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 temperatur
Trang 1The 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’Reilly1, Peter Bross2, Thomas J Corydon3, Simon E Olpin4, Jakob Hansen2, John M Kenney5,6, Shawn E McCandless7, Dianne M Frazier8, Vibeke Winter2, Niels Gregersen2, Paul C Engel1
and Brage Storstein Andresen2,3
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;3Department of Human Genetics, University of Aarhus, Denmark;4Department of Clinical Chemistry, Sheffield Children’s Hospital, UK;5Institute of Storage Ring Facilities, University of Aarhus, Denmark;6Department of Physics, East Carolina University, Greenville, NC, USA;7Department of Genetics, Case Western Reserve University, Cleveland, OH, USA;8Department 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 no reports of patients presenting
clinically with this mutation As a basis for judging its
potential consequences we have examined the protein
phe-notype of the Y42H mutation and the common
disease-associated K304E mutation Our studies of 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 the K304E variant To distinguish between effects of
temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant Substrate binding, interaction 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
(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-medium-chain
acyl-CoA dehydrogenase deficiency (MCADD; MIM 201450) is
the commonest fatty acid oxidation defect occurring in
Europe, affecting Caucasians of 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 sudden 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 occurs at a time of metabolic stress, associated with fasting or viral illness [2–4] In the past, up to 20% of patients died prior to diagnosis of the disease [3] However, with early diagnosis and treatment prognosis is very 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 reliable method for identification of acylcarnitines from dried blood spots by MS/MS [12–14] has led to newborn screening for this common disorder in
a number of US states, parts 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: brage@ki.au.dk
Abbreviations: MCAD, medium-chain acyl-CoA dehydrogenase;
ETF, electron transferring flavoprotein; SRCD, synchrotron 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)
Trang 2folding of the polypeptide into monomer is facilitated by the
Hsp60 chaperonin system and the tetramer is 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 Y42H, was
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 the 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 be present in Germany, New South Wales and
Spain ([24] and B S Andresen & N Gregersen,
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 of 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 985AfiG (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 was loaded onto a 100-mL
Q-Sepharose anion exchange column (2.5 cm diameter;
Pharmacia Biotech), pre-equilibrated with 20 mM KPi,
50 mM KCl pH 7.2 The column was washed with
pre-equilibration buffer for 1 column vol., then 4–5 column vols
of 20 mM KPi, 50 mM KCl pH 7.2, until no more contaminants eluted The enzyme was then eluted with
20 mMKPi, 200 mMKCl pH 7.2 The eluate was concen-trated and desalted utilizing an Amicon Centricon device (Mrcutoff 30 kDa) The protein was loaded onto a 20-mL Procion red HE-3B dye affinity column (Procion dyes were
a generous gift from C V Stead of the former Imperial Chemical Industries, Dyestuffs Division, Blackley, Man-chester, UK), linked to Sepharose (Pharmacia Biotech), pre-equilibrated with 50 mM KPi, 50 mM KCl pH 7.2 (1 cm diameter) The column was washed with the pre-equilibra-tion buffer, until no more contaminants eluted The enzyme was then eluted by adding 1 mL of 3.5 mMof 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 lM to 100 lM The activity was measured using the dye acceptor ferricenium method, as described by Lehman et al [26] The assay was carried out in 100 mM KPi buffer pH 7.6 at 25C The Kmand Vmaxvalues were determined by the Wilkinson method (nonlinear regression) The activity was also measured by a modified version of the method described by Thorpe [27] using recombinant 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 [35S]methionine (20 lCi per 50 lL reaction, 10 lCiÆlL)1; Amersham Biosciences) using the TnT coupled 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 previously [28,29] The translation product was mixed with isolated mitochondria and imported into mitochondria essentially as described previously [25] The mixture was then incubated at 26C or 41 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 soluble 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 visualized by phosphor imaging using an Amersham Biosciences Phosphorimager
Trang 3(STORM 840) and the bands were quantified using
IMAGE-QUANTsoftware
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-cm2flasks
at 5% (v/v) CO2 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)1streptomycin and 0.29 mgÆmL)1glutamine at
34C, 37 C and 39 C The cell pellets were lysed, and a
total protein amount of 30 lg was loaded and run on 12%
acrylamide denaturing SDS gels and 12% acrylamide native
gels, and analysed by Western blotting as described
previously [25] Measurements of the b-oxidation flux in
cultured fibroblasts using [9,10-3H] 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
34C, 37 C and 39 C for 72 h prior 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 mM KPi pH 7.2
buffer Samples at concentrations of 330 lgÆmL)1 and
50 lgÆmL)1 and buffer (for baseline 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 30C and 75 C)
and time (5 min equilibration at each temperature) The
baseline (buffer only) spectra were recorded before and
after the CD scan of each sample using the same cell as
that used for the sample and under the same conditions
(specifically temperature) Both the baselines and protein
scans were made in duplicate and the mean baseline
subtracted from the mean scan, before plotting The
spectra of the baseline-corrected 50-lgÆmL)1samples were
scaled by 330/50 (to remove the effect of concentration) so
that they could be directly compared to the 330 lgÆmL)1
data This was confirmed by comparing the data of all the
samples at 30C, 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 affinity chromatography The kinetics of
the catalysed reaction with octanoyl-CoA as substrate and
ferricenium as final electron acceptor was studied with each
purified protein The Kmwas determined by the Wilkinson
method to be 3.7 ± 0.3 l (Mean ± SE) for wild-type,
which compares well with the previously published results of 3.4 lM [33,34] However, the Km of the K304E mutant protein was determined to be 5.9 ± 0.7 lM, which is somewhat lower than the previously published value of
12 lM[33,34] We found that the Y42H mutant protein has approximately the same maximum velocity (Vmax¼ 24.2 ± 0.7· 103nmol ferricenium/mgÆmin)1) as the wild-type enzyme (Vmax¼ 24.6 ± 0.6 · 103), but the Y42H protein has a higher Km than wild-type (5.2 ± 0.5 lM), 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 (Vmax¼ 8.2 ± 0.3· 103)
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 relatively 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 whether the mutations affected the binding
of the prosthetic group FAD The peak-to-peak ratio
A280nm : 450nmfor the K304E mutant protein as purified is significantly increased (ratio K304E¼ 8.9; wild-type ¼ 7.2), showing that the binding of the prosthetic group is considerably impaired Y42H MCAD shows a slight increase in the peak-to-peak ratio (ratio Y42H¼ 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 temperature on biogenesis and stability of the Y42H MCAD, compared
to wild-type and K304E MCAD We performed 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
26C and 41 C (Fig 1) At 26 C the amounts of tetra-mer formed increased until 120 min There 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
Trang 4tetramers 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 41C (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 41C soluble Y42H protein reaches
a peak within the first 10 min, but in contrast to the wild-type protein the amount of soluble protein decreases over
Fig 1 Comparison of the biogenesis/stability of Y42H and K304E mutants 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 was imported into isolated rat liver mitochondria for 30 min at 26 C Aliquots were removed at the time points indicated The amounts of monomeric and tetrameric MCAD proteins were measured at 26 C and 41 C as described previously [25] Briefly, soluble and insoluble MCAD proteins were separated by centrifugation and the respective fractions, either soluble MCAD protein (present in the supernatant) or aggregating MCAD protein (present in the pellet) were measured
by quantification (phosphorimaging) of the MCAD monomeric band after SDS/PAGE The amounts of tetramers in the soluble fraction were measured by quantification (phosphorimaging) of the band corresponding to tetrameric MCAD protein after native PAGE The levels of MCAD protein were normalized to the total amount of radiolabelled MCAD protein (soluble and insoluble) in the corresponding lane of the SDS gel Results are representative of three separate experiments.
Trang 5time, concomitant with an increase in the amount of
insoluble protein Because this is a dynamic process we
cannot say from these experiments whether the increased
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 studies 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 stabithermostabi-lity
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)1a difference is observed between the variant
proteins (Fig 2A), with Y42H showing a decreased stability
at temperatures above 42C compared to wild-type, and K304E showing a more pronounced decrease At the higher protein concentration of 50 lgÆmL)1there is little difference observed in the thermostability between 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 This demonstrates that the thermal stability
of the MCAD enzyme is dependent on the protein concentration
To investigate whether the thermal stability depends on the total protein concentration in vitro or specifically on the concentration of MCAD polypeptide chains, the enzyme activity curves (Fig 2A) were repeated in the presence of
1 mgÆmL)1BSA (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 observed 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 activity curves shows that the loss of MCAD tetramer corresponds to the loss of activity (Fig 2D) SDS/PAGE of 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
D
E
Fig 2 Enzyme activity assays of wild-type, Y42H and K304E mutant protein at 3.3 lgÆmL)1and 50 lgÆmL)1for without BSA (A) and with
1 mgÆmL)1BSA (C) The activity at the higher concentration of 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 protein concentration The amount of protein, corresponding to the activity at 3.3 lgÆmL)1
is seen by Western blot analysis of (D) native gel showing tetramer formation and (E) showing the ratio of soluble (S ¼ supernatant fraction) and insoluble (P ¼ pellet fraction) protein by SDS/PAGE Error bars indicate the standard deviation of the mean result.
Trang 6tetramer falls together with the MCAD protein becoming
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)1at 35C showed no significant difference in
their spectra, indicating that the overall 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 temperatures, 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 35C after heating did not reproduce the initial characteristic spectrum
Interestingly, 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 and both mutant proteins are the same at
35C, 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 45C (Fig 3B) Taken together, these data confirm that MCAD thermal stability depends on the MCAD concentration and that thermal inactivation of the enzyme correlates with 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 described above, we analysed steady-state amounts of Y42H and K304E MCAD in immortalized lymphoblastoid cells cultured at 34C, 37 C and 39 C by 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 34C there is little difference between the amount of either tetramer or soluble protein present in the K304E/wild-type heterozy-gote, compared to the K304E/Y42H heterozyheterozy-gote, 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 37C and 39 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 are comparable to those observed from the K304E homozygote at 39C The effect of temperature on the mutant and wild-type proteins was investigated further by measuring the b-oxidation 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 temperature 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 34C to 37 C, and a further 14% loss with another 2C increase in temperature to 39 C Discussion
The Y42H mutation is of potential clinical importance, as it
is the second most prevalent mutation in the MCAD gene and compound heterozygosity for the K304E and Y42H mutations is the second most prevalent genotype in babies identified on the basis of an abnormal 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 returned to 35 C Although the mutant
protein temperature scans are almost identical, only wild-type is shown
for simplicity (B) Comparison of the CD data collected at 222 nm
at 50 lgÆmL)1and 330 lgÆmL)1protein concentrations Error bars
indicate the standard deviation of the mean result.
Trang 7carried out in the US, Australia, Germany and Spain ([11],
B S Andresen & N Gregersen, unpublished 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 Y42H 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
benign 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 is a cause for major concern that this method may detect benign variants of unknown clinical significance, creating unwarranted anxiety in parents and health care professionals [39] It is therefore of utmost importance that the consequences of the Y42H mutation are thoroughly investigated, to distinguish between a
benign MCAD variant that causes an abnormal acylcar-nitine pattern, but is unlikely to cause disease, and a disease-causing MCAD variant Therefore we have in the 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 data 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 mutation
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 cultured at 34 °C, 37 °C and 39 °C The tetrameric and the soluble MCAD protein was measured by native (A) and denaturing (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
K304E/K304E K304E/Y42H K304E/WT
Fig 5 Myristate oxidation from fibroblasts with genotypes K304E/
K304E, K304E/Y42H and K304E/WT as compared to WT/WT
con-trols cultured at 34 °C, 37 °C and 39 °C The results are expressed as
the percentage of the activity of normal control cell lines, and are the
average of two separate experiments (mean of five determinations),
using three different control lines Error bars indicate standard
devi-ation of the mean.
Trang 8protein [11] At low temperatures (31C) 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
31C to 37 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 folding mutant, like many of the
previously characterized disease-causing mutations in
MCAD [5,21,23] However, unlike the MCAD proteins
with folding mutations, overexpression of chaperonins
appeared to have very little or no effect on the Y42H
protein [11] A thermal stability curve of crude lysates from
E colioverexpressing mutant or wild-type MCAD
indica-ted a decrease in the thermal stability of Y42H protein as
compared to the wild-type suggesting that the temperature
effect might be due to a decreased stability of 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 compared
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 tetrameric 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 protein
To address this question we used the purified
recombin-ant MCAD varirecombin-ant proteins and investigated the thermal
stability of the native enzymes at different concentrations
This confirmed that both the Y42H and K304E proteins
were less stable than the wild-type, with K304E 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 three proteins becomes almost
indistinguishable We could show that the decisive factor is
the concentration of MCAD protein rather than the total
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 oligomeric 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 temperature 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 signal, 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 tetramer 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 high MCAD concentration This would explain the increased thermal stability 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 interaction network that stabilizes the fold of helices A, B and C and links it to the edge 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 interactions of carbon atoms in the aromatic ring of tyrosine with the neighbouring residues would be altered or abolished This could result in loosening of the 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 in the MCAD structure The illustrations were produced with VIEWER LITE 5.0 (Accelrys) using the PDB coordinates 1EGC (A) Tyr42 is localized at the surface of the MCAD tetramer In the space-filling model of the MCAD tetramer shown the four subunits are coloured white, magenta, 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 monomer is shown in schematic representation with FAD (yellow) and C8-CoA (blue) represented as sticks The side chains of Tyr42 (thick yellow sticks) and neighbouring residues are depicted (C) Blow up of the environment around Tyr42 The backbone of helices A, B and C is represented schematically The side chains of Tyr42 in helix B and residues in its environment are shown in space-filling representation.
Trang 9temperature and low MCAD concentration increased
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 34C, 37 C and 39 C, respectively,
showed a very clear temperature effect As the temperature
increased the amounts of both Y42H and K304E tetramer
decreased dramatically In fact the Y42H mutant protein
was almost undetectable at 39C 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 the concentration of the endogenous
MCAD proteins in these cells are many fold lower than
those investigated in the experiments performed with
purified enzymes The temperature sensitivity of the
K304E and Y42H proteins was also reflected as decreases
in b-oxidation activities of cultured fibroblasts with
increas-ing temperatures, indicatincreas-ing 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 observed in patients 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 are likely to experience a
further lowering of their 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 clinical symptoms as a result of metabolic
decompensation, but several individuals who are compound
heterozygotes for the Y42H and K304E mutations,
identi-fied by newborn screening, and who are followed by the
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 begun We are also aware of a similar clinical
presentation seen in a child identified when a younger
sibling had MCAD deficiency identified by MS/MS
new-born screening In this family both the affected newnew-born
and the older sibling were compound heterozygous for the
Y42H mutation and the G242R mutation (which is of
comparable severity to the K304E mutation [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 in 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 screening pro-grams who are heterozygous for the Y42H mutation and another mutation will shed more light on the risk of disease manifestation Until more knowledge is gained, these individuals should be considered as being at risk of disease manifestation
Acknowledgements
We are grateful to Bridget Wilcken for contributing patient fibroblasts.
We thank Linda Steinkrauss and Charles Stanley for sharing clinical information We thank Christian Knudsen for careful culturing of patient cells We are grateful to the Institute for Storage Ring Facilities (ISA), University of Aarhus, for access to the CD facility on the UV1 beamline at ASTRID, and especially for the help provided by Søren Vorrening This work was supported 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 Research Council and the March of Dimes (1-FY2003-688).
References
1 Schulz, H (1991) Oxidation of fatty acids In Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D.E & Vance, J., eds), pp 87–110 Elsevier Science Publishers, Amsterdam.
2 Roe, C.R & Ding, J (2001) Mitochondrial Fatty Acid Oxidation Disorders In The Metabolic and Molecular Bases of Inherited Disease, 8th edn (Scriver, C.R., Beaudet, A.L., Sly, W.S & Valle, D., eds), pp 2297–2326 McGraw-Hill, New York.
3 Iafolla, A.K., Thompson, R.J & Roe, C.R (1994) Medium chain acyl coenzyme A dehydrogenase deficiency: Clinical course in 120 affected children J Pediatr 124, 409–415.
4 Roe, CR & Coates, PM (1995) Mitochondrial Fatty Acid Oxi-dation Disorders In The Metabolic and Molecular Bases of Inherited Disease, 7th edn (Scriver, C.R., Beaudet, A.L., Sly, W.S.
& Valle, D., eds), pp 1501–1533 McGraw-Hill, New York.
5 Andresen, B.S., Bross, P., Udvari, S., Kirk, J., Gray, G., Kmoch, S., Chamoles, N., Knudsen, I., Winter, V., Wilcken, B., Yokota, I., Hart, K., Packman, S., Harpey, J.P., Saudubray, J.M., Hale, D.E., Bolund, L., Kølvraa, S & Gregersen, N (1997) The mole-cular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: Is there corre-lation between genotype and phenotype? Hum Mol Genet 6, 695–707.
6 Andresen, B.S., Bross, P., Jensen, T.G., Winter, V., Knudsen, I., Kølvraa, S., Jensen, U.B., Bolund, L., Duran, M., Kim, J.J., Curtis, D., Divry, P., Vianey-Saban, C & Gregersen, N (1993) A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD) Am J Hum Genet 53, 730–739.
7 Wilcken, B., Carpenter, K.H & Hammond, J (1993) Neonatal symptoms in MCAD deficiency Arch Dis Child 69, 292–294.
8 Marsden, D., Sege-Petersen, K., Nyhan, W.L., Roeschinger, W & Sweetman, L (1992) An unusual presentation of medium-chain acyl coenzyme A dehydrogenase deficiency Am J Dis Child 146, 1459–1462.
9 Ruitenbeek, W., Poels, P.J.E., Turnbull, D.M., Garavaglia, B., Chalmers, R.A., Taylor, R.W & Gabreels, F.J.M (1995) Rhab-domyolysis and acute encephalopathy in late onset medium chain
Trang 10acyl-CoA dehydrogenase deficiency J Neurol Neurosurg
Psy-chiatry 58, 209–214.
10 Yang, B.Z., Ding, J.H., Zhou, C., Dimachkie, M.M., Sweetman,
L., Dasouki, M.J., Wilkinson, J & Roe, C.R (2000) Identification
of a novel mutation in patients with medium-chain acyl-CoA
dehydrogenase deficiency Mol Genet Metab 69, 259–262.
11 Andresen, B.S., Dobrowolski, S.F., O’Reilly, L., Muenzer, J.,
McCandless, S.E., Fraizer, D.M., Udvari, S., Bross, P., Knudsen,
I., Banas, R., Chace, D.H., Engel, P., Naylor, E.W & Gregersen,
N (2001) Medium-chain acyl-CoA dehydrogenase (MCAD)
mutations identified by MS/MS-based prospective screening of
newborns differ from those observed in patients with clinical
symptoms: identification and characterization of a new, prevalent
mutation that results in mild MCAD deficiency Am J Hum.
Genet 68, 1408–1418.
12 Millington, D.S., Norwood, D.L., Kodo, N., Roe, C.R & Inoue,
F (1989) Application of fast atom bombardment with tandem
mass spectrometry and liquid chromatography/mass spectrometry
to the analysis of acylcarnitines in human urine, blood, and tissue.
Anal Biochem 180, 331–339.
13 Chace, D.H., Hillman, S.L., Van Hove, J.L.K & Naylor, E.W.
(1997) Rapid diagnosis of MCAD deficiency: quantitatively
ana-lysis of octanoylcarnitine and other acylcarnitines in newborn
blood spots by tandem mass spectrometry Clin Chem 43, 2106–
2113.
14 Zytkovicz, T.H., Fitzgerald, E.F., Marsden, D., Larson, C.A.,
Shih, V.E., Johnson, D.M., Strauss, A.W., Comeau, A.M., Eaton,
R.B & Grady, G.F (2001) Tandem mass spectrometric analysis
for amino, organic, and fatty acid disorders in newborn dried
blood spots: a two-year summary from the New England
New-born Screening Program Clin Chem 47, 1945–1955.
15 Gregersen, N., Blakemore, A.I., Winter, V., Andresen, B.,
Kolvraa, S., Bolund, L., Curtis, D & Engel, P.C (1991) Specific
diagnosis of medium chain acyl CoA (MCAD) deficiency in dried
blood spots by polymerase chain reaction (PCR) assay detecting a
point mutation (G985) in the MCAD gene Clin Chim Acta 203,
23–34.
16 Yokota, I., Coates, P., Hale, D.E., Rinaldo, P & Tanaka, K.
(1991) Molecular survey of a prevalent mutation, 985 A-to-G
transition, and identification of five infrequent mutations in the
medium-chain acyl-CoA dehydrogenase (MCAD) gene in 55
pa-tients with MCAD deficiency Am J Hum Genet 49, 1280–1291.
17 Pollitt, R.J & Leonard, J.V (1998) Prospective surveillance study
of medium chain acyl-CoA dehydrogenase deficiency in the UK.
Arch Dis Child 79, 116–119.
18 Saijo, T., Welch, W.J & Tanaka, K (1994) Intramolecular folding
and assembly of medium-chain acyl-CoA dehydrogenase
(MCAD) Demonstration of impaired transfer of K304E-variant
MCAD from its complex with hsp60 to the native tetramer.
J Biol Chem 269, 4401–4408.
19 Bross, P., Corydon, T.J., Andresen, B.S., Jorgensen, M.M.,
Bolund, L & Gregersen, N (1999) Protein misfolding and
degradation in genetic diseases Hum Mutat 14, 186–198.
20 Yokota, I., Saijo, T., Vockley, J & Tanaka, K (1992) Impaired
tetramer assembly of variant medium chain acyl Co-A
dehy-drogenase with a glutamate or aspartate substitution for lysine 304
causing instability of the protein J Biol Chem 267, 26004–26010.
21 Bross, P., Jespersen, C., Jensen, T.G., Andresen, B.S., Kristensen,
M.J., Winter, V., Nandy, A., Kra¨utle, F., Ghisla, S., Bolund, L.,
Kim, J.-J & Gregersen, N (1995) Effects of two mutations
detected in medium chain acyl-CoA dehydrogenase
(MCAD)-deficient patients on folding, oligomer assembly, and stability of
MCAD enzyme J Biol Chem 270, 10384–10390.
22 Bross, P., Andresen, B.S., Winter, V., Kraeutle, F., Jensen, T.G.,
Kolvraa, S., Rasched, I., Ghisla, S., Bolund, L & Gregersen, N.
(1993) Co-overexpression of bacterial GroESL chaperonins partly
overcomes non-productive folding and tetramer assembly of E coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation Biochim Biophys Acta 1182, 264–274.
23 Jensen, T.G., Andresen, B.S., Bross, P., Jensen, U.B., Holme, E., Kolvraa, S., Gregersen, N & Bolund, L (1992) Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eukaryotic cells Biochim Biophys Acta 1180, 65–72.
24 Zschocke, J., Schulze, A., Linder, M., Fiesel, S., Olgemo¨ller, K., Hoffmann, G.F., Penzien, J., Ruiter, J.P.N., Wanders, R.J.A & Mayatepek, E (2001) Molecular and functional characterization
of mild MCAD deficiency Hum Genet 108, 404–408.
25 Pedersen, C.B., Bross, P., Winter, V.S., Corydon, T.J., Bolund, L., Bartlett, K., Vockley, J & Gregersen, N (2003) Misfolding, degradation, and aggregation of variant proteins The molecular pathogenesis of short chain acyl-CoA dehydrogenase (SCAD) deficiency J Biol Chem 278, 47449–47458.
26 Lehman, T.C., Hale, D.E., Bhala, A & Thorpe, C (1990) An acyl-coenzyme A dehydrogenase assay utilizing the ferricenium ion Anal Biochem 186, 280–284.
27 Thorpe, C (1981) Acyl-CoA dehydrogenase from pig kidney Methods Enzymol 71, 366–374.
28 Gregersen, N (1979) Studies on the effects of saturated and unsaturated short-chain monocarboxylic acids on the energy metabolism of rat liver mitochondria Pediatr Res 13, 1227–1230.
29 Bross, P., Winter, V., Pedersen, C.B & Gregersen, N (2003) Investigation of folding and degradation of in vitro synthesized mutant proteins in mitochondria Meth Mol Biol 232, 285–293.
30 Bross, P., Jensen, T.G., Andresen, B.S., Kjeldsen, M., Nandy, A., Kolvraa, S., Ghisla, S., Rasched, I., Bolund, L & Gregersen, N (1994) Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme Bio-chem Med Metab Biol 52, 36–44.
31 Manning, N.J., Olpin, S.E., Pollitt, R.J & Webley, J (1990) A comparison of [9,10–3H] palmitic and [9,10–3H] myristic acids for the detection of defects of fatty acid oxidation in intact cultured fibroblasts J Inher Metab Dis 13, 58–68.
32 Olpin, S.E., Manning, N.J., Carpenter, K., Middleton, B & Pol-litt, R.J (1992) Differential diagnosis of hydroxydicarboxylic aciduria based on release of 3H2O from [9,10–3H] myristic and [9,10–3H] palmitic acids by intact cultured fibroblasts J Inher Metab Dis 15, 883–890.
33 Nandy, A., Kieweg, V., Krautle, F.G., Vock, P., Kuchler, B., Bross, P., Kim, J.J.P., Rasched, I & Ghisla, S (1996) Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active centre base arrangement of long-chain Acyl-CoA dehydrogenase Biochemistry 35, 12402– 12411.
34 Kieweg, V., Krautle, F., Nandy, A., Engst, S., Vock, P., Abdel-Ghandy, A.G., Bross, P., Gregersen, N., Rasched, I., Strauss, A.
& Ghisla, S (1997) Biochemistry characterization of purified, human recombinant Lys304fiGlu medium-chain acyl-CoA dehydrogenase containing the common disease-causing mutation and comparison with the normal enzyme Eur J Biochem 246, 548–556.
35 Andresen, B.S., Christensen, E., Corydon, T.J., Bross, P., Pilgaard, B., Wanders, R.J., Ruiter, J.P., Simonsen, H., Winter, V., Knudsen, I., Schroeder, L.D., Gregersen, N & Skovby, F (2000) Isolated 2-methylbutyrylglycinurina caused by short/bran-ched-chain acyl-CoA dehydrogenase deficiency: identification of a new enzyme defect, resolution of its molecular basis, and evidence for distinct acyl-CoA dehydrogenase in isoleucine and valine metabolism Am J Hum Genet 67, 1095–1103.