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Expression, purification, and characterization of human malonyl-CoA decarboxylase
Protein Expression and Purification 34 (2004) 261–269 www.elsevier.com/locate/yprep
Expression, purification, and characterization of human malonyl-CoA decarboxylase Demin Zhou,a Phoebe Yuen,a Donald Chu,a Vicki Thon,a Steve McConnell,a Steven Brown,a Andria Tsang,a Michael Pena,a Anna Russell,b Jie-Fei Cheng,b Alex M. Nadzan,b Miguel S. Barbosa,a Jason R.B. Dyck,c,d Gary D. Lopaschuk,c,d and Guang Yanga,* a
Department of Discovery Biology, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA Department of Discovery Chemistry, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA Cardiovascular Research Group, Departments of Pediatrics and Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alta., Canada d Metabolic Modulators Research Ltd., 2020 Research Transition Facility, University of Alberta, Edmonton, Alta., Canada b
c
Received 10 October 2003, and in revised form 11 November 2003
Abstract The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330–520 lM and a turnover rate ðkcat Þ of 13–28 s1 . For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4–10); whereas the full-length hMCD appeared to be stable only at pH P 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human malonyl-CoA decarboxylase; Expression and purification
Malonyl-CoA decarboxylase (MCD)1 catalyzes the conversion of malonyl-CoA to acetyl-CoA. Cytoplasmic malonyl-CoA, a physiological inhibitor of carnitine acyltransferases [1], plays a pivotal role in fatty-acid metabolism. Carnitine acyl-transferase is known to catalyze the esterification of carnitine and a long-chain fatty acid, the pre-requisite step required for the b-oxidation of * Corresponding author. Current address: Tanabe Research Laboratories USA, Inc, 4540 Towne Centre Court, San Diego, CA 92121, USA. Fax: 1-858-622-7096. E-mail address: [email protected] (G. Yang). 1 Abbreviations used: MCA, malonyl-coenzyme A or malonyl-CoA; MCD, malonyl-CoA decarboxylase; wt-hMCD, wild-type full-length precursor human MCD; D39-hMCD, truncated MCD with the first 39 amino acids on N-terminal deleted; IPTG, isopropyl-b-D -thiogalactoside; OD600 , optical density at 600 nm; PCR, polymerase chain reaction; DTT, dithiothreitol; pI, isoelectronic point.
1046-5928/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2003.11.023
fatty acids in mitochondria. Malonyl-CoA is biochemically synthesized by acetyl-CoA carboxylase, while hydrolyzed into acetyl-CoA and carbon dioxide by MCD. Mutant mice lacking the acetyl-CoA carboxylase II gene have a higher rate of fatty-acid oxidation, which is likely to be a consequence of lower levels of malonyl-CoA in the mitochondria compared with wild-type mice [2]. A significant role of MCD is suggested by severe phenotypic consequences arising from deficiency of the enzyme in humans, characterized by malonic aciduria, developmental delay, seizure disorder, and mental retardation [3–5]. These phenotypes overlap with genetic deficiency of fatty-acid oxidation enzymes [6], indicating that MCD indeed plays a critical role in fatty-acid metabolism. Abnormally high rates of fatty acid metabolism contribute to ischemic damage [7–9]. Pharmacological agents designed to directly inhibit fatty acid
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oxidation are efficacious in reducing ischemic heart injury. Since a decrease in the cytosolic malonyl-CoA is a key reason for the high fatty acid oxidation rates during and following ischemia, preventing this decrease by
development of cytoplasmic MCD inhibition represents a novel approach to treat ischemic heart disease. Malonyl-coenzyme A decarboxylase (MCD, EC 4.1.1.9) is ubiquitously distributed in all living
Fig. 1. Sequence alignment of mcd enzymes from human, mouse, rat, goose, C. elegans, S. meliloti, and Arabidopsis. The protein sequence of mammalian MCD (including goose MCD) is composed of three domains, the N-terminal putative mitochondrial targeting domain (highlighted in red, 39 amino acids for hMCD), the functional catalytic domain (in black, 454 amino acids for hMCD), and the peroxisomal targeting sequence at the C-terminal (SKL highlighted in blue). The retention signal sequences for mitochondria and peroxisome of the mammalian and goose MCD are underlined.
D. Zhou et al. / Protein Expression and Purification 34 (2004) 261–269
organisms including microorganisms, plants, and animals [10–14]. The MCD activity in mammalian cells was detected mainly in the mitochondria [13,15–17], whereas in the uropygial glands of birds, the large amount of decarboxylase activity of MCD appeared to locate in the soluble cytoplasm [18,19]. The cytoplasmic MCD enzyme of the uropygial glands was shown to be a 50-kDa protein, whereas the mitochondria enzyme was 3 kDa smaller [18,19]. Southern blot analyses of genomic DNA of goose uropygial gland revealed a single mRNA species of the MCD gene [18], indicating that the mature mitochondria enzyme is derived proteolytically from a 50 kDa precursor that accumulates in the cytoplasm of the specialized tissue. A molecular strategy using alternate promoters of the single MCD gene to generate two different transcripts was proposed to account for the subcellular localization of the avian MCD enzymes in the differentiated tissues [20]. Both the rat and human MCD were found to reside in the cytoplasm of the tissues tested [6,21]. However, no alternate transcript was found for either rat [22] or human MCD [6]. MCD enzyme was first purified from rat liver mitochondria [23,24] and uropygial gland of waterfowl [14]. Recently, it was cloned and purified from the bacteria Rhizobium trifolii [25], rat pancreatic b-cell [22,26], and goose (Anser anser) uropygial gland [18]. Identification of patients with MCD deficiency led to the cloning of the human MCD gene [27,28]. The ortholog genes of MCD in rat, mouse, and goose share 89, 84, and 71% amino acid sequence identity with the human MCD, respectively, based on the sequence alignment analyses (Fig. 1). Western blot analyses revealed that the highest mRNA expression level in human was in muscle and heart tissues, followed by liver, kidney, and pancreas, and detectable amounts in all other tissues examined (J.-F. Cheng, personal communication). Primary amino acid sequence analysis of the rat MCD [22] indicated a mitochondrial targeting sequence region consisting of a protease cleavage signal sequence at the N-terminus (amino acid residues 1–38) and a peroxisomal targeting sequence (SKL) at the C-terminal end. The human ortholog showed a similar primary sequence structure with a less defined N-terminal mitochondrial targeting sequence (amino acid residues 1– 39) due to the lack of potential to adopt an amphiphilic a-helical structure [27]. Limited biochemical characterization of the rat mitochondrial MCD [23] and the MCD from the uropygial gland of waterfowl [18,19] suggested that, unlike the Naþ -transporting decarboxylases such as methylmalonyl CoA decarboxylase, oxaloacetate decarboxylase, and glutaconyl CoA decarboxylase, the decarboxylase activity of MCD does not depend on any cofactors, e.g., biotin and metal ions. The reaction mechanism of the MCD catalyzed decarboxylation of malonyl-CoA is still poorly understood.
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In the present study, we describe a method for efficient expression and purification that generates large quantities of soluble full-length precursor (wt-hMCD) and N-terminal truncated (D39-hMCD, with the putative mitochondrial targeting sequence removed) human MCD enzyme. Initial steady-state kinetic analyses were carried out to characterize various isoforms of human MCD enzymes.
Materials and methods General Restriction endonucleases, pMAL vector, genenase I endopeptidase, and amylose resin were purchased from New England Biolabs (Beverly, MA). Taq DNA Polymerase kit was obtained from Roche Biosciences. The competent E. coli strain, TOP 10, and the TA cloning kit were obtained from Invitrogen (Carlsbad, CA), while the BL 21(DE3) competent E. coli strains were from Novagen. The oligonucleotide primers for PCR and sequencing were made at Allele (San Diego, CA). The mono Q and mono S pre-packed FPLC columns were purchased from Amersham Biosciences (Piscataway, NJ). All other reagents and chemicals were of the highest commercially available quality (Roche, Sigma, and others). Protein column chromatography was carried out on ATKAexplore system from Amersham Biosciences (Piscataway, NJ). DNA sequencing was carried out on a 3100 Genetic Analyzer of ABI PRISM system of Applied Biosystems (Foster City, CA). Enzyme assays were carried out on a Tecan Ultra MultiWell Plate Reader Series 4000 from Tecan (CH-8708 Maennedorf, Switzerland). Preparation of recombinant MBP-fusion gene constructs of human MCD A cDNA clone containing the human MCD gene was obtained by a conventional RT-PCR method using the mRNA template isolated from human heart tissue. The sequence of the open reading frame was confirmed by DNA sequencing. The full length (wt-hMCD) and the N-terminal truncated (D39-hMCD) (with the putative mitochondria pre-sequence of the first N-terminal 39 amino acids removed) gene constructs were amplified using the conventional PCR method using a pair of primers containing the EcoRI/SalI restriction cloning sites. The obtained PCR fragments containing the wthMCD gene and the D39-hMCD gene were confirmed with DNA sequencing and separately cloned via EcoRI and a SalI into the multiple cloning site of pMAL vector containing a strong Ptac promoter (New England Biolabs) immediately downstream from a malE gene encoding the sequence of the maltose-binding protein
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(MBP), and a linker gene encoding a sequence specifically recognized by the genenase I endopeptidase. Overexpression of recombinant MBP-fusion constructs in E. coli Expression of the MBP-fusion constructs was carried out in E. coli BL21 (DE3) in typical growth culture containing LB (MillerÕs LB broth, Invitrogen/Gibco) medium and 100 lg/ml ampicillin. Overexpression of recombinant enzyme was induced at mid-log growth phase of BL21 (DE3) (OD600 ¼ 0.6) using 0.3 mM IPTG at 30 °C. After addition of IPTG, the cultures were continuously grown for 5 h for MBP-wt-hMCD and overnight for MBP-D39-hMCD. Approximately 10–15 g of cell pellets was harvested from 1 L of growth culture. Purification of MBP-fusion human MCD enzymes Cell pellets harvested as described above were resuspended in a 150-ml lysis buffer consisting of 200 mM NaCl and 20 mM Tris–HCl (pH 7.5), and disrupted using a sonicator (Fisher Scientific) at 4 °C. The cell debris was removed by centrifugation at 8000g for 30 min at 4 °C. The supernatant of the cell lysate was subjected to an amylose affinity column. The amylose column was continuously washed with the lysis buffer until no detectable (Coomassie blue staining) proteins were present in the flow-through. The MBP-fusion enzymes were then eluted using lysis buffer containing 10 mM maltose to afford 80 mg/L and 200 mg/L of MBP-wt-hMCD and MBP-D39-hMCD, respectively. The purity of the MBP-fusion proteins was greater than 80% as judged by SDS–PAGE analysis (Figs. 2A and B, lane 4). Both MBP-wt-hMCD and MBP-D39-hMCD were soluble and catalytically active. Proteolytic cleavage of MBP-fusion tag The fusion protein MBP-D39-hMCD was cleaved with genenase I at a weight ratio of 50:1 (fusion protein:genenase I) in a cleavage buffer containing 200 mM NaCl, 20 mM Tris–HCl (pH 7.5), 10% glycerol, and 10 mM DTT. Incubation at 23 °C for 15 h showed complete separation of the D39-hMCD protein from the MBP-fusion tag. The resulting mixture of the cleavage reaction was dialyzed into buffer A (20 mM Tris–HCl (pH 7.5), 10 mM DTT, and 10% glycerol) containing no salt ions and then loaded onto a pre-packed Mono S cation exchange column. A linear salt gradient of 0–1 N NaCl over 10 min was applied to elute the bound MCD protein from the column. The unbound MBP-tag protein was eluted in the flow-through before the salt gradient, whereas the D39-hMCD proteins were eluted at approximately 0.2 N NaCl during the continuous salt gradient (Fig. 3) with an apparent recovery yield of 60%
Fig. 2. Expression and purification of full-length (wt-hMCD) and truncated (D39-hMCD) hMCD enyzmes. (A) SDS–PAGE Coomassie brilliant blue gel for MBP-D39-hMCD expression, genenase I digestion, and D39-hMCD purification: lane 1, molecular weight markers; lane 2, cell lysate from E. coli transformed with the pMAL vector containing the D39-hMCD gene; lane 3, flow-through of the amylose affinity column; lane 4, purified soluble MBP-D39-hMCD fusion protein; lane 5, genenase I catalyzed digestion of MBP-D39-hMCD fusion protein; lane 6, chromatography fractions containing the fusion tag, MBP; and lane 7, purified, soluble D39-hMCD protein. (B) SDS– PAGE Coomassie brilliant blue gel for MBP-wt-hMCD expression, genenase I digestion, and wt-hMCD purification: lane 1, molecular weight markers; lane 2, cell lysate from E. coli transformed with the pMAL vector containing the wt-hMCD gene; lane 3, flow-through of the amylose affinity column; lane 4, purified soluble MBP-wt-hMCD fusion protein; lane 5, genenase I catalyzed digestion of MBP-wthMCD fusion protein; lane 6, chromatography fractions containing the fusion tag, MBP; and lane 7, purified soluble wt-hMCD protein.
(6 mg >95% pure D39-hMCD from 10 mg MBP-D39hMCD). The protocol for genenase I-mediated cleavage of fusion protein MBP-wt-hMCD was almost the same as that for the cleavage of MBP-D39-hMCD except that the reaction buffer of the proteolytic digestion of MBPD39-hMCD consisted of 20 mM CAPS (pH 10.0), 200 mM NaCl, 10% glycerol, and 10 mM DTT and a 3-h incubation time afforded complete cleavage. After the protease cleavage, the digestion mixture was gradually dialyzed into a lower pH buffer C before loading onto a Mono Q anion exchange column. The gradient steps in the buffer exchange comprised a series of dialysis buffers with a change of 0.5 pH unit per buffer to a final pH of
D. Zhou et al. / Protein Expression and Purification 34 (2004) 261–269
Fig. 3. Elution profile of D39-hMCD from Mono S column chromatography of AKTAexplorer (Amersham). The unbound MBP-tag protein was eluted with the flow-through before the salt gradient, whereas the D39MCD proteins were eluted at a gradient containing 0.2 N NaCl from buffer B.
8.5. The final buffer C contains 20 mM Tris–HCl, pH 8.5, 10 mM DTT, and 10% glycerol. The wt-hMCD was eluted at approximately 0.3 N NaCl followed by the elution of the MBP tag at about 0.5 N NaCl during the continuous salt gradient. The estimated recovery yield was 20% (2 mg >95% pure wt-hMCD from 10 mg MBPwt-hMCD).
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accumulation of NADH can be continuously followed by monitoring the increase of fluorescence emission at 460 nm on a fluorescence plate reader calibrated using authentic acetyl-CoA from Sigma.2 For a typical 96-well plate assay, the increase in the fluorescence emission (kex ¼ 360 nm, kem ¼ 460 nm, for NADH) in each well was used to calculate the initial velocity of hMCD. Each 50 ll assay contained 10 mM phosphate-buffered saline (Sigma), pH 7.4, 0.05% Tween 20, 25 mM K2 HPO4 — KH2 PO4 (Sigma), 2 mM malate (Sigma), 2 mM NAD (Boehringer–Mannheim), 0.786 U MD (Roche Chemicals), 0.028 U CS (Roche Chemicals), 5–10 nM hMCD, and varying amounts of MCA substrate. Assays were initiated by the addition of MCA, and the rates were corrected for the background rate determined in the absence of hMCD. Kinetic data analysis Data were fitted to the appropriate rate equations using Grafit computer software (Erithacus Software). Initial velocity data conforming to Michaelis–Menten kinetics were fitted to Eq. (4). m ¼ VA=ðKa þ AÞ:
ð4Þ
In Eq. (4), m is the initial velocity, V is the maximum velocity, Ka is the apparent Michaelis constant, and A is the concentration of the MCA substrate.
Steady-state kinetic assay of enzymatic activity of human MCD Results The decarboxylase activity of hMCD could be measured spectrophotometrically by monitoring the acetylCoA formation using malic dehydrogenase (MD)/citrate synthase (CS) couple (Eqs. (1)–(3)). malonyl-CoA decarboxylase ðMCDÞ
Malonyl-CoA
!
acetyl-CoA ð1Þ
þ CO2 Malate þ NAD
malic dehydrogenase ðMDÞ
$
oxaloacetate
þ NADH Acetyl-CoA þ oxaloacetate þ CoA
ð2Þ citrate synthase ðCSÞ
!
citrate ð3Þ
The conversion of acetyl-CoA from malonyl-CoA was assayed using a modified protocol as previously described by Kim and Kolattukudy [23]. As shown in Eqs. (1)–(3), the establishment of the kinetic equilibrium between malate/NAD and oxaloacetate/NADH was catalyzed by malic dehydrogenase (Eq. (2)). The enzymatic reaction product of MCD, acetyl-CoA, shifted the equilibrium by condensing with oxaloacetate in the presence of citrate synthase (Eq. (3)), which resulted in a continuous generation of NADH from NAD. The
Expression of soluble recombinant human MCD in E. coli To demonstrate whether removal of the N-terminal mitochondria pre-sequence of the precursor human MCD is required for the human MCD enzyme to be fully functional, the recombinant human MCD genes of the full length and the truncated forms were constructed and expressed in E. coli. Initial attempts to express the full-length precursor hMCD (wt-hMCD) and the truncated mature hMCD (D39-hMCD) enzymes with a 6-His-tag on either N-terminus or C-terminus in E coli resulted in mainly inclusion bodies (data not shown). The GST-fusion and MBP-fusion constructs have been successfully used in the 2 In the same assay solution (50 ll final volume, as described in Materials and methods) containing all the assay components but the hMCD enzyme, varying amounts of acetyl-CoA, product of MCD reaction, were titrated and incubated at room temperature for 30 min to reach equilibrium. The NADH fluorescence change was measured on Tecan Ultra spectrophotometer. The concentration of acetyl-CoA showed a linear correspondence to the fluorescence signal generated by the coupling enzyme system. The extinction coefficient factor of acetylCoA under the assay condition (Tecan Ultra, Gain ¼ 40, kex ¼ 340, kem ¼ 465) was determined to be 30 fluorescence units/lM.
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expression of the full-length rat MCD [26] and the 39 N-terminal amino acid truncated form of human MCD [6], respectively. The pMAL vector, a high expression vector for MBP-fused construct, was also used in our effort to improve the solubility of MCD protein expressed in E. coli. The wt-hMCD gene and the truncated D39-hMCD gene amplified by routine RT-PCR using the mRNA template isolated from the human heart tissue were ligated with the malE gene in a pMAL-c2G vector. Between the malE sequence and the MCD gene a space linker DNA was inserted encoding a unique peptide sequence Pro-Gly-Ala-Ala-His-Tyr specifically recognized by the genenase I protease. The recombinant MBP-wt-hMCD and MBP-D39-hMCD genes were subsequently expressed in the BL21 cells (Figs. 2A and B). For expression of MBP-wt-hMCD, induction with 0.3 mM IPTG at 30 °C for 5 h led to maximum yield of soluble recombinant enzymes, whereas overnight incubation at room temperature is necessary to obtain maximum yield for the recombinant MBP-D39hMCD enzyme. A single-step purification using an amylose affinity column and 10 mM maltose elution solution described in the previous section afforded approximately 80 and 200 mg soluble MBP-wt-hMCD and MBP-D39-hMCD fusion enzyme, respectively, from 1 L of cell growth culture (Table 1). Dialysis against a Tris– HCl buffer (pH 7.5) to remove the free maltose from the fusion proteins led to a certain degree of precipitation of a concentrated MBP-wt-hMCD stock (>20 mg/ml), whereas there was little effect on the stability of the MBP-D39-hMCD stock with a concentration of >50 mg/ml concentration. The fact that both soluble MBP-wt-hMCD and MBP-D39-hMCD proteins were purified in high yields and showed the expected catalytic activity of malonyl-CoA decarboxylase (see below) suggests that the MBP-fusion has greatly enhanced the expression and stability of the MCD enzymes in E. coli. It appeared that the full-length wt-hMCD containing the putative mitochondria targeting sequence has a higher tendency than the truncated D39-hMCD to aggregate at neutral pH conditions even in the presence of the MBP-fusion tag.
Release of soluble human MCD enzymes from MBP-tag The recombinant fusion constructs of hMCD contain a unique Pro-Gly-Ala-Ala-His-Tyr peptide linker between the MBP-tag and hMCD that is specifically recognized by the endopeptidase, genenase I (see above). The proteolytic digestion was carried out according to the protocol described in Materials and methods. Initial digestions in a cleavage buffer (pH 7.5) containing no other additives for 3 h at 23 °C showed a complete release of hMCD proteins from the MBP-tag. However, the resulting wt-hMCD and D39-hMCD precipitated immediately after the cleavage. Analyses of the amino acid sequence of hMCD revealed that nearly 40% of the residues are highly hydrophobic, and a total of 7 cysteine residues are located in both wt-hMCD and D39hMCD proteins. These could lead to polymerization and aggregation of the released wt-hMCD and D39hMCD under aqueous and oxidative environments. One direct way to prevent the problem is to supplement the digestion buffer with DTT and glycerol. It was found that supplementation of 10 mM DTT and 10% glycerol in the digestion buffer completely prevented aggregation of the released D39-hMCD (Fig. 2A, lanes 5 and 7). The presence of 10 mM DTT and 10% glycerol in the reaction buffer, however, greatly slowed protease cleavage and extended the digestion time from 3 to 15 h for MBPD39-hMCD. Isolation of the soluble D39-hMCD protein from the MBP-tag was carried out using a Mono S cation exchange column as described previously. The obtained >95% pure D39-hMCD (as judged by SDS– PAGE gel as shown in Fig. 2A, lane 7) appeared to be stable even in the absence of the DTT and glycerol supplement. It was found that at a final concentration as high as 20 mg/ml, exchanging into a 20 mM Tris–HCl (pH 7.5) buffer containing no DTT and glycerol did not cause precipitation of the D39-hMCD enzyme. The purified enzyme was soluble and stable in a broad range of pH conditions (pH 4–10) as indicated in our crystallization effort using various crystal screening kits (data not shown). Enzyme assay showed that the D39-hMCD enzyme displayed an MCD activity comparable to the fusion MBP-D39-hMCD enzyme (Table 2). A plausible
Table 1 Summary of preparation of the four isoforms of human malonyl-CoA decarboxylase from 1 L E. coli culture media Total proteins (mg)
Total enzyme activity (Ua )
Specific enzyme activity (Ua /mg)
Estimated yield (%)
Estimated purity (%)
Full-length hMCD Purified MBP-fusion construct Proteolytic digestion process
80 16
0.65 0.33
0.0081 0.021
100 20
80 95
Truncated D39-hMCD Purified MBP-fusion construct Proteolytic digestion process
200 120
2.8 4.0
0.014 0.033
100 60
80 95
a
1 U is defined as 1 mmol product formed per minute.
D. Zhou et al. / Protein Expression and Purification 34 (2004) 261–269 Table 2 Kinetic parameters of human malonyl-CoA decarboxylases using malic dehydrogenase/citrate synthase coupled assay method Human MCD enzymes
kcat (s1 )
Km (lM)
kcat =Km (M1 min1 )
MBP-wtMCD wt-MCD MBP-D39MCD D39MCD
13 0.3 19 0.4 21 0.5 28 0.5
520 30 360 20 360 30 330 30
(1.5 0.1) 106 (3.2 0.2) 106 (3.5 0.3) 106 (5.1 0.5) 106
reason for the high solubility displayed by the purified D39-hMCD may be that the presence of 10 mM DTT and 10% glycerol in the cleavage buffer reduced the misfolding of apo-hMCD by slowing down the release rate of D39-hMCD protein from MBP-tag by inhibiting the endopeptidase activity of genenase I. The purification protocol developed provided an efficient system (60% recovery yield, Table 1) to generate a large amount of pure, active, and soluble apo-D39-hMCD, which greatly facilitates the high throughput screening and crystallization studies. For the proteolytic cleavage of the fusion full-length hMCD enzyme, MBP-wt-hMCD, the addition of DTT and glycerol did not prevent precipitation of the fulllength apo-wt-hMCD. The 39 N-terminal amino acids of the putative mitochondrial targeting sequence are apparently amphipathic containing highly hydrophobic and basic amino acid residues. The theoretical pI of the sequence is 12.5, which shifts the pI of the apo-hMCD from pI 8.0 for D39-hMCD to pI 9.1 for wt-hMCD. It was believed that the pI shift is a key contributor to the precipitation of the full-length wt-hMCD when released from the MBP-tag at neutral pH condition. The pH of the cleavage buffer was thus adjusted to pH 10 to stabilize the full-length wt-hMCD during the digestion. As expected, no aggregations were observed during the proteolytic digestion. The apo-wt-MCD appeared to be highly pH sensitive. Lowering the pH to less than 8.0 resulted in mostly aggregation, while a sudden drop of the pH from 10.0 to 8.5 also led to a significant amount of precipitation. To bring the final pH to 8.5 before chromatography (described in Materials and methods), a step-wise gradual exchange against a series of buffer solutions in which the pH was reduced 0.5 U per step was applied. The purified apo-wt-hMCD showed at least 95% purity as judged by SDS–PAGE (Fig. 2B, lane 7) and the expected enzymatic activity of malonyl-CoA decarboxylase (Table 2). The recovery yield for the fulllength wtMCD was low (20%, Table 1) due to multiple manipulation steps. Steady state kinetic characterization of human MCD enzymes Using the fluorogenic coupled-enzyme assay, steadystate kinetic analyses were conducted on the four iso-
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forms of the human MCD enzymes, MBP-wt-hMCD, MBP-D39-hMCD, wt-hMCD, and D39-hMCD. The extinction coefficient factors of acetyl-CoA under the assay condition (Tecan Ultra, Gain ¼ 40, kex ¼ 340, kem ¼ 465) were determined to be 30 fluorescence unit/ lM2 . Consistent with the literature, the human MCD enzyme appeared to require no cofactors or divalent metal ions to be fully functional. From Table 1, all the isoforms of hMCD showed compatible kinetic parameters with MBP-wt-hMCD being the least active. The apo full-length hMCD, wt-hMCD, showed an almost identical Km value and a slightly reduced catalytical efficiency ðkcat =Km Þ as those of the N-terminal truncated D39-hMCD. The N-terminal 39 amino acid residues of the precursor hMCD, the postulated leader sequence of mitochondria, thus, play little role in the hydrolytic decarboxylation of malonyl-CoA. It is interesting to note that extension at the N-terminal of hMCD as by the MBP-fusion at the N-terminal of the enzyme resulted in little effect on the kinetic behavior of the enzyme.
Discussion The cytosolic malonyl-CoA level has been implicated in signaling the cellular fuel partitioning and metabolic signal transduction in various tissues such as liver, adipose, heart, and pancreatic islet [29–33]. Identification of the cellular factors that regulate the cytosolic MCA level is of great interest for pharmacological intervention in related diseases. MCD is the cellular enzyme known to be directly involved in the cellular metabolism of MCA. The primary amino acid sequence of the full-length human MCD (Fig. 1) is composed of a putative, 39 amino acid mitochondria targeting sequence, the mature MCD domain of 454 amino acids, and a peroxisomal targeting sequence (SKL). Western blot analyses of the intracellular distribution of malonyl-CoA decarboxylase in rat and human liver using an affinity purified antihuman MCD fusion (MBP-D186-hMCD) antibody showed the accumulation of MCD in both cytoplasm and peroxisome [6]. Cytosolic enzymatic activity of MCD was also demonstrated in the rat skeletal muscle and heart [21], whereas in rat liver, the MCD activity was mostly compartmentalized in mitochondria [15]. In mammals, besides an alternate transcript of MCD, it is plausible that the cytosolic MCD activity is from the precursor MCD. Our in vitro data are consistent with this hypothesis. We demonstrated that the recombinant human MCD enzyme could be expressed in E. coli at a high level in a soluble form via an MBP-fusion construct that can be efficiently purified by amylose-affinity chromatography. The shift of the isoelectronic point of the enzyme caused by the N-terminal pre-sequence in the precursor hMCD
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appears to play a key role in protein stability. The precursor full-length hMCD was folded and functionally active in vitro. The success in the expression and purification of both the precursor and mature recombinant MCD enzymes in vitro and the observed, almost identical kinetic properties of the two enzyme forms are consistent with the hypothesis that the precursor MCD could regulate the cytosolic MCA level. The pH sensitivity of the precursor human MCD appears to provide a plausible mechanism for the cellular regulation of the cytoplasmic enzymatic activity of MCD since cellular pH has been implicated in regulating several cellular events such as apoptosis [34]. It is also known that for cellular matrix proteins synthesized in cytoplasm, in addition to a targeting pre-sequence, a partially folded conformation (facilitated by molecular chaperones) is also required for efficient import into subcellular compartments [35–37]. By altering the pH in vitro, we have demonstrated that the full-length precursor hMCD can adopt different conformations. A fully folded and functional precursor hMCD could certainly be retained in the cytoplasm and play a role in the regulation of cytosolic MCA level in cells.
[9]
[10]
[11] [12]
[13] [14]
[15]
[16]
[17] [18]
Acknowledgments We thank Mrs. Jia Zhang and Dr. Jose Pardinas for DNA sequencing.
[19]
[20]
References [1] J.D. McGarry, K.F. Woeltje, M. Kuwajima, D.W. Foster, Regulation of ketogenesis and the renaissance of carnitine palmitoyltransferase, Diabetes Metab. Rev. 5 (1989) 271–284. [2] L. Abu-Elheiga, M.M. Matzuk, K.A. Abo-Hashema, S.J. Wakil, Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2, Science 291 (2001) 2613–2616. [3] E.A. Haan, R.D. Scholem, H.B. Croll, G.K. Brown, Malonyl coenzyme A decarboxylase deficiency. Clinical and biochemical findings in a second child with a more severe enzyme defect, Eur. J. Pediatr. 144 (1986) 567–570. [4] G.B. MacPhee, R.W. Logan, J.S. Mitchell, D.W. Howells, E. Tsotsis, D.R. Thorburn, Malonyl coenzyme A decarboxylase deficiency, Arch. Dis. Child 69 (1993) 433–436. [5] S. Yano, L. Sweetman, D.R. Thorburn, S. Mofidi, J.C. Williams, A new case of malonyl coenzyme A decarboxylase deficiency presenting with cardiomyopathy, Eur. J. Pediatr. 156 (1997) 382– 383. [6] K.A. Sacksteder, J.C. Morrell, R.J. Wanders, R. Matalon, S.J. Gould, MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency, J. Biol. Chem. 274 (1999) 24461–24468. [7] B. Liu, A.S. Clanachan, R. Schulz, G.D. Lopaschuk, Cardiac efficiency is improved after ischemia by altering both the source and fate of protons, Circ. Res. 79 (1996) 940–948. [8] Q. Liu, J.C. Docherty, J.C. Rendell, A.S. Clanachan, G.D. Lopaschuk, High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by
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D. Zhou et al. / Protein Expression and Purification 34 (2004) 261–269 [28] D.R. FitzPatrick, A. Hill, J.L. Tolmie, D.R. Thorburn, J. Christodoulou, The molecular basis of malonyl-CoA decarboxylase deficiency, Am. J. Hum. Genet. 65 (1999) 318–326. [29] J.D. McGarry, N.F. Brown, The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis, Eur. J. Biochem. 244 (1997) 1–14. [30] M. Prentki, B.E. Corkey, Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain Acyl-CoA implicated in multiple tissue defects of obesity and NIDDM?, Diabetes 45 (1996) 273–283. [31] A.K. Saha, T.G. Kurowski, N.B. Ruderman, A malonyl-CoA fuel-sensing mechanism in muscle: effects of insulin, glucose, and denervation, Am. J. Physiol. 269 (1995) E283–E289. [32] G.D. Lopaschuk, D.D. Belke, J. Gamble, T. Itoi, B.O. Schonekess, Regulation of fatty acid oxidation in the mammalian heart in health and disease, Biochim. Biophys. Acta 1213 (1994) 263–276.
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[33] M. Prentki, New insights into pancreatic beta-cell metabolic signaling in insulin secretion, Eur. J. Endocrinol. 134 (1996) 272– 286. [34] S. Matsuyama, J.C. Reed, Mitochondria-dependent apoptosis and cellular pH regulation, Cell Death Differ. 7 (2000) 1155– 1165. [35] R.J. Deshaies, B.D. Koch, W.M. Werner, E.A. Craig, R. Schekman, A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides, Nature 332 (1988) 800–805. [36] W.J. Chirico, M.G. Waters, G. Blobel, 70K heat shock related proteins stimulate protein translocation into microsomes, Nature 322 (1988) 805–810. [37] A. Artigues, A. Iriarte, M. Martinez-Carrion, Binding to chaperones allows import of a purified mitochondrial precursor into mitochondria, J. Biol. Chem. 272 (1997) 16852–16861.
Expression, purification, and characterization of human malonyl-CoA decarboxylase Demin Zhou,a Phoebe Yuen,a Donald Chu,a Vicki Thon,a Steve McConnell,a Steven Brown,a Andria Tsang,a Michael Pena,a Anna Russell,b Jie-Fei Cheng,b Alex M. Nadzan,b Miguel S. Barbosa,a Jason R.B. Dyck,c,d Gary D. Lopaschuk,c,d and Guang Yanga,* a
Department of Discovery Biology, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA Department of Discovery Chemistry, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA Cardiovascular Research Group, Departments of Pediatrics and Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alta., Canada d Metabolic Modulators Research Ltd., 2020 Research Transition Facility, University of Alberta, Edmonton, Alta., Canada b
c
Received 10 October 2003, and in revised form 11 November 2003
Abstract The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330–520 lM and a turnover rate ðkcat Þ of 13–28 s1 . For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4–10); whereas the full-length hMCD appeared to be stable only at pH P 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human malonyl-CoA decarboxylase; Expression and purification
Malonyl-CoA decarboxylase (MCD)1 catalyzes the conversion of malonyl-CoA to acetyl-CoA. Cytoplasmic malonyl-CoA, a physiological inhibitor of carnitine acyltransferases [1], plays a pivotal role in fatty-acid metabolism. Carnitine acyl-transferase is known to catalyze the esterification of carnitine and a long-chain fatty acid, the pre-requisite step required for the b-oxidation of * Corresponding author. Current address: Tanabe Research Laboratories USA, Inc, 4540 Towne Centre Court, San Diego, CA 92121, USA. Fax: 1-858-622-7096. E-mail address: [email protected] (G. Yang). 1 Abbreviations used: MCA, malonyl-coenzyme A or malonyl-CoA; MCD, malonyl-CoA decarboxylase; wt-hMCD, wild-type full-length precursor human MCD; D39-hMCD, truncated MCD with the first 39 amino acids on N-terminal deleted; IPTG, isopropyl-b-D -thiogalactoside; OD600 , optical density at 600 nm; PCR, polymerase chain reaction; DTT, dithiothreitol; pI, isoelectronic point.
1046-5928/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2003.11.023
fatty acids in mitochondria. Malonyl-CoA is biochemically synthesized by acetyl-CoA carboxylase, while hydrolyzed into acetyl-CoA and carbon dioxide by MCD. Mutant mice lacking the acetyl-CoA carboxylase II gene have a higher rate of fatty-acid oxidation, which is likely to be a consequence of lower levels of malonyl-CoA in the mitochondria compared with wild-type mice [2]. A significant role of MCD is suggested by severe phenotypic consequences arising from deficiency of the enzyme in humans, characterized by malonic aciduria, developmental delay, seizure disorder, and mental retardation [3–5]. These phenotypes overlap with genetic deficiency of fatty-acid oxidation enzymes [6], indicating that MCD indeed plays a critical role in fatty-acid metabolism. Abnormally high rates of fatty acid metabolism contribute to ischemic damage [7–9]. Pharmacological agents designed to directly inhibit fatty acid
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oxidation are efficacious in reducing ischemic heart injury. Since a decrease in the cytosolic malonyl-CoA is a key reason for the high fatty acid oxidation rates during and following ischemia, preventing this decrease by
development of cytoplasmic MCD inhibition represents a novel approach to treat ischemic heart disease. Malonyl-coenzyme A decarboxylase (MCD, EC 4.1.1.9) is ubiquitously distributed in all living
Fig. 1. Sequence alignment of mcd enzymes from human, mouse, rat, goose, C. elegans, S. meliloti, and Arabidopsis. The protein sequence of mammalian MCD (including goose MCD) is composed of three domains, the N-terminal putative mitochondrial targeting domain (highlighted in red, 39 amino acids for hMCD), the functional catalytic domain (in black, 454 amino acids for hMCD), and the peroxisomal targeting sequence at the C-terminal (SKL highlighted in blue). The retention signal sequences for mitochondria and peroxisome of the mammalian and goose MCD are underlined.
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organisms including microorganisms, plants, and animals [10–14]. The MCD activity in mammalian cells was detected mainly in the mitochondria [13,15–17], whereas in the uropygial glands of birds, the large amount of decarboxylase activity of MCD appeared to locate in the soluble cytoplasm [18,19]. The cytoplasmic MCD enzyme of the uropygial glands was shown to be a 50-kDa protein, whereas the mitochondria enzyme was 3 kDa smaller [18,19]. Southern blot analyses of genomic DNA of goose uropygial gland revealed a single mRNA species of the MCD gene [18], indicating that the mature mitochondria enzyme is derived proteolytically from a 50 kDa precursor that accumulates in the cytoplasm of the specialized tissue. A molecular strategy using alternate promoters of the single MCD gene to generate two different transcripts was proposed to account for the subcellular localization of the avian MCD enzymes in the differentiated tissues [20]. Both the rat and human MCD were found to reside in the cytoplasm of the tissues tested [6,21]. However, no alternate transcript was found for either rat [22] or human MCD [6]. MCD enzyme was first purified from rat liver mitochondria [23,24] and uropygial gland of waterfowl [14]. Recently, it was cloned and purified from the bacteria Rhizobium trifolii [25], rat pancreatic b-cell [22,26], and goose (Anser anser) uropygial gland [18]. Identification of patients with MCD deficiency led to the cloning of the human MCD gene [27,28]. The ortholog genes of MCD in rat, mouse, and goose share 89, 84, and 71% amino acid sequence identity with the human MCD, respectively, based on the sequence alignment analyses (Fig. 1). Western blot analyses revealed that the highest mRNA expression level in human was in muscle and heart tissues, followed by liver, kidney, and pancreas, and detectable amounts in all other tissues examined (J.-F. Cheng, personal communication). Primary amino acid sequence analysis of the rat MCD [22] indicated a mitochondrial targeting sequence region consisting of a protease cleavage signal sequence at the N-terminus (amino acid residues 1–38) and a peroxisomal targeting sequence (SKL) at the C-terminal end. The human ortholog showed a similar primary sequence structure with a less defined N-terminal mitochondrial targeting sequence (amino acid residues 1– 39) due to the lack of potential to adopt an amphiphilic a-helical structure [27]. Limited biochemical characterization of the rat mitochondrial MCD [23] and the MCD from the uropygial gland of waterfowl [18,19] suggested that, unlike the Naþ -transporting decarboxylases such as methylmalonyl CoA decarboxylase, oxaloacetate decarboxylase, and glutaconyl CoA decarboxylase, the decarboxylase activity of MCD does not depend on any cofactors, e.g., biotin and metal ions. The reaction mechanism of the MCD catalyzed decarboxylation of malonyl-CoA is still poorly understood.
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In the present study, we describe a method for efficient expression and purification that generates large quantities of soluble full-length precursor (wt-hMCD) and N-terminal truncated (D39-hMCD, with the putative mitochondrial targeting sequence removed) human MCD enzyme. Initial steady-state kinetic analyses were carried out to characterize various isoforms of human MCD enzymes.
Materials and methods General Restriction endonucleases, pMAL vector, genenase I endopeptidase, and amylose resin were purchased from New England Biolabs (Beverly, MA). Taq DNA Polymerase kit was obtained from Roche Biosciences. The competent E. coli strain, TOP 10, and the TA cloning kit were obtained from Invitrogen (Carlsbad, CA), while the BL 21(DE3) competent E. coli strains were from Novagen. The oligonucleotide primers for PCR and sequencing were made at Allele (San Diego, CA). The mono Q and mono S pre-packed FPLC columns were purchased from Amersham Biosciences (Piscataway, NJ). All other reagents and chemicals were of the highest commercially available quality (Roche, Sigma, and others). Protein column chromatography was carried out on ATKAexplore system from Amersham Biosciences (Piscataway, NJ). DNA sequencing was carried out on a 3100 Genetic Analyzer of ABI PRISM system of Applied Biosystems (Foster City, CA). Enzyme assays were carried out on a Tecan Ultra MultiWell Plate Reader Series 4000 from Tecan (CH-8708 Maennedorf, Switzerland). Preparation of recombinant MBP-fusion gene constructs of human MCD A cDNA clone containing the human MCD gene was obtained by a conventional RT-PCR method using the mRNA template isolated from human heart tissue. The sequence of the open reading frame was confirmed by DNA sequencing. The full length (wt-hMCD) and the N-terminal truncated (D39-hMCD) (with the putative mitochondria pre-sequence of the first N-terminal 39 amino acids removed) gene constructs were amplified using the conventional PCR method using a pair of primers containing the EcoRI/SalI restriction cloning sites. The obtained PCR fragments containing the wthMCD gene and the D39-hMCD gene were confirmed with DNA sequencing and separately cloned via EcoRI and a SalI into the multiple cloning site of pMAL vector containing a strong Ptac promoter (New England Biolabs) immediately downstream from a malE gene encoding the sequence of the maltose-binding protein
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(MBP), and a linker gene encoding a sequence specifically recognized by the genenase I endopeptidase. Overexpression of recombinant MBP-fusion constructs in E. coli Expression of the MBP-fusion constructs was carried out in E. coli BL21 (DE3) in typical growth culture containing LB (MillerÕs LB broth, Invitrogen/Gibco) medium and 100 lg/ml ampicillin. Overexpression of recombinant enzyme was induced at mid-log growth phase of BL21 (DE3) (OD600 ¼ 0.6) using 0.3 mM IPTG at 30 °C. After addition of IPTG, the cultures were continuously grown for 5 h for MBP-wt-hMCD and overnight for MBP-D39-hMCD. Approximately 10–15 g of cell pellets was harvested from 1 L of growth culture. Purification of MBP-fusion human MCD enzymes Cell pellets harvested as described above were resuspended in a 150-ml lysis buffer consisting of 200 mM NaCl and 20 mM Tris–HCl (pH 7.5), and disrupted using a sonicator (Fisher Scientific) at 4 °C. The cell debris was removed by centrifugation at 8000g for 30 min at 4 °C. The supernatant of the cell lysate was subjected to an amylose affinity column. The amylose column was continuously washed with the lysis buffer until no detectable (Coomassie blue staining) proteins were present in the flow-through. The MBP-fusion enzymes were then eluted using lysis buffer containing 10 mM maltose to afford 80 mg/L and 200 mg/L of MBP-wt-hMCD and MBP-D39-hMCD, respectively. The purity of the MBP-fusion proteins was greater than 80% as judged by SDS–PAGE analysis (Figs. 2A and B, lane 4). Both MBP-wt-hMCD and MBP-D39-hMCD were soluble and catalytically active. Proteolytic cleavage of MBP-fusion tag The fusion protein MBP-D39-hMCD was cleaved with genenase I at a weight ratio of 50:1 (fusion protein:genenase I) in a cleavage buffer containing 200 mM NaCl, 20 mM Tris–HCl (pH 7.5), 10% glycerol, and 10 mM DTT. Incubation at 23 °C for 15 h showed complete separation of the D39-hMCD protein from the MBP-fusion tag. The resulting mixture of the cleavage reaction was dialyzed into buffer A (20 mM Tris–HCl (pH 7.5), 10 mM DTT, and 10% glycerol) containing no salt ions and then loaded onto a pre-packed Mono S cation exchange column. A linear salt gradient of 0–1 N NaCl over 10 min was applied to elute the bound MCD protein from the column. The unbound MBP-tag protein was eluted in the flow-through before the salt gradient, whereas the D39-hMCD proteins were eluted at approximately 0.2 N NaCl during the continuous salt gradient (Fig. 3) with an apparent recovery yield of 60%
Fig. 2. Expression and purification of full-length (wt-hMCD) and truncated (D39-hMCD) hMCD enyzmes. (A) SDS–PAGE Coomassie brilliant blue gel for MBP-D39-hMCD expression, genenase I digestion, and D39-hMCD purification: lane 1, molecular weight markers; lane 2, cell lysate from E. coli transformed with the pMAL vector containing the D39-hMCD gene; lane 3, flow-through of the amylose affinity column; lane 4, purified soluble MBP-D39-hMCD fusion protein; lane 5, genenase I catalyzed digestion of MBP-D39-hMCD fusion protein; lane 6, chromatography fractions containing the fusion tag, MBP; and lane 7, purified, soluble D39-hMCD protein. (B) SDS– PAGE Coomassie brilliant blue gel for MBP-wt-hMCD expression, genenase I digestion, and wt-hMCD purification: lane 1, molecular weight markers; lane 2, cell lysate from E. coli transformed with the pMAL vector containing the wt-hMCD gene; lane 3, flow-through of the amylose affinity column; lane 4, purified soluble MBP-wt-hMCD fusion protein; lane 5, genenase I catalyzed digestion of MBP-wthMCD fusion protein; lane 6, chromatography fractions containing the fusion tag, MBP; and lane 7, purified soluble wt-hMCD protein.
(6 mg >95% pure D39-hMCD from 10 mg MBP-D39hMCD). The protocol for genenase I-mediated cleavage of fusion protein MBP-wt-hMCD was almost the same as that for the cleavage of MBP-D39-hMCD except that the reaction buffer of the proteolytic digestion of MBPD39-hMCD consisted of 20 mM CAPS (pH 10.0), 200 mM NaCl, 10% glycerol, and 10 mM DTT and a 3-h incubation time afforded complete cleavage. After the protease cleavage, the digestion mixture was gradually dialyzed into a lower pH buffer C before loading onto a Mono Q anion exchange column. The gradient steps in the buffer exchange comprised a series of dialysis buffers with a change of 0.5 pH unit per buffer to a final pH of
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Fig. 3. Elution profile of D39-hMCD from Mono S column chromatography of AKTAexplorer (Amersham). The unbound MBP-tag protein was eluted with the flow-through before the salt gradient, whereas the D39MCD proteins were eluted at a gradient containing 0.2 N NaCl from buffer B.
8.5. The final buffer C contains 20 mM Tris–HCl, pH 8.5, 10 mM DTT, and 10% glycerol. The wt-hMCD was eluted at approximately 0.3 N NaCl followed by the elution of the MBP tag at about 0.5 N NaCl during the continuous salt gradient. The estimated recovery yield was 20% (2 mg >95% pure wt-hMCD from 10 mg MBPwt-hMCD).
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accumulation of NADH can be continuously followed by monitoring the increase of fluorescence emission at 460 nm on a fluorescence plate reader calibrated using authentic acetyl-CoA from Sigma.2 For a typical 96-well plate assay, the increase in the fluorescence emission (kex ¼ 360 nm, kem ¼ 460 nm, for NADH) in each well was used to calculate the initial velocity of hMCD. Each 50 ll assay contained 10 mM phosphate-buffered saline (Sigma), pH 7.4, 0.05% Tween 20, 25 mM K2 HPO4 — KH2 PO4 (Sigma), 2 mM malate (Sigma), 2 mM NAD (Boehringer–Mannheim), 0.786 U MD (Roche Chemicals), 0.028 U CS (Roche Chemicals), 5–10 nM hMCD, and varying amounts of MCA substrate. Assays were initiated by the addition of MCA, and the rates were corrected for the background rate determined in the absence of hMCD. Kinetic data analysis Data were fitted to the appropriate rate equations using Grafit computer software (Erithacus Software). Initial velocity data conforming to Michaelis–Menten kinetics were fitted to Eq. (4). m ¼ VA=ðKa þ AÞ:
ð4Þ
In Eq. (4), m is the initial velocity, V is the maximum velocity, Ka is the apparent Michaelis constant, and A is the concentration of the MCA substrate.
Steady-state kinetic assay of enzymatic activity of human MCD Results The decarboxylase activity of hMCD could be measured spectrophotometrically by monitoring the acetylCoA formation using malic dehydrogenase (MD)/citrate synthase (CS) couple (Eqs. (1)–(3)). malonyl-CoA decarboxylase ðMCDÞ
Malonyl-CoA
!
acetyl-CoA ð1Þ
þ CO2 Malate þ NAD
malic dehydrogenase ðMDÞ
$
oxaloacetate
þ NADH Acetyl-CoA þ oxaloacetate þ CoA
ð2Þ citrate synthase ðCSÞ
!
citrate ð3Þ
The conversion of acetyl-CoA from malonyl-CoA was assayed using a modified protocol as previously described by Kim and Kolattukudy [23]. As shown in Eqs. (1)–(3), the establishment of the kinetic equilibrium between malate/NAD and oxaloacetate/NADH was catalyzed by malic dehydrogenase (Eq. (2)). The enzymatic reaction product of MCD, acetyl-CoA, shifted the equilibrium by condensing with oxaloacetate in the presence of citrate synthase (Eq. (3)), which resulted in a continuous generation of NADH from NAD. The
Expression of soluble recombinant human MCD in E. coli To demonstrate whether removal of the N-terminal mitochondria pre-sequence of the precursor human MCD is required for the human MCD enzyme to be fully functional, the recombinant human MCD genes of the full length and the truncated forms were constructed and expressed in E. coli. Initial attempts to express the full-length precursor hMCD (wt-hMCD) and the truncated mature hMCD (D39-hMCD) enzymes with a 6-His-tag on either N-terminus or C-terminus in E coli resulted in mainly inclusion bodies (data not shown). The GST-fusion and MBP-fusion constructs have been successfully used in the 2 In the same assay solution (50 ll final volume, as described in Materials and methods) containing all the assay components but the hMCD enzyme, varying amounts of acetyl-CoA, product of MCD reaction, were titrated and incubated at room temperature for 30 min to reach equilibrium. The NADH fluorescence change was measured on Tecan Ultra spectrophotometer. The concentration of acetyl-CoA showed a linear correspondence to the fluorescence signal generated by the coupling enzyme system. The extinction coefficient factor of acetylCoA under the assay condition (Tecan Ultra, Gain ¼ 40, kex ¼ 340, kem ¼ 465) was determined to be 30 fluorescence units/lM.
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expression of the full-length rat MCD [26] and the 39 N-terminal amino acid truncated form of human MCD [6], respectively. The pMAL vector, a high expression vector for MBP-fused construct, was also used in our effort to improve the solubility of MCD protein expressed in E. coli. The wt-hMCD gene and the truncated D39-hMCD gene amplified by routine RT-PCR using the mRNA template isolated from the human heart tissue were ligated with the malE gene in a pMAL-c2G vector. Between the malE sequence and the MCD gene a space linker DNA was inserted encoding a unique peptide sequence Pro-Gly-Ala-Ala-His-Tyr specifically recognized by the genenase I protease. The recombinant MBP-wt-hMCD and MBP-D39-hMCD genes were subsequently expressed in the BL21 cells (Figs. 2A and B). For expression of MBP-wt-hMCD, induction with 0.3 mM IPTG at 30 °C for 5 h led to maximum yield of soluble recombinant enzymes, whereas overnight incubation at room temperature is necessary to obtain maximum yield for the recombinant MBP-D39hMCD enzyme. A single-step purification using an amylose affinity column and 10 mM maltose elution solution described in the previous section afforded approximately 80 and 200 mg soluble MBP-wt-hMCD and MBP-D39-hMCD fusion enzyme, respectively, from 1 L of cell growth culture (Table 1). Dialysis against a Tris– HCl buffer (pH 7.5) to remove the free maltose from the fusion proteins led to a certain degree of precipitation of a concentrated MBP-wt-hMCD stock (>20 mg/ml), whereas there was little effect on the stability of the MBP-D39-hMCD stock with a concentration of >50 mg/ml concentration. The fact that both soluble MBP-wt-hMCD and MBP-D39-hMCD proteins were purified in high yields and showed the expected catalytic activity of malonyl-CoA decarboxylase (see below) suggests that the MBP-fusion has greatly enhanced the expression and stability of the MCD enzymes in E. coli. It appeared that the full-length wt-hMCD containing the putative mitochondria targeting sequence has a higher tendency than the truncated D39-hMCD to aggregate at neutral pH conditions even in the presence of the MBP-fusion tag.
Release of soluble human MCD enzymes from MBP-tag The recombinant fusion constructs of hMCD contain a unique Pro-Gly-Ala-Ala-His-Tyr peptide linker between the MBP-tag and hMCD that is specifically recognized by the endopeptidase, genenase I (see above). The proteolytic digestion was carried out according to the protocol described in Materials and methods. Initial digestions in a cleavage buffer (pH 7.5) containing no other additives for 3 h at 23 °C showed a complete release of hMCD proteins from the MBP-tag. However, the resulting wt-hMCD and D39-hMCD precipitated immediately after the cleavage. Analyses of the amino acid sequence of hMCD revealed that nearly 40% of the residues are highly hydrophobic, and a total of 7 cysteine residues are located in both wt-hMCD and D39hMCD proteins. These could lead to polymerization and aggregation of the released wt-hMCD and D39hMCD under aqueous and oxidative environments. One direct way to prevent the problem is to supplement the digestion buffer with DTT and glycerol. It was found that supplementation of 10 mM DTT and 10% glycerol in the digestion buffer completely prevented aggregation of the released D39-hMCD (Fig. 2A, lanes 5 and 7). The presence of 10 mM DTT and 10% glycerol in the reaction buffer, however, greatly slowed protease cleavage and extended the digestion time from 3 to 15 h for MBPD39-hMCD. Isolation of the soluble D39-hMCD protein from the MBP-tag was carried out using a Mono S cation exchange column as described previously. The obtained >95% pure D39-hMCD (as judged by SDS– PAGE gel as shown in Fig. 2A, lane 7) appeared to be stable even in the absence of the DTT and glycerol supplement. It was found that at a final concentration as high as 20 mg/ml, exchanging into a 20 mM Tris–HCl (pH 7.5) buffer containing no DTT and glycerol did not cause precipitation of the D39-hMCD enzyme. The purified enzyme was soluble and stable in a broad range of pH conditions (pH 4–10) as indicated in our crystallization effort using various crystal screening kits (data not shown). Enzyme assay showed that the D39-hMCD enzyme displayed an MCD activity comparable to the fusion MBP-D39-hMCD enzyme (Table 2). A plausible
Table 1 Summary of preparation of the four isoforms of human malonyl-CoA decarboxylase from 1 L E. coli culture media Total proteins (mg)
Total enzyme activity (Ua )
Specific enzyme activity (Ua /mg)
Estimated yield (%)
Estimated purity (%)
Full-length hMCD Purified MBP-fusion construct Proteolytic digestion process
80 16
0.65 0.33
0.0081 0.021
100 20
80 95
Truncated D39-hMCD Purified MBP-fusion construct Proteolytic digestion process
200 120
2.8 4.0
0.014 0.033
100 60
80 95
a
1 U is defined as 1 mmol product formed per minute.
D. Zhou et al. / Protein Expression and Purification 34 (2004) 261–269 Table 2 Kinetic parameters of human malonyl-CoA decarboxylases using malic dehydrogenase/citrate synthase coupled assay method Human MCD enzymes
kcat (s1 )
Km (lM)
kcat =Km (M1 min1 )
MBP-wtMCD wt-MCD MBP-D39MCD D39MCD
13 0.3 19 0.4 21 0.5 28 0.5
520 30 360 20 360 30 330 30
(1.5 0.1) 106 (3.2 0.2) 106 (3.5 0.3) 106 (5.1 0.5) 106
reason for the high solubility displayed by the purified D39-hMCD may be that the presence of 10 mM DTT and 10% glycerol in the cleavage buffer reduced the misfolding of apo-hMCD by slowing down the release rate of D39-hMCD protein from MBP-tag by inhibiting the endopeptidase activity of genenase I. The purification protocol developed provided an efficient system (60% recovery yield, Table 1) to generate a large amount of pure, active, and soluble apo-D39-hMCD, which greatly facilitates the high throughput screening and crystallization studies. For the proteolytic cleavage of the fusion full-length hMCD enzyme, MBP-wt-hMCD, the addition of DTT and glycerol did not prevent precipitation of the fulllength apo-wt-hMCD. The 39 N-terminal amino acids of the putative mitochondrial targeting sequence are apparently amphipathic containing highly hydrophobic and basic amino acid residues. The theoretical pI of the sequence is 12.5, which shifts the pI of the apo-hMCD from pI 8.0 for D39-hMCD to pI 9.1 for wt-hMCD. It was believed that the pI shift is a key contributor to the precipitation of the full-length wt-hMCD when released from the MBP-tag at neutral pH condition. The pH of the cleavage buffer was thus adjusted to pH 10 to stabilize the full-length wt-hMCD during the digestion. As expected, no aggregations were observed during the proteolytic digestion. The apo-wt-MCD appeared to be highly pH sensitive. Lowering the pH to less than 8.0 resulted in mostly aggregation, while a sudden drop of the pH from 10.0 to 8.5 also led to a significant amount of precipitation. To bring the final pH to 8.5 before chromatography (described in Materials and methods), a step-wise gradual exchange against a series of buffer solutions in which the pH was reduced 0.5 U per step was applied. The purified apo-wt-hMCD showed at least 95% purity as judged by SDS–PAGE (Fig. 2B, lane 7) and the expected enzymatic activity of malonyl-CoA decarboxylase (Table 2). The recovery yield for the fulllength wtMCD was low (20%, Table 1) due to multiple manipulation steps. Steady state kinetic characterization of human MCD enzymes Using the fluorogenic coupled-enzyme assay, steadystate kinetic analyses were conducted on the four iso-
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forms of the human MCD enzymes, MBP-wt-hMCD, MBP-D39-hMCD, wt-hMCD, and D39-hMCD. The extinction coefficient factors of acetyl-CoA under the assay condition (Tecan Ultra, Gain ¼ 40, kex ¼ 340, kem ¼ 465) were determined to be 30 fluorescence unit/ lM2 . Consistent with the literature, the human MCD enzyme appeared to require no cofactors or divalent metal ions to be fully functional. From Table 1, all the isoforms of hMCD showed compatible kinetic parameters with MBP-wt-hMCD being the least active. The apo full-length hMCD, wt-hMCD, showed an almost identical Km value and a slightly reduced catalytical efficiency ðkcat =Km Þ as those of the N-terminal truncated D39-hMCD. The N-terminal 39 amino acid residues of the precursor hMCD, the postulated leader sequence of mitochondria, thus, play little role in the hydrolytic decarboxylation of malonyl-CoA. It is interesting to note that extension at the N-terminal of hMCD as by the MBP-fusion at the N-terminal of the enzyme resulted in little effect on the kinetic behavior of the enzyme.
Discussion The cytosolic malonyl-CoA level has been implicated in signaling the cellular fuel partitioning and metabolic signal transduction in various tissues such as liver, adipose, heart, and pancreatic islet [29–33]. Identification of the cellular factors that regulate the cytosolic MCA level is of great interest for pharmacological intervention in related diseases. MCD is the cellular enzyme known to be directly involved in the cellular metabolism of MCA. The primary amino acid sequence of the full-length human MCD (Fig. 1) is composed of a putative, 39 amino acid mitochondria targeting sequence, the mature MCD domain of 454 amino acids, and a peroxisomal targeting sequence (SKL). Western blot analyses of the intracellular distribution of malonyl-CoA decarboxylase in rat and human liver using an affinity purified antihuman MCD fusion (MBP-D186-hMCD) antibody showed the accumulation of MCD in both cytoplasm and peroxisome [6]. Cytosolic enzymatic activity of MCD was also demonstrated in the rat skeletal muscle and heart [21], whereas in rat liver, the MCD activity was mostly compartmentalized in mitochondria [15]. In mammals, besides an alternate transcript of MCD, it is plausible that the cytosolic MCD activity is from the precursor MCD. Our in vitro data are consistent with this hypothesis. We demonstrated that the recombinant human MCD enzyme could be expressed in E. coli at a high level in a soluble form via an MBP-fusion construct that can be efficiently purified by amylose-affinity chromatography. The shift of the isoelectronic point of the enzyme caused by the N-terminal pre-sequence in the precursor hMCD
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appears to play a key role in protein stability. The precursor full-length hMCD was folded and functionally active in vitro. The success in the expression and purification of both the precursor and mature recombinant MCD enzymes in vitro and the observed, almost identical kinetic properties of the two enzyme forms are consistent with the hypothesis that the precursor MCD could regulate the cytosolic MCA level. The pH sensitivity of the precursor human MCD appears to provide a plausible mechanism for the cellular regulation of the cytoplasmic enzymatic activity of MCD since cellular pH has been implicated in regulating several cellular events such as apoptosis [34]. It is also known that for cellular matrix proteins synthesized in cytoplasm, in addition to a targeting pre-sequence, a partially folded conformation (facilitated by molecular chaperones) is also required for efficient import into subcellular compartments [35–37]. By altering the pH in vitro, we have demonstrated that the full-length precursor hMCD can adopt different conformations. A fully folded and functional precursor hMCD could certainly be retained in the cytoplasm and play a role in the regulation of cytosolic MCA level in cells.
[9]
[10]
[11] [12]
[13] [14]
[15]
[16]
[17] [18]
Acknowledgments We thank Mrs. Jia Zhang and Dr. Jose Pardinas for DNA sequencing.
[19]
[20]
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