van
der Vies 1991). To facilitate expression and assembly
of El, our approach was to overexpress chaperonin
GroEL and GroES in the same host. We cotransformed
two plasmids into an ESts£.coli host, CG-712 (Wynn
et al. 1992). The first plasmid pHl coexpressed the
human MBP-Ela and the human EIßsubunits and
confers ampicillin resistance. The second plasmid
pGroESL coexpressed GroES and GroEL and produces
chloramphenicol resistance. The double transformants
were selected for growth in both antibiotics. Figure
3A shows that the single transformant with pHl ex
presses little, if any, El activity. Cotransformation
with the second plasmid pGroESL results in >500-fold
increase in El specific activity. Significant albeit
smaller increases in El specific activity were also ob
served in ELtsdouble transformants, compared with
single transformants. Coomassie blue stain of SDS
polyacrylamide gels showed that the increase in MBP
Eia and EIßsubunit levels in soluble E. coli lysates
coincided with a marked increase in expression of
GroEL and a smaller increase in GroES in the double
transformants (Fig. 3B). The results established that
chaperonin GroEL and GroES promote efficient fold
ing and assembly of human El in bacteria.
Altered kinetics of El assembly
in Type IA MSUD
To approach the problem of putative assembly de
fects in Ela-deficient (Type IA) MSUD, we utilized
the double transformation bacterial expression devel
oped in our laboratory. We replaced the MBP fusion
with the histidine-tagged system (Janknecht et al.
1991). The His affinity tag consisting of six histidine
residues was linked to the amino terminus of mature
human Eia sequence through a specific linker recog-
DISEASE 1769S
A.
ES'Host ES-Host EL' Host EL" Host
f pH1 + pH1
+ pGroESL
+ pH l + pH l
+pGroESL
B.
ES-Ho« ES-Ho« EL"Ho« EL"Ho«
«pHl ,pH1 „pH1 *pH1
* pGroESL . pQroESL
Downloaded from jn.nutrition.org by guest on July 13, 2011
— Elß
— groES
FIGURE 3 Increase in El activity and subunit concen
trations by overexpression of chaperonins GroEL and GroES
in £.coii. ESts(CG-712) and EL" (CG-714) mutant strains
were singly transformed with the pH l plasmid encoding the
MBP-Ela fusion protein and E1/3or doubly transformed with
pHl and pGroESL plasmid that overexpresses GroEL and
GroES. Cultures were induced with 0.5 mM IPTG and grown
for 15 hours with single transformants at 26 °Cand double
transformants at 37°C.Cell suspensions were sonicated and
clarified by centrifugation at 1 X IO4g for 20 minutes. Cell
lysates are analyzed for El activity assay (A) or SDS-PAGE
with Coomassie blue staining (B). Reproduced from Wynn
et al. (1992) with permission.
nized by tobacco etch virus (TEV) protease. The
pGroESL contains the heat-shock sensitive elements
(HSE), which allowed us to induce chaperones by
growing ESts E. coli host cultures at 42°Cfor 4 hours
before the expression of El. The rationale was to prime
the cell with sufficient quantities of chaperonin pro
teins before the induced synthesis of recombinant
proteins by isopropyl thiogalactoside (IPTG). This
permitted the proper folding and assembly to occur
in the cell. High levels of His-tagged El in the bacterial
lysate was purified by Ni-NTA affinity chromatogra-
phy. Coomassie blue stains of the column fractions
eluted with an imidazole gradient showed that un-
tagged E1/3 copurifies with His-tagged Eia as a cata-
lytically active El protein. The E1/3 does not contain
1770S SUPPLEMENT
a His-tag; the copurification with His-tagged Eia on
the Ni-NTA column therefore indicated proper as
sembly into the a2/?2structure. In addition, an inactive
His-tagged Ela-GroEL complex was eluted earlier than
His-tagged El, which may represent an intermediate
in the chaperone-mediated folding pathway.
Using this system, a low level of Y393N Eia as
sembly with E1(8in doubly transformed £.coli com
pared with normal Eia was observed, which sug
gested that the kinetics of El assembly with the mu
tant Eia may be altered. To test this hypothesis, we
have developed pulse-chase experiments to study the
kinetics of El assembly. Briefly, EstsE. coli cells were
doubly transformed with a His-tagged El expression
vector carrying either normal or mutant Eia cDNA
along with the second vector pGroESL. The cells were
heat-shocked at 42°Cfor 4 hours, followed by in
duction at 37°Cwith IPTG for 5 minutes. The cells
were pulsed with 35S-cysteine/35S-methionine for 1
minute and chased with nonradioactive amino acids.
Samples were taken at different intervals from 1 to
120 minutes, and lysates were purified by Ni-NTA
affinity chromatography. The eluted radioactive pep-
tides (His-tagged Eia and associated E1/3)were ana
lyzed by SDS-polyacrylamide gel electrophoresis.
Autoradiograms were obtained by fluorography. The
kinetics of E1/3association with normal and mutant
His-tagged Eia as revealed by the autoradiograms
were compared. The Eia mutations were classified
into four groups with respect to the rate of assembly
of E1/3 with His-tagged Eia. The first group repre
sented by the N222S mutation (in exon 6) snowed a
normal rate of assembly with association occurring
as early as 10 minutes of the chase. The second group
exemplified by G245R (in exon 7) showed moderate
or medium assembly rate compared with normal. The
third group consisting of Y393N and Y368C muta
tions (in exon 9) exhibited a very slow rate or little
association of E1/3when compared with normal. The
last or fourth group associated with T265R (in exon
7) mutation shows an unstable and rapidly degraded
His-tagged Eia, with little or no assembly with EIß.
The kinetics of association of EIßwith His-tagged
normal or mutant Eia was determined by densitom-
etry of the autoradiograms.
The results of expression studies of normal and
mutant El can be depicted in a model, in which na
scent mature Eia and E1/3are folded by interactions
with two separate GroEL/GroES complexes. The
properly folded Eia and EIß each are released from
their individual GroEL scaffolds and assemble ini
tially into an inactive aßintermediate. The steps
from chaperone-mediated folding to the production
of inactive aßintermediates appear to be fast (10-
20 minutes). The aßintermediate is assembled into
an active a^ßi,which appears to be a slow process
requiring several hours. The mutations in Eia can
prevent the release of folded peptides from GroEL,
resulting in the accumulation of unproductive
GroEL-Ela intermediates. Alternatively, the muta
tion may impede the association of released folded
Eia with E1/3. Studies are in progress to elucidate
the precise steps in the chaperone-mediated folding
and assembly pathway of El.
Stable correction of MSLID
by retroviral gene transfer
Current treatment of MSUD relies primarily on di
etary restriction of BCAAs. Dietary therapy is not
completely satisfactory because it requires strict ad
herence and often leads to complication (Chuang and
Shih 1994). One in five classic MSUD patients dies
while on dietary treatment. Moreover, MSUD is a
paradigm for developing gene therapy to correct the
mitochondrial multienzyme complex deficiencies. A
majority of gene therapy protocols to date are confined
Downloaded from jn.nutrition.org by guest on July 13, 2011
to single locus diseases.
As the first step to somatic gene therapy for MSUD,
we recently carried out retroviral gene transfer of nor
mal Eia cDNA into cultured lymphoblasts from a
Mennonite patient (Koyata et al. 1993). As described
above, Mennonite cells are deficient in the Eia subunit
(Y393N-Ela). Our goal was to achieve a stable inte
gration of normal Eia cDNA into genomes of a host
cell by using a retroviral vector. We initially inserted
both normal human Eia and E2 precursor cDNAs into
the LXSN retroviral vector. The LN retroviral vectors
contain important features that increase the packaging
efficiency through the ^+ signal and to reduce the
possibility of helper virus production. The recombi
nant Eia and E2 retroviral vectors were packaged in
ecotropic packaging cells GP -t-E86. The murine pack
aging cell line is unique in that the gap and pol genes
are on one plasmid, and the env gene is on a second,
which further reduces the possibility of helper virus
contamination through recombination. To produce
amphotropic virus capable of infecting human cells,
transduced GP + E86 cells were cocultivated with
PA317 cells, resulting in the production of high titer
viruses at 2-6 X 10s colony-forming units/ml. Cul
tured lymphoblasts from a Mennonite MSUD patient
were placed above the cocultivated packaging cells and
were infected with a high efficiency of 80-100%. The
transduced lymphoblasts were selected for G-418 an
tibiotic resistance. Transduced lymphoblasts from the
Mennonite patient (LMK)were assayed on week 6, for
the rate of decarboxylation using a-keto [1-14C]iso-
valerate as the substrate. The transduction of LMK
cells with LSN-Ela that contained Eia cDNA restored
the rate of decarboxylation to approximately 100% of
normal (Fig. 4A). LXSN (no insert) and LSN-E2 (car
rying E2 cDNA) did not significantly affect the rate of
decarboxylation and were negative controls. The res-
 MAPLE SYRUP URINE
100
— E1ß
FIGURE 4 (A), decarboxylation of a-keto [1-UC] isolav-
eric acid by untransduced and transduced MSUD lympho-
blasts. Cultured lymphoblasts from a Mennonite MSUD pa
tient (LMK) were transduced with the retroviral vectors and
analyzed for decarboxylation of a-keto [1-14C]isovalerate by
the intact cell assay 6 weeks after infection. The decarbox
ylation rate in untransduced normal lymphoblasts (0.18
±0.09 nmol CO2/min/mg protein) is 100%. The activity of
the pyruvate dehydrogenase complex as assayed with [1-14C]
pyruvate as substrate is normal in both untransduced and
transduced cells (data not shown). The rate of decarboxyl
ation was measured weekly and found stable from 6 through
14 weeks. (A), Western blot analysis of lysates prepared from
untransduced and transduced MSUD lymphoblasts. Trans
duced LMK lymphoblasts were cultured for 11 weeks with
out G-418 selection before preparation of the cell lysate.
Each lane contains 200 ng of protein. After SDS-polyacryl-
amide gel electrophoresis, proteins were electrotransfered
to polyvinylidene difluoride membranes. The filters were
probed with either Eia (top) or E Iß(bottom) antibodies ra-
diolabeled by coupling with 115I-protein A. Eia and Ela-P
depict the unphosphorylated and phosphorylated forms of
the Eia subunit, respectively. (JB)was reproduced from Ko-
yata et al. (1993) with permission.
DISEASE 1771S
toration of decarboxylation activity in transduced
LMK cells was stable without antibiotic selection for
up to 14 weeks, which was the duration of the study.
Northern blot analysis of transduced MSUD cells
showed the overproduction of recombinant Eia
mRNA of the expected 5.0-kb size. The genomic DNA
from the untransduced and transduced cells were di
gested with EcoRI and subjected to Southern blotting
using the full-length El«cDNA as a probe. The results
show the integration of the expected 0.6-kb and 1.5-
kb EcoRI fragments into the genome of the transduced
LMK cells. The level of integration is one copy of pro-
viral DNA per cell. Lysates prepared from untrans
duced and transduced lymphoblasts were analyzed by
Western blotting using anti-El a or anti-El ß antibodies
as a probe. Figure 4B (top) shows that transduction of
LMK cell with LSN-E1a resulted in an increase in the
level of both phosphorylated (Ela-p) and dephosphor-
ylated Eia subunits. The E1/3 subunit, which was
barely detectable in untransduced LMK cells, was re
Downloaded from jn.nutrition.org by guest on July 13, 2011
stored by LSN-Ela to a level of approximately 50% of
normal (Fig. 4B, bottom). LXSN (no insert) and LSN-
E2 did not affect the E1/3 level and again serve as a
negative control. The less-than-full level of restoration
of El/3 was perplexing and requires further studies.
However, our results clearly demonstrated the feasi
bility of stable correction of Ela-deficient MSUD
(Type IA) by retroviral gene transfer. The BCKAD
complex is widely distributed in different tissues and
organs. Therefore, introduction of the normal gene
into a variety of MSUD cell types including hepato-
cytes, T cells and myoblasts will be effective in de
grading BCAAs. Information derived from these stud
ies can be applied to animal models, e.g., MSUD in
Hereford calves (Harper et al. 1986).
LITERATURE CITED
Chuang, D. T. & Shih, V. E. (1995) Disorders of branched-chain
amino acid and keto acid metabolism. In: The Metabolic and
Molecular Basis of Inherited Disease (Scriver, C. R., Beaudet,
A. L., Sly, W. S. & Valle D., eds), 7th ed. McGraw-Hill, New
York, NY. pp. 1239-1277.
Davie, J. R., Wynn, R. M., Cox, R. P. &. Chuang, D. T. (1992)
Expression and assembly of a functional El component |i>.•.<..!of
mammalian branched-chain a-ketoacid dehydrogenase complex
in Escherichia coli. }. Biol. Chem. 267: 16601-16606.
Ellis, R. J. & van der Vies, S. M. (1991) Molecular chaperones.
Ann. Rev. Biochem. 60: 321-347.
Fisher, C. R., Chuang, J. L., Cox, R. P., Fisher, C. W., Star, R. A. &
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