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: brage@ki.au.dk 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. 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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. that the temperature- induced change in the fold of the structure is indistinguishable between the three proteins at this protein concentration. Furthermore,