Báo cáo khoa học: The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitiv pptx

The Y42H mutation in medium-chain acyl-CoA dehydrogenase,
which is prevalent in babies identified by MS/MS-based newborn
screening, is temperature sensitive
Linda O’Reilly
1
, Peter Bross
2
, Thomas J. Corydon
3
, Simon E. Olpin
4
, Jakob Hansen
2
, John M. Kenney
5,6
,
Shawn E. McCandless
7
, Dianne M. Frazier
8
, Vibeke Winter
2
, Niels Gregersen
2
, Paul C. Engel
1
and Brage Storstein Andresen
2,3
1
Department of Biochemistry and the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland;
2

Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Science, Skejby Sygehus, Aarhus,
Denmark;
3
Department of Human Genetics, University of Aarhus, Denmark;
4
Department of Clinical Chemistry, Sheffield Children’s
Hospital, UK;
5
Institute of Storage Ring Facilities, University of Aarhus, Denmark;
6
Department of Physics, East Carolina University,
Greenville, NC, USA;
7
Department of Genetics, Case Western Reserve University, Cleveland, OH, USA;
8
Department of
Pediatrics, University of North Carolina at Chapel Hill, NC, USA
Medium-chain acyl-CoA dehydrogenase (MCAD) is a
homotetrameric flavoprotein which catalyses the initial step
of the b-oxidation of medium-chain fatty acids. Mutations in
MCAD may cause disease in humans. A Y42H mutation is
frequently found in babies identified by newborn screening
with MS/MS, yet there are n o reports of patients presenting
clinically with this mutation. As a basis for judging its
potential consequences we have examined the protein phe-
notype o f t he Y42H mutation and the common d isease-
associated K304E mutat ion. Our studies o f the intracellular
biogenesis of the variant proteins at different temperatures in
isolated mitochondria after in vitro translation, together with
studies of cultured patient cells, indicated that steady-state

levels of the Y42H variant in comparison to wild-type were
decreased at higher temperature though to a lesser extent
than for t he K304E variant. To d istinguish between effects of
temperature on folding/assembly and t he stability o f the
native enzyme, the thermal stability of the variant proteins
was studied after expression and purification by dye affinity
chromatography. T his s howed that, compared w ith t he wild-
type e nzyme, the thermostability of the Y42H variant was
decreased, but not to the same degree as that of the K304E
variant. Substrate binding, i nteraction with the natural
electron acceptor, and the binding of the prosthetic group,
FAD, were only slightly affected by the Y42H mutation. Our
study suggests that Y42H is a temperature sensitive muta-
tion, which is mild at low temperatures, but may have
deleterious effects at increased temperatures.
Keywords: chaperones; newborn screening; protein folding;
thermostability.
Medium-chain acyl-CoA dehydrogenase (MCAD)
(EC 1 .3.99.3) is a homotetrameric enzyme that catalyses
the initial oxidation step in the b-oxidation of medium-
chain fatty acids in mitochondria [1]. Medium-chain acyl-
CoA dehydrogenase deficiency (MCADD; MIM 201450) is
the commonest fatty acid o xidation defect occurring in
Europe, affecting Caucasians o f North-western European
origin, with an incidence as high as 1 : 8000 live births [2].
Symptoms can be quite broad, ranging from hypoglycaemia
and lethargy to seizures, coma and s udden death. Some
genetically predisposed patients remain asymptomatic
throughout life [3–5]. The disease can present at any time
of life, from the neonatal period [6,7] to adulthood [8–10].

Clinical presentation usually oc curs at a time of metabolic
stress, associated with fasting or v iral illness [2–4]. In t he
past, up to 20% of patients died prior to diagnosis of the
disease [3]. However, w ith e arly diagnosis and treatment
prognosis is v ery favourable [11]. Treatment is simple,
consisting primarily of the avoidance of fasting and the
institution of an emergency treatment regimen at times of
intercurrent infection or other metabolic stress. Develop-
ment of a rapid and r eliable method for identification of
acylcarnitines from dried b lood spots b y MS/MS [1 2–14]
has led to newborn screening f or this common disorder i n
a number of US s tates, par ts of Australia and some
European countries [11,14]. MCAD deficiency is an auto-
somal recessive disorder. The most common mutation,
985AfiG (K304E) is homozygous in 80% of patients
presenting clinically, and a further 18% are compound
heterozygous with the 985AfiG mutation in one allele and
one of a variety of rare mutations in the other allele
[5,11,15–17]. MCAD is normally translated in the cytosol,
and then transported into the mitochondria, where the
Correspondence to B. Storstein Andresen, Research Unit for
Molecular Medicine ( MMF), Skejby Sygehus, 8200 Aarhus N,
Denmark. Fax: +45 8949 6018, Tel.: + 45 8949 5146,
E-mail:
Abbreviations: MCAD, medium-chain acyl-CoA dehydrogenase;
ETF, electr on transferring flavoprot ein; SRCD, s ynchrotron radiation
CD.
Enzyme: medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3).
(Received 2 June 2004, revised 22 July 2004, accepted 23 August 2004)
Eur. J. Biochem. 271, 4053–4063 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04343.x

folding of t he polypeptide into monomer is facilitated by the
Hsp60 chaperonin system and the t etramer i s formed [18].
We and others have previously reported that the K304E
mutation influences this biogenesis process at several steps
by affecting the folding of the monomer, impairing
oligomerization and destabilizing the tetramer [18–23].
The folding/tetramerization defect of the K304E mutant
protein can be partly overcome by increasing the amount of
chaperonins or by lowering the culture temperature when
the recombinant protein is expressed in Escherichia coli
[21,22]. Similarly, many of the other disease-causing muta-
tions in MCAD that have been characterized seem to
influence folding and can be rescued to a varying extent by
chaperonin co-overexpression and/or lowering the growth
temperature [5,11]. Recently a new prevalent mutation
199TfiC, causing the missense mutation Y 42H, w as
identified [11,24]. It was found in newborns heterozygous
for the prevalent 985AfiG mutation who showed an
abnormal acylcarnitine profile in an MS/MS screen of
blood spots, and the carrier frequency in the US was
determined to be 1/500 [11]. The carrier frequency of the
985AfiG (K304E) mutation in t he same area ranges from
1/80 to 1/100, making Y42H the second most prevalent
mutation of MCAD deficiency [ 11]. Y42H has also been
found to b e present in Germany, New South Wales and
Spain ([24] and B . S . Andresen & N. G regersen, unpub-
lished results). Despite the fact that the Y42H mutation is so
prevalent, and gives rise to an abnormal acylcarnitine profile
in blood spots, it has so far not been reported in clinically
manifesting patients [11,24]. Therefore, the clinical implica-

tions of this mutation have remained unresolved. The aim of
the present study was to gain more knowledge about the
molecular pathology o f this mutation by investigation of the
effect of the Y42H mutation on MCAD structure, function
and intracellular biogenesis using both in vitro techniques
and patient cells.
Experimental procedures
Expression vectors for wild-type, Y42H and K304E MCAD
The MCAD proteins were overexpressed in E. coli JM109
cells using the pWT vector or derivatives of this vector
where 199TfiC (Y42H) or 985A fiG (K304E) mutations
have been introduced by PCR-directed mutagenesis. The
pWT plasmid carries a gene encoding the mature part of
human MCAD preceded by an artificial initiator methion-
ine under control of the lac promoter [18]. All expression
vectors were sequenced to ensure that no PCR based errors
were present.
Protein purification
Using the pWT vector MCAD mutant proteins and wild-
type proteins were overexpressed in E. coli JM109 cells.
Six litres growth of E. coli in Luria–Bertani medium were
harvested, lysed by sonication, and centrifuged at 10 000 g
for 30 min. The supernatant w as loa ded onto a 100-mL
Q-Sepharose anion exchange column (2.5 cm diameter;
Pharmacia Biotech), pre-equilibrated with 20 m
M
KPi,
50 m
M
KCl pH 7.2. The column was washed with pre-

equilibration buffer for 1 column vol., then 4–5 column vols
of 20 m
M
KPi, 50 m
M
KCl pH 7.2, until no m ore
contaminants eluted. The enzyme was then e luted with
20 m
M
KPi, 200 m
M
KCl pH 7.2. The eluate was concen-
trated and d esalted utilizing an A micon Centricon device
(M
r
cutoff 30 kDa). The protein was loaded onto a 20-mL
Procion red HE-3B dye affinity column (Procion dyes were
a g enerous gift from C. V. Stead of the former Imperial
Chemical Industries, Dyestuffs Division, Blackley, Man-
chester, UK), linked t o Sepharose (Pharmacia Biotech), pre-
equilibrated w ith 5 0 m
M
KPi, 50 m
M
KCl p H 7 .2 (1 cm
diameter) The column was washed with th e pre-equilibra-
tion buffer, until no more contaminants eluted. The enzyme
was then eluted by adding 1 mL of 3.5 m
M
of the substrate

octanoyl-CoA. An aliquot of each fraction was analysed by
SDS/PAGE, and the pure fractions were pooled.
PAGE and Western blotting
SDS/PAGE, native PAGE, and Western blotting were
performed essentially as described previously [25], using
ECL+ reagents (Amersham Pharmacia Biotech).
Enzyme kinetics parameters
Kinetics measurements were performed using increasing
concentrations of substrate octanoyl-CoA (Sigma Chemical
Co.), from 1 l
M
to 100 l
M
. The activity was measured
using the dye acceptor ferricenium method, as described by
Lehman et al . [26]. The assay was carried out in 100 m
M
KPi buffer pH 7.6 at 25 °C. The K
m
and V
max
values were
determined by the Wilkinson method (nonlinear regression).
The activity w as also measured by a m odified version of
the method described b y Thorpe [27] u sing r ecombinant
human electron transferring flavoprotein (ETF) as electron
acceptor.
MCAD biogenesis in isolated rat liver mitochondria
In vitro transcription and translation of wild-type and
precursor MCAD cDNAs in pcDNA3.1+ were performed

in the presence of [
35
S]methionine (20 lCi per 50 lL
reaction, 10 lCiÆlL
)1
; Amersham Bio sciences) using the
TnT co upled reticulocyte lysate kit (Promega) according to
the manufacturer’s protocol. The translation was stopped
by the addition of cycloheximide (0.15 lgÆmL
)1
final
concentration). Rat liver mitochondria were isolated as
described p revio usly [28,29]. The translation p roduct was
mixed w ith i solated m itochondria and imported into
mitochondria essentially as d escribed previously [25]. The
mixture was then incubated at 26 °Cor41°C, and
intramitochondrial biogenesis was followed by withdrawing
aliquots at different time points (0–180 min). Samples were
treated as described previously [25]. The supernatant
fraction, which contained s oluble matrix components,
including MCAD enzyme protein and complexes thereof,
was analysed by native (nondenaturing) PAGE (4–15%
Tris/HCl Criterion gels from Bio-Rad) and by SDS/PAGE
(12.5% Tris/HCl Criterion gels from Bio-Rad) as described
previously [25]. The pellet fraction, which contains insoluble
MCAD protein, was analysed by SDS/PAGE. Radio-
labelled MCAD protein was v isualized by phosphor
imaging using an Amersham Biosciences Phosphorimager
4054 L. O’Reilly et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(STORM 840) and the bands were quantified using

IMAGE-
QUANT
software.
Analysis of human cells with different MCAD genotypes
Primary patient lymphoblast cells were immortalized by
Epstein–Barr virus transformation and cultured as des-
cribed elsewhere [30]. Cells were cultured in 75-cm
2
flasks
at 5% (v/v) CO
2
in RPMI 1640 medium (In Vitro,
Copenhagen, Denmark) containing 10% (v/v) fetal bovine
serum (Life Technologies, Inc.), 100 UÆmL
)1
penicillin,
0.1 mgÆmL
)1
streptomycin and 0.29 mgÆmL
)1
glutamine at
34 °C, 37 °Cand39°C. The cell pellets were lysed, and a
total p rotein amount of 30 lg was loaded and run on 12%
acrylamide denaturing SDS gels and 12% acrylamide native
gels, a nd analysed by Western b lotting a s d escribed
previously [25]. Measurements o f the b-oxidation flux in
cultured fib roblasts using [9,10-
3
H] myristate (Amersham
International) were performed using the method of Man-

ning and Olpin [31,32]. Patient fibroblasts, along with
controls, were seeded into 24-well plates and incubated at
34 °C, 37 °Cand39°C for 72 h p rior to assay at these
temperatures.
Structure analysis of MCAD by synchrotron radiation CD
(SRCD)
CD studies were preformed using the UV 1 beamline at the
Institute for Storage Ring Facilities at the University of
Aarhus. Samples were prepared in 20 m
M
KPi pH 7.2
buffer. Samples at concentrations of 330 lgÆmL
)1
and
50 lgÆmL
)1
and buffer (for b aseline correction) were
placed in a 0.5-mm light path Suprasil quartz cell (Helma)
for CD spectroscopy. CD spectroscopy of all the samples
were made under the same conditions as a function of
temperature (at fixed points between 30 °Cand75°C)
and time (5 min equilibration at each temperature). The
baseline (buffer only) spectra were recorded before and
after t he CD scan of each sample using the same cell as
that u sed for the sample and under the s ame c onditions
(specifically temperature). Both the baselines and protein
scansweremadeinduplicateandthemeanbaseline
subtracted from the mean scan, before plotting. The
spectra of the baseline-corrected 50-lgÆmL
)1

samples were
scaled by 330/50 (to remove the effe ct of concentration) so
that they could be d irectly compared to the 330 lgÆmL
)1
data. This was confirmed by comparing the data of a ll the
samples at 30 °C, where they exhibited indistinguishable
CD spectra.
Results
Purification of recombinant MCAD proteins and
determination of enzyme parameters
Wild-type MCAD and the mutant proteins K304E and
Y42H, expressed in E. coli, were purified utilizing anion
exchange, and dye affin ity chromatography. The kinetics of
the catalysed reaction with octanoyl-CoA as s ubstrate and
ferricenium as final electron acceptor was studied with each
purified protein. The K
m
was determined by the Wilkinson
method to be 3.7 ± 0.3 l
M
(Mean ± SE) f or wild-type,
which compares well with the previously published results of
3.4 l
M
[33,34]. However, the K
m
of the K304E mutant
protein was determined to be 5.9 ± 0.7 l
M
,whichis

somewhat lower than the previously published value of
12 l
M
[33,34]. We found that the Y42H mutant protein has
approximately the same maximum velocity (V
max
¼
24.2 ± 0.7 · 10
3
nmol ferricenium/mgÆmin
)1
) as the wild-
type enzyme (V
max
¼ 24.6 ± 0 .6 · 10
3
), but the Y42H
protein has a higher K
m
than wild-type (5.2 ± 0.5 l
M
),
indicating that the substrate binding is slightly impaired.
The maximum velocity of the K304E mutant protein
was only one third of the wild-type value (V
max
¼ 8.2 ±
0.3 · 10
3
).

When the activity values with the natural electron
acceptor ETF are expressed as a percentage of the specific
activity with the artificial electron acceptor ferricenium, the
K304E mutant protein shows a r elatively higher activity
(16%) with the natural electron acceptor than the wild-type
protein (9%), whereas the Y42H mutant protein shows a
slightly decreased relative activity (7%) with the natural
electron acceptor compared to wild-type. The mutant and
wild-type proteins were subjected to spectral scans in order
to investigate w hether the m utations affected the binding
of the prosthetic group FAD. The peak-to-peak r atio
A
280nm : 450nm
for the K304E mutant protein as purified is
significantly increased (ratio K304E ¼ 8.9; wild-t ype ¼
7.2), s howing t hat t h e bin ding o f the prosthetic group is
considerably impaired. Y42H MCAD shows a slight
increase in the peak-to-peak ratio (ratio Y42H ¼ 7.5),
indicating that FAD binding is also slightly affected by this
mutation, but to a much lesser extent than for the K304E
mutant protein.
MCAD biogenesis in isolated mitochondria
We have previously used combined in vitro transcription/
translation and import into mitochondria to study wild-type
and mutant acyl-CoA dehydrogenases [35]. Recently we
have developed this system further and used it to charac-
terize the biogenesis and turnover of wild-type and a series
of SCAD proteins [25]. Since the preliminary results from
overexpression of the Y42H MCAD in E. coli had shown
that the steady-state activity levels were affected by the

growth temperature [11], we decided to use this eukaryotic
system [25] to investigate the influence of t emperature on
biogenesis and s tability o f t he Y42H MCAD , c ompared
to wild-t ype a nd K3 04E MCAD. We p erformed in vitro
transcription/translation of the MCAD variants, imported
the products into purified rat liver mitochondria, and
monitored the time course of folding and formation/
stability of the tetramer. The studies were performed at
26 °Cand41°C (Fig. 1). At 26 °C the amounts of tetra-
mer formed increased until 120 min. T here is an obvious
difference between the amounts of K304E tetramers being
formed compared to wild-type, whereas the rate of tetramer
formation is only slightly decreased for the Y42H mutant
protein. Considering the fraction of soluble MCAD protein
that represents tetrameric enzyme, it appears that relatively
less Y42H tetramer is formed compared to wild-type. This
could be interpreted as indicating that folding of the
monomers into an assembly-competent conformation is
slowed for the Y42H protein, and/or that formation of
Ó FEBS 2004 The MCAD Y42H mutation is temperature sensitive (Eur. J. Biochem. 271) 4055
tetramers from the assembly competent monomers is also
slightly decreased. The observation that the amount of
soluble nontetrameric K304E protein increases over time,
and that it makes up a much bigger fraction at the last time
points than observed for the wild-type protein is consistent
with previous studies showing that the K304E protein has a
defect both in monomer folding and tetramer assembly [19].
In the studies performed at 41 °C (Fig. 1) it can be seen
that for the wild-type the amount of soluble protein reached
a peak within the first 10–30 min and the amount of

tetramer formed from the pool of soluble protein increased
for the first 60 min. At 41 °C soluble Y42H protein reaches
a peak within the first 10 min, but in contrast to the wild-
type protein the amount of soluble pro tein decreases over
Fig. 1. Comparison of the biogenesis/stability of Y42H and K304E m utants to that of wild-type at 26 °C and 41 °C. In vitro transcription/translation
of MCAD precursor proteins was performed using [
35
S]methionine. The product of translation w as imported into isolated rat liver mitochondria
for 3 0 min at 26 °C. Aliquots were removed at the time points in dicated. The amounts of monomeric and tet rameric MCAD prot eins were
measured at 26 °Cand41°C as described previously [25]. Briefly, soluble a nd insoluble MCAD proteins were separated by centrifugation and the
respective fractions, either soluble M CAD protein (present in the supernatant) or aggregating MCAD protein (present in the p ellet) were measured
by quantification (phosphorimaging) of the MCAD monomeric band afte r SDS/PAGE. The amounts of tetramers in the soluble fraction were
measured by quantification (phosphorimaging) of the band corresponding to tetrameric MCAD protein a fter native PAGE. The levels of MCAD
protein were normalized to the total am ount of radiolabelled MCAD protein (soluble and insoluble) in the corresponding lane of the SDS gel.
Results are representative of three separate experiments.
4056 L. O’Reilly et al. (Eur. J. Biochem. 271) Ó FEBS 2004
time, concomitant with an increase in t he amount of
insoluble protein. Because this is a dynamic process we
cannot say f rom these experiments w hether the i ncreased
temperature destabilizes the structure of the monomers,
thereby causing them to aggregate, and/or if increased
temperature destabilizes the tetramers. This tendency of a
generally slowed, and temperature dependent biogenesis
that is observed for the Y42H mutant protein is much more
pronounced for the K304E mutant (Fig. 1), consistent with
previous s tudies that indicated a combined defect in
monomer folding and tetramer formation/stability of
K304E MCAD [19].
Thermal stability of purified Y42H mutant protein,
as determined by enzyme activity curves

Because the experiments described above could not unam-
biguously delineate if the temperature sensitivity of the
Y42H mutant protein is caused by decreased thermostabi-
lity and/or biogenesis, we investigated the thermal stability
by generating thermostability curves with the purified
recombinant MCAD proteins. Preliminary thermal inacti-
vation profiles of crude extracts from E. coli cells over-
expressing Y42H or K304E mutant proteins, respectively,
have previously demonstrated that the thermal inactivation
profiles of K304E and Y42H are shifted to lower temper-
atures [11,21]. In the present study the residual enzyme
activity levels were measured at two MCAD pro-
tein concentrations (3.3 lgÆmL
)1
and 50 lgÆmL
)1
). At
3.3 lgÆmL
)1
a d ifference i s observed between the variant
proteins (Fig. 2A), with Y42H showing a decreased stability
at temperatures above 42 °C c ompared to w ild-type, and
K304E showing a more pronounced decrease. At the higher
protein concentration of 50 lgÆmL
)1
there is little difference
observed i n t he ther mostability b etween the various pro-
teins. Interestingly, all MCAD variant proteins show an
increased thermal stability at the higher concentration and a
further elevation of the concentration ( 0.33 mgÆmL

)1
;
Fig. 2B) further enhances the thermal stability likewise. At
the same time, the differences in thermostability become less
pronounced. T his demonstrates that the t hermal stability
of the MCAD e nzyme is depend ent on the protein
concentration.
To investigate whether the thermal stability depends on
the total protein concentration in vitro or specifically on the
concentration o f M CAD polypeptide chains, the enzyme
activity curves (Fig. 2A) were repeated in the presence of
1mgÆmL
)1
BSA (Fig. 2C). The results clearly show that the
presence of a high concentration of unrelated protein does
not alter the thermal stability, and therefore the concentra-
tion dependence of the thermostability ob served depends on
the specific presence of MCAD protein.
In Western blots of native polyacrylamide gels with
samples for the 3.3 lgÆmL
)1
the enzyme a ctivity curves
shows that the loss of MCAD tetramer corresponds to the
loss of activity (Fig. 2 D). SDS/PAGE o f these samples
(Fig. 2E) reveals that as tetramer and enzyme activity is lost
the amount of soluble protein (in the supernatant fraction)
decreases, and the amount of insoluble/aggregated protein
(in the pellet fraction) increases correspondingly. These
results indicate that the temperature-dependent decay of
activity concurs with loss of tetramer, and that loss of

ABC
D
E
Fig. 2. Enzyme activity assays of wild-type, Y42H and K304E mutant protein at 3.3 lgÆmL
)1
and 50 lgÆmL
)1
for without BSA (A) and with
1mgÆmL
)1
BSA (C). The activity at the higher concentration o f 0.33 mgÆmL
)1
(B) is also shown. Note that the graphs for Y42H and the wild-type
mutant proteins are completely overlapping at this high p rotein concentration. The amount of protein, corresponding to the activity at 3.3 lgÆmL
)1
is seen by Western blot analysis of ( D) native ge l showing tetramer formation and (E) showin g the ratio of soluble (S ¼ supernatant fraction) and
insoluble (P ¼ pellet fraction) prote in by SDS/PAG E. Error bars ind icate the standard d eviation of the me an result.
Ó FEBS 2004 The MCAD Y42H mutation is temperature sensitive (Eur. J. Biochem. 271) 4057
tetramer falls together with the MCAD protein be coming
insoluble and aggregating.
SRCD of wild-type and mutant MCAD proteins
To determine whether the K304E and Y42H mutations
have an effect on the secondary structure of MCAD, the
purified variant proteins were analysed by SRCD [36],
which is more sensitive than conventional CD spectroscopy
at low wavelengths (< 190 nm) [37,38]. Analysis of the
three variant MCAD proteins at a protein concentration of
330 lgÆmL
)1
at 35 °C showed no significant difference in

their s pectra, indicating that the o verall fold of the three
proteins is very similar at this temperature (Fig. 3A, data
not shown for the mutant proteins). The spectra exhibited
features typical of a protein fold dominated by alpha-helical
secondary structures. At this protein concentration
(330 lgÆmL
)1
) at increasing t emperatures, an identical
temperature-dependent change in the spectra was observed,
indicating that the temperature-induced change in the fold
of the structure is indistinguishable between the three
proteins at this protein concentration. Furthermore, this
temperature-induced structural change was irreversible,
since a return to 35 °C a fter heating did not reproduce the
initial characteristic spectrum.
Interest ingly, the identical spectral (and hence structural)
behaviour does not strictly hold at a lower protein
concentration (50 lgÆmL
)1
). Although the CD spectra of
the wild-type a nd both m utant proteins a re the same a t
35 °C, an accelerated temperature -induced change in the
spectrum (and hence fold) of K304E MCAD compared to
wild-type and Y42H MCAD could be monitored at 45 °C
(Fig. 3B). Taken together, these data confirm that MCAD
thermal stability depends on the MCAD concentration and
that thermal i nactivation of t he enzyme correlates w ith a
change in the fold of the native structure leading to a
reduction in the alpha-helical secondary structure content.
Steady-state amounts of endogenous MCAD proteins

in human cells
To investigate the relevance of the results obtained in the
model systems descr ibed a bove, we analysed steady-state
amounts of Y42H and K304E MCAD in immortalized
lymphoblastoid cells cultured at 34 °C, 37 °Cand39°Cby
Western blotting. Cells homozygous or heterozygous for the
K304E mutation, cells compound heterozygous for
the K304E and Y42H mutations and cells homozygous
for the wild-type allele were used (Fig. 4). At 34 °Cthereis
little difference between the amount of either tetramer or
soluble protein present in the K304E/wild -type heterozy-
gote, compared t o the K304E/Y42H heterozygote, indica-
ting that at this temperature there is little difference between
wild-type and the Y42H variant. However, if the tempera-
ture is raised to 37 °Cand39°C, the difference becomes
more obvious with much less MCAD protein present for the
K304E/Y42H heterozygote. In fact, both the levels of
soluble MCAD protein present in the SDS gel and the
amounts of MCAD tetramers present in the native gels
from the K304E/Y42H heterozygote a re comparable to
those observed from the K304E homozygote at 39 °C.
The effect of temperature on the mutant and wild-type
proteins was investigated further by measuring the
b-o xidation in fibroblast cells using myristic acid as
substrate. The results are shown in Fig. 5. As expected,
the K304E homozygote cells had the lowest activity level.
However, this level remains relatively unaffected by the
increasing tempe rature. The wild-type/K304E heterozygote
cells showed the highest activity level, and again were
relatively stable with increasing temperature. However, the

K304E/Y42H heterozygote cells showed the most thermo-
lability, with a 27% loss in activity when the temperature
was increased from 34 °Cto37°C, and a further 14% loss
with another 2 °C increase in temperature to 39 °C.
Discussion
The Y42H mutation is of potential clinical importance, as it
is the s econd most prevalent mutation in the MCAD gene
and c ompound heterozygosity for the K304E and Y42H
mutations is the second most prevalent genotype in babies
identified o n the basis of a n a bnormal acylcarnitine pro-
file in the MS/MS-based newborn screening programs,
Fig. 3. SRCD . (A) Temperature scans of wild-type (330 lgÆmL
)1
)
from 35 °C) 75 °C, and re turned to 35 °C. Although th e mutant
protein te mperature scans are almost ide ntical, only wild-type is show n
for simplicity. (B) Comparison of the CD data collected at 222 nm
at 50 lgÆmL
)1
and 330 lgÆmL
)1
protein concentrations. Error bars
indicate the standard deviation of the mean result.
4058 L. O’Reilly et al. (Eur. J. Biochem. 271) Ó FEBS 2004
carried out in the US, Australia, Germany and Spain ([11],
B. S. Andresen & N. Gregersen, unp ublished data). How-
ever, the possible pathological significance associated with
this mutation is unclear, as there have been no reports of
patients presenting clinically with the Y 42H mutation so far.
This is surprising given the high frequency of this mutation,

and might suggest that Y42H rarely precipitates clinically
manifested disease, and therefore could be regarded as a
ÔbenignÕ variant. Alternatively, patients with this mutation
may not be recognized because they exhibit a different
clinical presentation. Given the widespread use of MS/MS-
based newborn screening, it i s a cause for major c oncern
that this method ma y detect Ôbenign Õ variants of unknown
clinical significance, creating unwarranted anxie ty in parents
and health care professionals [39]. It is therefore of utmost
importance that t he consequences of the Y 42H mutation
are t horoughly i nvestigated, to distinguish b etween a
ÔbenignÕ MCAD variant that causes an abnormal acylcar-
nitine pattern, but is unlikely to cause disease, and a Ôdisease-
causingÕ MCAD variant. Therefore we have in t he present
study investigated how the Y42H mutation affected the
MCAD protein using studies in both in vitro systems and
patient cells.
Our investigation of purified recombinant protein showed
that the Y42H mutation only had a minimal effect on the
catalytic activity of the enzyme, the prosthetic group
binding, or interaction with the natural electron acceptor.
Together these d ata show that the Y42H mutation
compromises the enzymatic function to a minor degree. It
is unlikely that these changes alone could explain the
biochemical abnormality observed in newborns with the
Y42H muta tion.
Instead is seems that the biogenesis and/or stability of the
Y42H mutant enzyme is more significantly affected. This
was also indicated in previous experiments as overexpres-
sion in E. coli revealed that the temperature at which the

mutant variant was expressed was decisive for the amounts
of steady-state enzyme activity produced from the Y42H
A
B
Fig. 4. Western blot analysis of steady-state amounts of MCAD protein in lymphoblasts with genotypes K304E/K304E, K304E/Y42H, K304E/WT
and WT/WT cultur ed at 34 °C, 37 °C and 39 °C. ThetetramericandthesolubleMCADproteinwasmeasuredbynative(A)anddenaturing(B)
PAGE in combination with Western blotting. The blot was also secondarily stained for ETF, showing the a and b subunits, as a loading control.
0
20
40
60
80
100
Temperature
Myristate Oxidation % of Controls
34°C37°C39°C
K304E/K304E
K304E/Y42H
K304E/WT
Fig. 5. Myristate oxidation from fibro blasts with genotypes K304E/
K304E, K304E/Y42H and K304E/WT as compared to WT/WT con-
trols cultured at 34 °C, 37 °Cand39°C. The results are expressed as
the percentage of t he activ ity of norma l control c ell lines, an d are the
average of two separate experiments (mean o f fi ve determin ations),
using three different control lines. Error bars indicate standard devi-
ation o f th e m ea n.
Ó FEBS 2004 The MCAD Y42H mutation is temperature sensitive (Eur. J. Biochem. 271) 4059
protein [11]. At low temperatures (31 °C) the residual
enzyme activity levels from cells expressing the Y42H
mutant was close (80–90%) to that of cells expressing the

wild-type, but when the temperature was increased from
31 °Cto37°C, this activity was significantly decreased
(35–40% of wild-type). This impact of culture temperature
on enzyme activity levels for the Y42H mutant protein
could indicate that it is a Ôfolding mutantÕ, like many of the
previously characterized disease-causing mutations in
MCAD [5,21,23]. However, unlike t he MCAD proteins
with folding mutations, overexpression of chaperonins
appeared to have very little o r no e ffect on the Y42H
protein [11]. A thermal stability curve of crude lysates from
E. coli overexpressing mutant or wild-type M CAD indica-
ted a dec rease in the thermal stability of Y42H protein as
compared to the wild-type suggesting that the temperature
effect might be due to a decreased s tability o f the active
enzyme once it has acquired the native structure [11].
In the present study we investigated the biogenesis/
stability of the Y42H mutant protein further and c ompared
it to wild-type and K304E mutant MCAD. Our results with
the coupled in vitro transcription/translation of MCAD
proteins followed by import into rat liver mitochondria
corroborated previous studies of the K304E mutant protein
[19], showing that this mutation has a drastic effect on
formation of t etrameric MCAD protein, probably as a
result of a combined defect in the folding of monomers and
in assembly/stability of the tetramer. It was clear from the
present studies that the amounts of tetramers formed for the
Y42H mutant variant were slightly decreased compared to
wild-type and the effect was exacerbated at increased
temperature.
The in vitro translation studies clearly demonstrated that

temperature has an effect on the amounts of tetrameric
Y42H protein formed, however, they could not distinguish
between a defect in folding/tetramer assembly and decreased
stability of the assembled tetrameric mutant pro tein.
To address this question we used the purified recombin-
ant MCAD variant proteins and investigated the thermal
stability of the native enzymes at different concentrations.
This confirmed t hat both t he Y42H and K 304E proteins
were less stable than the wild-type, with K304 E being the
most unstable. Interestingly, the thermal stability of MCAD
is very much dependent on the concentration of the MCAD
protein, and at high concentrations the differences between
the thermal stabilities of the thre e proteins becomes almost
indistinguishable. We could show that the decisive factor is
the concentration of MCAD protein rather than the t otal
protein concentration because addition of large amounts of
BSA had no effect. MCAD is a homotetrameric protein,
actually a dimer of dimers, and the transition between the
different o ligomeric states (monomers, dimers, tetramers)
could be reversible and thus concentration dependent
whereas refolding of denatured monomers appears not to
occur in vitro under the conditions applied, and therefore
thermal unfolding is practically irreversible. Using SRCD
analysis to study thermal stability of the MCAD variants we
show that the secondary structure of MCAD is maintained
up to the t emperature where a gross change in the folding
(leading to a loss of alpha-helical structure) occurs. Cooling
the samples did not in any way recover the s ignal, thus
confirming that the folding change is irreversible.
One could thus envisage that by increasing the stress

placed on the MCAD tetramer, i.e. by increasing the
temperature, the t etramer has an increased tendency to
dissociate into dimers and monomers. At low MCAD
concentrations, the probability of an MCAD monomer/
dimer meeting other monomers/dimers and re-forming the
tetramer is lower than at h igh MCAD concentration. This
would explain the increased thermal s tability observed at
high MCAD concentrations. The effect of the Y42H
mutation may thus be primarily on stability of the native
structure resulting in both temperature and concentration
sensitivity.
From the crystal structure [40] it can be seen that
tyrosine-42 is placed in the small helix B with the side chain
pointing to the surface of the tetramer (Fig. 6). The
aromatic ring of tyrosine-42 is packed between residues
and this structure appears to be part of an i nteraction
network that stabilizes the fold of helices A, B and C and
links it to the e dge of the b-sheet domain. Substitution of
tyrosine-42 with histidine may be expected to disturb these
interactions. The side chain of histidine is somewhat smaller
than that of tyrosine and hydrophobic interaction s of
carbon atoms in the aromatic ring of tyrosine with the
neighbouring residues would b e altered or abolished. This
could result in loosening of th e stability of the structure in
this part of the monomer. Although tyrosine-42 is distant
from subunit interaction interfaces, increased breathing of
the helix A, B, C fold and its anchoring to the b-sheet
domain could result in an increased tendency of the tetramer
to dissociate. At high MCAD concentrations and low
temperature, reassociation would dominate, whereas at high

Fig. 6. Location of the Y42H mutation i n the MCAD structure. The
illustrations were produced with
VIEWER LITE
5.0 ( Ac celrys ) using
the PDB coordinates 1E GC. (A) Tyr42 is localized at the surface of t he
MCAD tetramer. In t he space-filling model of the MCAD tetramer
shown the four subunits are coloured white, m agenta, red and orange
and Tyr42 in the magenta and red subunits is highlighted in green.
(B) Tyr42 is localized in the short helix B in a turn between helices A
and C. The backbone of one MCAD m onome r is sho wn in schema tic
representation with FAD (yellow) and C8-CoA (blue) represented as
sticks. The side chains of Tyr42 (thick yellow sticks) and ne ighbouring
residues are depicted. (C) Blow up of the enviro nment around Tyr42.
The backbone of helices A, B and C is represented sc hematica lly. The
side chains of Tyr42 in helix B and residues in its environment are
shown in space-filling representation.
4060 L. O’Reilly et al. (Eur. J. Biochem. 271) Ó FEBS 2004
temperature and low MCAD c oncentration i ncreased
unfolding might occur.
To test the relevance of these physicochemical observa-
tions we investigated the levels and activity of Y42H
MCAD in patient cells at different temperatures. In these
cells physiological concentrations of MCAD are present
and bias due to over- or under expression is excluded. Our
investigation of the steady-state amounts of Y42H and
K304E MCAD in immortalized lymphoblastoid cells and
fibroblasts cultured at 34 °C, 37 °Cand39°C, respectively,
showed a very clear temperature effect. As the temperature
increased the amounts of both Y42H and K304E tetramer
decreased dramatically. I n fact the Y42H mutant protein

was almost undetectable at 39 °C. Y42H also showed the
greatest reduction in the enzyme activity level. The reason
for the temperature sensitivity of the Y42H and K304E to
become so pronounced in fibroblasts and lymphoblasts is
most probably that t he concentration of t he endogenous
MCAD proteins in these cells are many fold l ower than
those i nvestigated in t he experiments p erformed with
purified enzymes. The t emperature sensitivity of the
K304E and Y42H proteins was also r eflected a s decre ases
in b-oxidation activities of cultured fibroblasts with increas-
ing temperatures, indicating that this effect is observable in
intact cells. Interestingly, the activity of the Y42H/K304E
heterozygote approached that of a K304E homozygote
demonstrating that at increased temperature the Y42H/
K304E genotype can result in b-oxidation levels dropping
almost to the levels o bserved in p atients with clinically
manifested disease. These observations clearly show that
the steady-state amounts of functional MCAD enzyme in
human cells compound heterozygous for the Y42H and
K304E mutations is highly dependent on temperature.
In conclusion our results show that Y42H is indeed a mild
mutation, but that its effect becomes more pronounced at
higher temperatures. These data suggest that individuals
with the Y42H/K304E genotype a re likely to experience a
further lowering of t heir MCAD enzyme activity in relation
to increased body temperature as may be experienced
during intercurrent infection. It is not easy to judge if this
will lead to c linical symptoms as a result of metabolic
decompensation, but several individuals who are compound
heterozygotes for the Y42H and K304E mutations, identi-

fied b y newborn s creening, and who are followed by t he
authors (SEM and DMF), have been admitted to the
hospital with significant lethargy and vomiting during
intercurrent illnesses. None have had documented hypogly-
cemia. However, the clinical protocols followed by our clinic
institute intravenous glucose therapy before frank hypogly-
cemia develops in the setting of vomiting and lethargy. In
one case, the fingerstick blood sugar was falling from the
baseline of 90 mgÆdL
)1
to 60 mgÆdL
)1
, as intravenous
therapy was be gun. We are also aware of a similar clinical
presentation seen in a child identified when a youn ger
sibling had MCAD deficiency identified by MS/MS new-
born s creening. In this family both the affected newborn
and the older sibling w ere compound heterozygous for the
Y42H mutation and t he G242R mutation (which is of
comparable severity to the K304E m utation [5]). Clinical
follow up revealed that the older sibling had suffered from
a vomiting illness at 1 year of age, had become lethargic
and ill quickly, and this episode had resulted i n hospital
admission. This occurred prior to the child being diagnosed
with MCAD deficiency. These cases suggest that the Y42H
mutation may not be clinically neutral. We expect that
experience gained from careful clinical follow up of the
individuals identified by MS/MS newborn s creening pro-
grams who are heterozygous for the Y42H mutation and
another mutation will shed more light on the risk of disease

manifestation. Until m ore knowledge i s gained, these
individuals should be considered as being at risk of disease
manifestation.
Acknowledgements
We are grateful to Bridget Wilcken for c ontributing patient fibroblasts.
We thank Linda Steinkrauss and Charles Stan ley for sharing clinical
information. We thank Christian Knudsen f or careful culturing of
patient cells. We are grateful to the Institute for Storage Ring Facilities
(ISA), University of Aarhus, for a ccess to the CD facility on the UV1
beamline at ASTRID, and especially for the help provided by Søren
Vorrening. This wo rk was sup ported by grants from the Enterprise
Ireland International Collaboration Programme, the Faculty of
Science, University College Dublin (through the award of a demonst-
ratorship), the Danish Medical ResearchCouncilandtheMarchof
Dimes (1-FY2003-688).
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