Expression, purification, and characterization of human acetyl-CoA carboxylase 2

Protein Expression and PuriWcation 53 (2007) 16–23 www.elsevier.com/locate/yprep

Expression, puriWcation, and characterization of human acetyl-CoA carboxylase 2 Ki Won Kim a, Harvey Yamane b, James Zondlo b, James Busby a, Minghan Wang a,¤ a

Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA b Department of Protein Science, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA Received 23 August 2006, and in revised form 27 November 2006 Available online 9 December 2006

Abstract The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and puriWed to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure–activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770–3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, puriWcation, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1–148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1–133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and puriWcation of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1–20 and 1–26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a Km value of 2 M for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower Km value for ATP (Km D 52 M). Although this diVerence could be ascribed to diVerent assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher aYnities for the substrates and could be more similar to the native enzyme. © 2006 Elsevier Inc. All rights reserved. Keywords: Acetyl-CoA carboxylase; Expression; Baculovirus

Acetyl-CoA carboxylase (ACC)1 catalyzes the conversion of acetyl-CoA to malonyl-CoA, the committed step in the biosynthesis of long chain fatty acids. The prokaryotic ACC is composed of three separate functional proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT) [1]. In fungi and *

Corresponding author. Fax: +1 805 499 0953. E-mail address: [email protected] (M. Wang). 1 Abbreviations used: ACC, acetyl-CoA carboxylase; AUC, area under the curve; BC, biotin carboxylase; BCCP, biotin carboxyl carrier protein; CT, carboxyltransferase; CPT1, carnitine palmitoyltransferase 1; HPLC, high-performance liquid chromatography; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 1046-5928/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.11.021

mammals, the enzyme is one polypeptide with distinct BC, BCCP, and CT domains [2]. The BC domain is at the N-terminus followed by the BCCP domain immediately downstream; the CT domain is at the C-terminus [2]. Biotin is covalently attached to a lysine residue within the BCCP domain and mediates the capture and subsequent transfer of a carboxyl group. The ACC reaction occurs in two separate steps. First, the BC domain catalyzes the ATP-dependent carboxylation of biotin to form carboxybiotin. Then the CT domain mediates the transfer of the carboxyl group from biotin to acetyl-CoA to form malonyl-CoA. The net reaction is the production of malonyl-CoA from acetylCoA with ATP and bicarbonate as cofactors.

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

There are two isoforms of mammalian ACC encoded by two separate genes, ACC1 and ACC2, with a molecular weight of »265 and »280 kDa, respectively [3,4]. ACC1 is primarily expressed in lipogenic tissues such as liver and adipose [3,5], whereas ACC2 is the dominant ACC in skeletal muscle and heart [4,5]. ACC2 is also present in liver but at a much lower level than ACC1 [5,6]. ACC1 is a cytosolic enzyme important in fatty acid biosynthesis, while ACC2 is associated with the mitochondrial outer membrane and regulates the rate-limiting step of fatty acid -oxidation [7]. ACC1 deletion is embryonically lethal [8], suggesting that it is important for development. Liver-speciWc ACC1 knockout mice are viable and have reduced hepatic triglyceride accumulation but no change in glucose homeostasis [9]. These data suggest that ACC1 is critical for lipogenesis. ACC2 knockout mice have increased fatty acid oxidation, reduced fat storage, and signiWcantly improved insulin sensitivity [10–12]. These animals are protected from dietinduced obesity [10–12], suggesting that ACC2 activity is critical in the regulation of energy homeostasis. Simultaneous inhibition of both ACC1 and ACC2 with antisense oligonucleotides resulted in favorable metabolic changes [13]. Together, these data indicate that ACCs are important enzymes in maintaining energy homeostasis. Studies of mammalian ACCs have been heavily reliant on enzymes puriWed directly from tissues. To boost ACC1 production from rat liver, rats have to be fasted for 48 h and refed with a high carbohydrate diet to induce ACC1 activity [14,15]. Moreover, the Wnal ACC1 preparation is often contaminated with a signiWcant level of ACC2 [16]. ACC2 is usually puriWed from rat heart [17], but often contaminated with ACC1 [18,19] due to broader tissue expression patterns of rat ACCs [20]. The yield of ACCs from rat tissues are very low, with ACC1 at 20–30 g/g of liver and ACC2 at 2 g/g of heart [17]. Most importantly, obtaining suYcient quantities of puriWed human ACCs for kinetic studies could be challenging due to limited supply of human tissues. To fully characterize human ACCs, there is a need to produce recombinant enzymes. Recently, two reports described the production of recombinant human ACCs in baculovirus and HEK293 cells [21,22]. Although full-length ACC1 was expressed and puriWed, neither group was successful in expressing fulllength ACC2 [21,22]. Gu et al. made a fusion ACC2 with the N-terminal 275 amino acids (aa) replaced with the corresponding ACC1 region (1–133 aa) [21]. Cheng and co-workers expressed a truncated version of human or rat ACC2 lacking the N-terminal 148 aa [22]. Neither case is ideal because it is not clear if the deleted N-terminal regions are important for the correct folding of the catalytic domains as acknowledged by Cheng et al. [22]. We have independently developed a method to express human ACC2 that lacks only the N-terminal membrane attachment sequence (lacking 1–20 or 1–26 aa). The enzyme is expressed at high levels in the soluble fraction of cell lysates and puriWed to homogeneity. Based on the higher aYnities of our enzyme for the substrates, we con-

17

clude the longer enzyme produced in our system is likely a better representation of the native conformation. Materials and methods General [1-14C]acetyl-CoA (51.0 mCi/mmol) and [2-14C]malonylCoA (52.0 mCi/mmol) were purchased from GE Healthcare (Piscataway, NJ). Q Sepharose HP and Superdex 200 columns were from GE Healthcare (Piscataway, NJ). Centricon Plus-70 (100 kDa MWCO) was purchased from Millipore. Peroxidase-linked streptavidin was from Biomeda Corp. (Foster City, CA). Vector construction A full-length cDNA clone encoding the human ACC2 (2458 aa) was assembled using three separate fragments ampliWed by PCR from human skeletal muscle cDNA. The sequence is identical to a GenBank human ACC2 sequence (Accession # AY382667.1) except for the following diVerences: T120 and I422. Two PCR fragments comprising residues 21-2458 and 27-2458, respectively, were ampliWed for protein expression in baculovirus. The sequence was cloned into a modiWed version of the pFastBac shuttle vector for expression using BEVS (Invitrogen). On the baculovirus expression construct, the coding region starts with a 6£His tag followed by a caspase cleavage site that was fused to the N-terminus of the ACC2 fragments. The expressed protein starts with the sequence of MAHHHHHHDEVD before ACC2 (21-2458) or (27-2458). Expression of recombinant human ACC2 Recombinant baculovirus for human ACC2 (21-2458) and ACC2 (27-2458), respectively, was generated and ampliWed in SF9 insect cells (Invitrogen). The resulting high-titer virus was used to infect a Wave (Wave Biotech) bioreactor culture of Trichoplusia ni (Orbigen) growing in ExCell 405 Medium (SAFC Biosciences) at 25 °C. At a cell density of 1.8£ 106 cells/ ml, the bioreactor culture was supplemented with 20M D-biotin, and infected to 1% v/v with virus stock. The incubation continued until cell harvest at 50 h post-infection. To examine expression, cell lysates were incubated at 42 °C for 30 min in the presence of 20 mM DTT and resolved by a 10% NuPage Bis–Tris gel (Invitrogen). The gel was run at 200 V for 90 min and stained with Imperial Protein Stain (Pierce) to visualize the protein bands. The same cell lysates were also resolved on a 4–12% NuPage Bis–Tris gel and transferred to nitrocellulose membrane. The membrane was washed with TBST (20 mM Tris–HCl, 150 mM NaCl, 0.5% Tween 20, pH 7.6), blocked in 5% IgG free BSA in TBST overnight, and incubated with peroxidase-linked streptavidin at 1:10,000 dilution in 5% IgG free BSA in TBST for 1 h at room temperature. The blot was developed with an ECL reagent (Amersham).

18

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

PuriWcation of recombinant human ACC2 Trichoplusia ni cells infected with recombinant baculovirus were harvested by centrifugation and resuspended in 20 mM Tris–HCl (pH 7.5) containing 150 mM NaCl, 5 mM imidazole, 10% glycerol, and 20 mM -mercaptoethanol. Cells were lysed by nitrogen cavitation in a Parr bomb. The cell lysate was subjected to centrifugation at 20,000g for 2 h at 4 °C and the supernatant was Wltered through a 0.45 m membrane. The clariWed lysate was incubated with metal aYnity resin for 2 h at 4 °C. After washing with 20 mM Tris–HCl (pH 7.5) containing 500 mM NaCl, 5 mM imidazole, 10% glycerol, and 20 mM -mercaptoethanol, bound proteins were eluted with 20 mM Tris–HCl (pH 7.5) containing 150 mM NaCl, 150 mM imidazole, 10% glycerol, and 20 mM -mercaptoethanol. The eluent was diluted 4-fold with equilibration buVer [20 mM Tris–HCl (pH 7.5), 10% glycerol, and 5 mM DTT] and applied to a Q Sepharose HP column equilibrated with the same buVer. Elution was performed with a linear gradient of 0–0.5 M NaCl in the equilibration buVer. Peak fractions containing ACC2 were pooled, concentrated in a Centricon Plus-70 (100 kDa MWCO) device, and loaded on a Superdex 200 gel Wltration column equilibrated with 20 mM Tris–HCl (pH 7.5), 0.3 M NaCl, 10% glycerol, and 5 mM DTT. Peak fractions containing ACC2 were pooled and the protein concentration was determined using Bradford method with BSA as standard. The purity of the protein sample was conWrmed by SDS–PAGE under reduced condition. The enzyme was stored at ¡80 °C in 20 mM Tris–HCl (pH 7.5) containing 0.3 M NaCl, 20% glycerol, and 5 mM DTT. For comparison purpose, ACC2 (21-2458) was puriWed with metal aYnity resin for kinetic and IC50 studies. Enzymatic assay and determination of kinetic parameters The enzymatic assay was run in a Wnal volume of 100 l containing 50 mM Hepes (pH 7.5), 2.0 mM MgCl2, 2.0 mM ATP, 2.0 mM potassium citrate, 12.5 mM sodium bicarbonate, 1 mg/ml bovine serum albumin, and 5 M [1-14C]acetylCoA. The reaction was initiated by the addition of 10 nM puriWed enzyme and incubated at room temperature for 30 min. The reaction was stopped by adding 50 l of 100% cold methanol and placed on ice for 10 min. The reaction was transferred to a HPLC vial and loaded on the HPLC machine for analysis. For kinetic studies, the reaction was run at [1-14C]acetyl-CoA and ATP concentrations ranging from 0.35 to 30 M and from 1 M to 1 mM, respectively. The enzyme amount and reaction time were chosen so that the conversion of acetyl-CoA to malonyl-CoA was less than 20%. The initial velocity was determined in the linear range of malonyl-CoA formation. The other components in the reaction stayed the same. Malonyl-CoA was isolated on a Beckman Gold System HPLC instrument (Fullerton, CA). After the reaction, 100 l of the reaction mixture was injected onto a pre-column (Security Guard) connected to a reverse phase column (Luna 5 m C18 100A 250 £ 4.6 mm),

both purchased from Phenomenex (Torrance, CA). Malonyl-CoA and acetyl-CoA were separated with the following elution scheme at a Xow rate of 1 ml/min: 100% buVer A at 0–1 min, an linear gradient of 0–30% buVer B in buVer A at 1–5 min, 30% buVer B in buVer A at 5–11 min, a linear gradient of 30–0% buVer B in buVer A at 11–12 min, and 100% buVer A at 12–15 min. BuVer A consisted of 10 mM of KH2PO3 (pH 7.5) and buVer B consisted of 100% methanol. An online -Ram Model 2 radiometric detector from IN/ US Systems (Tampa, FL) was used to detect the radiolabeled malonyl-CoA and acetyl-CoA peaks. A malonylCoA standard curve was constructed by injecting 100 l of 0.1–100 M [2-14C]malonyl-CoA into the HPLC instrument. The area under the malonyl-CoA peak from the malonyl-CoA standards was used to generate a linear regression standard curve using Beckman’s 32 Karat software. The amount of malonyl-CoA from each reaction was quantiWed using the corresponding area of the peak and the standard curve. For IC50 determinations, enzyme assays were carried out as described above except in the presence of increasing concentrations of the inhibitor. For enzyme stability studies, puriWed enzyme was stored at ¡80 °C in aliquots in 20 mM Tris–HCl (pH 7.5) with 0.3 M NaCl buVer containing 10, 20, and 30% glycerol, respectively. At diVerent time points, individual tubes were thawed and the enzyme was assayed under the conditions described above. The speciWc activity of the puriWed enzyme on day 0 is deWned as 100% and the speciWc activities during storage were calculated as % relative to the day 0 speciWc activity. Kinetic data analysis Kinetic constants were determined by carrying out reactions at varying substrate concentrations. The initial rate for each substrate concentration was determined using the 1st order rate equation [At] D [A0](1¡e¡kt), where [At], product concentration at time t; [A0], substrate concentration at time 0; k, rate constant. Using GraWt™ 5.0.3 software (Erithacus Software, Horley Surrey, UK), Km and Vmax values were calculated by applying reaction rate to the Michaelis–Menten equation [v D Vmax . [S]/(Km + [S])] (v, reaction rate at a speciWc substrate concentration; Vmax, the maximum reaction rate; [S], substrate concentration), where the rate is plotted as a function of the substrate concentration. The Km value calculation incorporated the use of linear Wtting with the Scatchard rearrangement. Results Expression of human ACC2 in Trichoplusia ni cells infected with baculovirus The cDNA for human ACC2 in our study is identical to a GenBank human ACC2 sequence (Accession # AY382667.1) except for the following diVerences: T120 and I422. There is only one diVerence between our sequence and

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

that used by Cheng et al. [22]: V2141 (I2141 in the sequence used by Cheng et al.; the numbering is for the entire open reading frame). Both V2141 and I2141 are found in other human ACC2 sequences in the public database. It has been described that the N-terminal 20 aa of ACC2 encodes a hydrophobic segment that mediates its insertion into the mitochondrial membrane [7]. In order to express soluble human ACC2, the N-terminal 20 or 26 aa were deleted. In addition, a 6£His tag was fused to the N-terminus of the truncated fragments for convenience of puriWcation (see Materials and methods). T. ni cells were infected with the baculovirus constructs for ACC2 (21-2458) and ACC2 (27-2458), respectively, and cells were harvested after growth at 25 °C in the presence of D-biotin to ensure full biotinylation of the recombinant enzyme. The soluble fractions of cell lysates were prepared and resolved by SDS– PAGE (Fig. 1a). The ACC2 protein band corresponding to »280 kDa is visible in the soluble fractions of the infected cell lysates, suggesting there is expression of ACC2 in the

a

kDa

1

2

3 hACC2

250 160 105

19

infected cells. The same samples were resolved by SDS– PAGE and subject to Western blotting with peroxidaselinked streptavidin. Since ACC2 is expected to be biotinylated, the recombinant ACC2 should be recognized by streptavidin. As shown in Fig. 1b, a band with the size of »280 kDa was detected in the samples infected by the ACC2 expression constructs, indicating the expression of ACC2. There is a band of similar size in the control lane but with a much lower intensity. It could be the endogenous ACC in the insect cells. Since ACC2 (27-2458) gave rise to a slightly higher expression level than ACC2 (21-2458), we used ACC2 (27-2458) for puriWcation and further studies. PuriWcation of human ACC2 expressed by Trichoplusia ni cells Trichoplusia ni cells infected by baculovirus was grown at 25 °C for 50 h before the cell paste was harvested. PuriWcation was carried out in three separate steps: Ni aYnity chromatography, Q Sepharose HP ion-exchange chromatography, and Superdex 200 size-exclusion chromatography. Samples from each step were analyzed for enzymatic activity using a HPLC assay and purity by SDS–PAGE under reduced conditions. As shown in Fig. 2, the purity of the recombinant protein improved during puriWcation. The

75

kDa

1

2

3

4

50 250

35

hACC2

160 30 105 75

b

kDa 250

1

2

50

3 hACC2

160

35 30

105 75

50 35

Fig. 2. PuriWcation of human ACC2 from Trichoplusia ni cells expressing ACC2 (27-2458). Samples from all puriWcation steps were resolved by SDS–PAGE on a 4–10% gel. A total of 3 g protein was loaded in each lane. The human ACC2 band is indicated. Lane 1, soluble fraction of ACC2 (27-2458) lysate; lane 2, Ni resin pool; lane 3, Q Sepharose pool; lane 4, Superdex 200 eluent (Wnal puriWed protein).

30

Fig. 1. Expression of human ACC2 in Trichoplusia ni cells with baculovirus. (a) The soluble fractions of lysates from cells infected with control or human ACC2 baculovirus were resolved on a 10% SDS–polyacrylamide gel under reduced condition. A total of 15 g protein was loaded in each lane. The band with the estimated size of human ACC2 is indicated. Lane 1, control; lane 2, ACC2 (21-2458); lane 3, ACC2 (27-2458). (b) The same samples in (a) were resolved on a 4–12% SDS–polyacrylamide gel under reduced condition with 15 g protein per lane and the proteins were transferred to a nitrocellulose membrane. The membrane was blotted using peroxidase-linked streptavidin and the band corresponding to human ACC2 is indicated. Lane 1, control; lane 2, ACC2 (21-2458); and lane 3, ACC2 (27-2458).

Table 1 Summary of human ACC2 puriWcation from Trichoplusia ni cells infected with baculovirus Total Total activity SpeciWc activity Recovery proteins (mg) (nmol/min) (nmol/min/mg) (%) Cell lysate Ni resin Q Sepharose HP Superdex 200

6319 60.48 22.40 13.77

6068a 460 230 130

0.96 7.52 9.17 9.21

100 7.58 3.79 2.14

a Multiple product peaks were observed on HPLC with the crude lysate, including the malonyl-CoA peak. When calculating the ACC activity in the lysate, only the malonyl-CoA peak was quantiWed.

20

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

a

0.0175 0.0150

Acetyl-CoA

Volts

0.0125 0.0100 0.0075 0.0050 0.0025 0.0000 -0.0025 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

9

10

11

12

13

14

15

Minutes 0.025

Volts

0.002

Malonyl-CoA

0.015

0.010

0.005

0.000

0

1

2

3

4

5

6

7

8

Minutes 0.025

Malonyl-CoA

Volts

0.020

Acetyl-CoA

0.015

0.010

0.005

0.000

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Minutes

b

1600

Acetyl-CoA

AUC (103x volts x min)

1400

Malonyl-CoA

1200 1000

R2 = 0.9955 for Acetyl-CoA

800

R2 = 0.9968 for Malonyl-CoA

600 400 200 0 0

1

2

3

4

5

6

7

8

9

10

Amount (μM)

Fig. 3. Development of a HPLC assay for ACC2. (a) HPLC proWles of acetyl-CoA and malonyl-CoA. Individual acetyl-CoA and malonyl-CoA peaks were identiWed by injecting 100 l of 2.5 M [1-14C]acetyl-CoA and 3.0 M [2-14C]malonyl-CoA in 50 mM Hepes buVer (pH 7.5), respectively. A mixture of 2.5 M [1-14C]acetyl-CoA and 3.0 M [2-14C]malonyl-CoA in 50 mM Hepes buVer (pH 7.5) was also injected at 100 l to demonstrate the separation of the two peaks on HPLC. (b) Standard curve for malonyl-CoA quantitation. Peak areas were quantiWed after injecting 100 l of 0.1–100 M [2-14C]malonylCoA into the HPLC instrument. A linear curve was generated using AUC values and the corresponding [2-14C]malonyl-CoA concentrations.

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

Wnal puriWed ACC2 has a speciWc activity of 9.21 nmol/min/ mg (Table 1). The identity of the recombinant enzyme was conWrmed by micro-sequencing (data not shown).

glycerol can stabilize the enzyme for a period up to 4 weeks (Fig. 4). Kinetic properties of recombinant human ACC2 and IC50 determination

Development of a HPLC assay for ACC2 A method was developed to separate acetyl-CoA and malonyl-CoA on HPLC. We developed a condition where the acetyl-CoA and malonyl-CoA peaks appear at diVerent elution times (Fig. 3a). When both molecules were mixed and injected, the two peaks were separated (Fig. 3a). Next, we incubated acetyl-CoA and the puriWed ACC2 (27-2458) in reactions containing all the components (see Materials and method) and demonstrated that the acetyl-CoA and malonyl-CoA peaks could be separated in the same fashion (data not shown). The identity of the malonyl-CoA peak from the ACC reaction was conWrmed by mass spectrometry (data not shown). To quantify the malonyl-CoA produced in the ACC reaction, we generated a standard curve correlating malonyl-CoA amounts and the corresponding area under the curve values (AUCs). As shown in Fig. 3b, there is a good linear relationship between the AUC and malonyl-CoA amount, suggesting that this analytical method is sensitive and accurate for the quantitation of malonyl-CoA. Characterization of puriWed human ACC2 To assess the stability of the puriWed enzyme, aliquots of the same preparation were stored at ¡80 °C in 10, 20, and 30% glycerol, respectively. Samples were tested at diVerent time points during storage. The enzyme rapidly lost activity in 10% glycerol. However, both 20 and 30%

To understand the enzymatic properties of the recombinant human ACC2, kinetic parameters were determined using the Michaelis–Menton kinetics (Table 2). The recombinant enzyme has a Km of 2 M for acetyl-CoA, about 30fold less than that reported for a truncated version of human ACC2 [22]. This Wnding suggests that the longer recombinant enzyme produced in our system has higher aYnity for acetyl-CoA. In addition, our ACC2 has a smaller Km value for ATP (52 M) compared with that (120 M) for the truncated ACC2 [22]. The Kcat value for the longer human ACC2 in our study is signiWcantly smaller than that reported by Cheng et al. [22]. This could be due to the diVerent assay conditions such as reaction temperature. We also determined the kinetic parameters with the longer construct ACC2 (21-2458) and the values are comparable to those with ACC2 (27-2458) (Table 2). The inhibitory eVect of a small molecule compound on ACC2 activity was determined in the presence of increasing concentrations of the compound. The determined IC50 value is 51 nM (Fig. 5). In addition, the IC50 values of several known ACC inhibitors were determined using both versions of recombinant human ACC2 (Table 3). TOFA has a good potency against the recombinant human ACC2 but MEDICA-16 is only a modest inhibitor of human

120

POC

10% Glycerol 100

20% Glycerol 30% Glycerol

80

POC

21

60 40 20

110 100 90 80 70 60 50 40 30 20 10 0 -12 -11 -10

Compound A IC50 =51nM

-9

-8

-7

-6

-5

-4

-3

log [Compound A], M

0 0

4

8

12

16

20

24

28

32

Days

Fig. 4. Stability of puriWed human ACC2. PuriWed human ACC2 was stored at ¡80 °C in 10, 20, and 30% glycerol, respectively. Enzymatic activity was determined during storage and expressed as percent of the initial speciWc activity. POC, percent of control. Data are expressed as means § SD; n D 3.

Fig. 5. Inhibition of recombinant human ACC2 by a small molecule compound. The enzymatic activity of recombinant human ACC2 was determined in the presence of increasing concentrations of the compound. The activity in the absence of the compound is deWned as total control (POC D 100%) and all activities are expressed as percent of the total control (in POC). The curve is representative of three independent experiments.

Table 2 Kinetic parameters of recombinant human ACC2 Substrate

Kcat (min¡1)

Acetyl-CoA ATP

ACC2 (27-2458) 11.5 § 2.0 9.3 § 2.0

Values are means § SD (n D 3).

Km (M) ACC2 (21-2458) 17.8 § 1.6 13.7 § 0.5

ACC2 (27-2458) 2.0 § 0.2 52.3 § 4.4

ACC2 (21-2458) 2.6 § 0.8 43.7 § 3.5

22

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

Table 3 Summary of IC50 values of diVerent ACC inhibitors Construct

Compound A (nM)

TOFA (M)

Malonyl-CoA (M)

MEDICA-16 (M)

ACC2 (27-2458) ACC2 (21-2458)

55 § 9 74 § 13

7§1 8§1

22 § 3 17 § 5

367 § 66 558 § 63

Values are means § SD (n D 3).

ACC2. Based on our study, the calculated Ki for malonylCoA is about 6.3 M, comparable to a value reported with the rat native enzyme [19]. The calculated Ki for MEDICA16 is about 105 M, comparable to a reported value with the native enzyme from rat liver [23]. Discussion ACC1 and ACC2 play pivotal roles in the biosynthesis and -oxidation of fatty acids, respectively. Biochemical studies of human ACCs have been hampered by the lack of recombinant enzymes. Recently, two independent research groups reported the expression and puriWcation of human ACC1 and ACC2. Although both groups expressed and puriWed full-length human ACC1, they were not successful in expressing full-length ACC2 [21,22]. One of the challenges acknowledged by the authors is the diYculty to achieve solubility of full-length ACC2 [21,22]. To resolve this issue, they took separate approaches that both involved deletion of the N-terminal region of ACC2. Gu et al. replaced the N-terminal 275 aa of human ACC2 with the corresponding 133 aa of human ACC1 [21], whereas Cheng et al. deleted the Wrst 148 aa of human or rat ACC2 to improve expression and solubility [22]. Although both strategies resulted in the production of an active enzyme [21,22], deletion of the N-terminal regions of ACC2 is not ideal. When aligning the amino acid sequences of human ACCs, it is apparent that ACC2 is very similar to ACC1 in all regions except for the longer N-terminus (142 aa longer than ACC1) [3,4]. The membrane attachment function does not fully explain this large diVerence because the Wrst 20 aa hydrophobic segment is suYcient to mediate the attachment of ACC2 to the mitochondrial outer membrane [7]. Consistent with this notion, the next 20–140 aa form a hydrophilic segment [7], suggesting that it is not involved in membrane association but rather likely to be required for the structural integrity of the enzyme. This segment is deleted in both recombinant enzymes in the recent reports [21,22]. If this region is important in the structural integrity of ACC2, the truncated or fusion enzyme could exhibit properties signiWcantly diVerent from the native enzyme. Indeed, we found drastic diVerences in kinetic parameters between the longer human ACC2 in our study and the truncated enzyme in a recent report [22]. Our data indicate that the Km value for acetyl-CoA is much lower than that reported for the truncated enzyme [22]. There is no speciWc activity data in the report by Gu et al. [21]. Hence, it is not clear how the properties of our ACC2 compare with those of the fusion enzyme [21]. It is possible that the diVerent Km values determined in our study when compared with those by Cheng et al. [22] are due to the diVerence in assay

protocols. Although similar buVer conditions were used in both studies, the enzymatic reaction was carried out at 37 °C by Cheng et al., whereas our reaction was performed at 25 °C. It is possible that the acetyl-CoA pocket may vary slightly with the temperature. There are several diVerences between our expression system and those reported [21,22]. We used the full-length human ACC2 that included the N-terminal hydrophilic region except for the membrane attachment sequence (1–20 aa) and 6 extra aa. It is very unlikely that deletion of the extra 6 aa could impact the overall ACC2 structure. Our approach ensures the integrity of the ACC2 enzyme so that the recombinant protein is most likely to mimic the structure of the native enzyme. Since there is only one diVerence between the sequence used in our study and that by Cheng et al. (V2141 vs. I2141), it is unlikely to account for the signiWcant diVerence in the Km values for acetyl-CoA. The longer ACC2 in our study is tagged at the N-terminus, while the truncated ACC2 is tagged at the C-terminus [22]. It is not known if the C-terminal tagging could aVect the correct folding of the CT domain. One other notable diVerence is that we used T. ni cells (rather than Sf9 cells) and a growth condition at 25 °C in the presence of D-biotin. This may have helped the correct folding. In our expression system, we observed great solubility and high level expression (Fig. 1). Further, we developed an accurate HPLC assay that is more sensitive than the traditional CO2 Wxation method, which could lead to high variability in assays. The CO2 Wxation method involves a step where a strong acid was added to the reaction to evaporate the remaining radio-labeled bicarbonate. We have found that the added acid leads to malonyl-CoA degradation (data not shown). If this results in the loss of the total radioactivity in the dried material at the last step of the assay, signiWcant errors will be introduced in the enzymatic activity readout, which could lead to larger determined Km values. In summary, we have expressed and puriWed full-length human ACC2 in which only the membrane attachment sequence (1–26 aa) was deleted. This enzyme exhibited tighter substrate binding than a truncated human ACC2 in a recent report [22]. Although the diVerence in the kinetic parameters could be attributable to multiple factors, the longer recombinant ACC2 produced in our system is likely more similar to the native enzyme. Acknowledgments We thank Mark Grilo for conWrming the malonyl-CoA peak on HPLC using mass spectrometry. We also thank John Robinson for assistance in micro-sequencing.

K.W. Kim et al. / Protein Expression and PuriWcation 53 (2007) 16–23

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