©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Overexpression and Characterization of the Human Peroxisomal Acyl-CoA Oxidase in Insect Cells (*)

(Received for publication, August 29, 1994; and in revised form, December 15, 1994)

Ruiyin Chu Usha Varanasi Su Chu Yulian Lin Nobuteru Usuda M. Sambasiva Rao Janardan K. Reddy (§)

From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H(2)O(2). The inducibility of this H(2)O(2)-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (20 mg/1 times 10^9 cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, K and V(max) values for palmitoyl-CoA were found to be 10 µM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B + C complex which could subsequently be inactivated by dissociating with detergent.


INTRODUCTION

In animal cells, mitochondria as well as peroxisomes oxidize fatty acids via beta-oxidation(1) . The physiological significance of this division of labor is not entirely clear, but it appears that ordinary fatty acids up to 18 carbons in length are oxidized mainly by the mitochondrial beta-oxidation system, whereas the long and very long chain fatty acids are processed predominantly in peroxisomes(1) . The peroxisomal beta-oxidation system consists of three proteins: H(2)O(2)-generating fatty acyl-CoA oxidase, enoyl-CoA hydratase/3hydroxyacyl-CoA dehydrogenase bifunctional enzyme, and 3-ketoacyl-CoA thiolase(2, 3) . Rat liver contains three forms of peroxisomal fatty acyl-CoA oxidase: (a) a palmitoyl-CoA oxidase (ACOX), (^1)inducible by peroxisome proliferators, that oxidizes esters of medium, long, and very long chain fatty acids; (b) a noninducible pristanoyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids such as pristanic acid; and (c) a noninducible trihydroxycoprostanoyl-CoA oxidase, which oxidizes the CoA esters of bile acid intermediates di- and trihydroxycoprostanoic acids(1) . Among these three oxidases, the inducible ACOX is well characterized and is implicated in the oxidative DNA damage and hepatocarcinogenesis resulting from exposure to peroxisome proliferators(4, 5) . In contrast to rat, peroxisomes in human liver and kidney contain only two forms of acyl-CoA oxidases, and the first enzyme, ACOX, cross-reacts with polyclonal antibodies raised against the rat enzyme(6) . The CoA esters of 2-methyl branched-chain fatty acids and of bile acid intermediates di- and trihydroxycoprostanic acids are oxidized in human tissues by one single peroxisomal branched-chain acyl-CoA oxidase which does not cross-react with antibodies raised against rat pristanoyl-CoA oxidase (6) . The highly inducible ACOX of rat and human has a molecular mass of 140 kDa and consists of two subunits of 72 kDa (component A), which can be proteolytically cleaved into 51-kDa (component B), and 21-kDa (component C) products within the peroxisome matrix(3) .

Previously we have shown that the human ACOX gene is located on chromosome 17q25, spans 33 kilobases, consists of 14 exons, and encodes an open reading frame of 660 amino acids(7) . The transcription of the human ACOX gene in liver appears relatively poor when compared to rat(8) . The human ACOX gene exhibits several large transcripts in the liver, kidney, and skeletal muscle whose significance remains unclear(7) . In normal human liver, the acyl-CoA oxidase activity is only one-fifth of that reported in rat liver(9) . So far, the human acyl-CoA oxidase has been only partially purified. Since liver homogenates invariably contain branched-chain acyl-CoA oxidase activity, reliable estimations of K values are not possible by assaying partially purified preparations(10) . The structural and functional characterization of human ACOX is essential in view of the role of this enzyme in oxidative stress caused by the sustained induction of peroxisome proliferation in liver and its potential role in a peroxisomal deficiency disorder designated pseudo-neonatal adrenoleukodystrophy (characterized by the absence of ACOX resulting in the impaired degradation of very long chain fatty acids)(11) . Furthermore, in other peroxisomal genetic disorders such as Zellweger and Zellweger-like syndromes, the translated ACOX protein is not processed into the 52- and 21-kDa subunits(12) . A novel subtype of peroxisomal acyl-CoA oxidase deficiency with detectable protein has been reported recently(13) . Knowledge of the ACOX enzyme properties is essential to understand the molecular mechanisms of these peroxisomal acyl-CoA oxidase deficiency disorders and to develop new approaches for clinical therapy.

We report here the construction of a full-length human liver ACOX cDNA and the production of relatively large quantities of functionally active recombinant human ACOX by a baculovirus-based expression system. We describe the enzymatic properties of the purified ACOX protein (component A) and also present evidence that ACOX subunits B and C, when expressed separately in insect cells, lack enzyme activity, but when these B + C components are expressed together, they form a heterodimeric complex which exhibits ACOX activity.


EXPERIMENTAL PROCEDURES

Amplification and Construction of Human ACOX cDNA

Total cellular RNA from human liver was isolated by the guanidinium isothiocyanate method(14) . Each of the cDNA fragments was amplified by reverse transcription-PCR using a reverse transcriptase RNA PCR kit (Perkin-Elmer Corp.) and appropriate pairs of primers (Table 1). The reverse transcription mixture was in a volume of 20 µl and contained 10 mM Tris-HCl, pH 8.3, 90 mM KCl, 1 mM MgCl(2), 0.2 mM of each dNTP, 0.1 µM downstream primer, 1 µg of total RNA, and 5 units of thermostable rTth DNA polymerase. The reaction mixture was incubated at 70 °C for 15 min, and then the volume was increased to 100 µl by adding H(2)O together with 8 µl of 10times chelating buffer, 2 µl of 25 mM MgCl(2) solution, and 10 µl of 10 µM upstream primer. The reaction mixture was cycled 35 times, 1 min at 94 °C, 1 min at 52 °C, and 1-2 min at 72 °C. Four overlapping DNA fragments (fragment 1, 470 bp; fragment 2, 837 bp; fragment 3, 572 bp; fragment 4, 608 bp) were obtained, respectively, by PCR. Amplified fragments were analyzed by agarose-gel electrophoresis and cloned into pGEMT vector (Promega). For each of the fragments, at least two clones were sequenced, and the one without any artifact was chosen for further experiments. Fragments 1 and 2, with an overlapping region of 202 bp (324-685, type I exon 3 and part of exon 4) were subjected to recombinant PCR with ExpS1 and AS2 primers. The generated fragment 1 + 2 (1161 bp) was cloned into the pGEMT vector. Fragment 3 + 4 (1025-2233) was created by ligation of fragments 3 and 4 using the EcoRV site. The full-length cDNA was obtained by ligating the combined fragments 1 + 2/3 + 4 using the NcoI site at 1045 by partial digestion. The entire full-length cDNA fragment was sequenced with specific primers to confirm its authenticity.



Expression of Human ACOX in Sf9 Insect Cells

We initially designed the full-length human ACOX cDNA fragments with restriction sites at the 5` and 3` ends that would allow insertion into the baculovirus transfer vector, pVL1392 (Invitrogen), thereby allowing the direct expression of protein in Sf9 cells. The full-length human ACOX cDNA with EcoRI and BamHI sites introduced by PCR primers at the 5` and 3` ends was first subcloned into the transfer vector pVL1392(15) . The construct was then linearized by EcoRI digestion, and an EcoRI site-tailed linker starting with an ATG codon followed by 6 consecutive histidine residues was inserted. The resulting plasmids were sequenced with BV1 primer (16) . DNA from the colony that contains a single copy of the ``6 histidine'' affinity tag in the right orientation was used to construct recombinant virus(17) . The availability of a 6 histidine tag greatly enabled the purification of the protein and circumvented the need for heat treatment and chromatographic procedures. Purification of ACOX was performed by a one-step elution method (see below). The truncated cDNA fragments encoding components B and C were obtained by amplification of the full-length cDNA and subcloned into the pVL1392 vector. The linker sequence encoding the 6 histidines was also inserted into the transfer vectors in front of the coding region of component B and C. The recombinant viruses were obtained by co-transfection of Sf9 insect cells with the transfer vector DNA and linearized wild type AcNPV DNA as reported previously(17) . In the recombinant baculovirus, fragments harboring A, B, and C components with the 6 histidine tag were designated as hisA-NPV, hisB-NPV, and hisC-NPV, respectively. Two recombinant virus expression components B and C without the 6 histidine tag were subsequently constructed and designated as B-NPV and C-NPV, respectively. The purified recombinant virus stocks were used at a mutiplicity of infection (m.o.i.) of 10 to infect Sf9 insect cells which were at an 80% confluence in a T-flask or at a density of 1.5 times 10^6 cells/ml in a spinner flask. Cell cultures were maintained at 26 °C and were harvested at selected intervals after infection.The expression of full-length or truncated human ACOX was determined by SDS-PAGE, using 10% polyacrylamide gels (18) and immunoblotting(19) . Immunoblotting was performed by employing rabbit anti-rat ACOX antibodies and alkaline phosphatase-coupled goat anti-rabbit IgG antibodies (Bio-Rad) as the primary and secondary antibodies, respectively.

Purification of the Recombinant Human ACOX

For a scale-up of full-length ACOX protein production, Sf9 insect cells were maintained in spinner flasks and infected with recombinant virus at a density of 3 times 10^6 cells/ml. At 72 h postinfection the cells were harvested, rinsed in phosphate-buffered saline, resuspended in 50 mM potassium phosphate buffer, pH 7.4, together with 1 mM PMSF, and lysed by sonication for 5 min. The cell lysates were then pelleted at 10,000 times g for 20 min. The supernatant was collected and then absorbed to 10 ml of packed Ni-NTA affinity resin on ice for 30 min. The resin was then packaged into a 20-ml syringe column, washed twice with 50 ml of binding buffer (50 mM potassium phosphate, pH 7.8, 0.1 M NaCl, 1 mM PMSF) and with 75 ml of washing buffer (50 mM potassium phosphate, pH 6.0, 0.1 M NaCl, 1 mM PMSF), and once with the same buffer containing 50 mM imidazole. The protein was eluted from the resin using 20 ml of the washing buffer containing 500 mM imidazole, pH 6.0. The eluted fractions containing ACOX were dialyzed against 20 mM potassium phosphate, pH 7.5, 1 mM PMSF overnight at 4 °C. The dialyzed ACOX solution was clarified by centrifugation at 16,000 times g and then analyzed by SDS-PAGE. To purify component B and C protein, monolayer-cultured Sf9 cells were infected with appropriate viruses and harvested at 72 h after infection. The same procedures were employed to package, wash, and elute the Ni-NTA affinity column. The eluted peak fractions were pooled together, concentrated by Centricon-10 (Amicon) spin columns, and used for enzyme activity assay and SDS-PAGE analysis.

Enzyme Assay

ACOX activity was assayed by measuring the palmitoyl-CoA-dependent H(2)O(2) production as described by Osumi et al.(3) . The reaction mixture was in a total volume of 1.0 ml with the following constituents: 50 mM potassium phosphate, pH 7.4, 0.82 mM 4-aminoantipyrine, 10.6 mM phenol, 10 µM FAD, 5 IU of horseradish peroxidase, 20 µM palmitoyl-CoA, and the enzyme. The reaction was carried out at 30 °C, and the formation of H(2)O(2) was measured by following the increase in absorbance at 500 nM. The molar extinction coefficient was 6,390 M cm at pH 7.4. One unit of the enzyme was defined as the amount which catalyzes the formation of 1 µmol of product/min.

Other Methods

Immunomorphological procedures were the same as reported previously(20) . Protein concentration was determined by the method of Bradford(21) . Plasmid isolation, endonuclease digestion, ligation, and DNA sequencing were performed according to standard protocols(22) .


RESULTS

Construction of a Full-length Human ACOX cDNA

Using human liver total cellular RNA as template, four cDNA fragments overlapping the entire coding region of human ACOX were obtained by reverse transcription-PCR. The strategy for engineering a cDNA construct encoding the 1980-nucleotide open reading frame of the human ACOX involved joining and ligating together four fragments derived from the PCR products (Fig. 1). Sequencing of the four constituent DNA fragments revealed that it exactly matched the corresponding exonic sequences of the ACOX gene(7) .


Figure 1: Schematic representation of PCR cloning of human ACOX cDNA. Panel A, human ACOX cDNA structure based on the ACOX gene structure according to Varanasi et al.(7) ; the coding region is boxed. Components B and C of ACOX are derived from the full-length protein by a post-translational proteolytic cleavage of component A(3) . Panel B, four overlapping clones used to construct the full-length coding region of ACOX cDNA. The primers are indicated by arrows, and the sequences are as showed in Table 1. S, sense; AS, antisense; Fr, fragment; Exp, expression. Panel C, construction of truncated human ACOX cDNA encoding components B and C by PCR.



A Histidine Residue within 14th Exon Is Missing in Human ACOX

When compared with the rat ACOX sequence which encodes a 661-amino acid residue(23) , the human ACOX cDNA encodes for a 660-amino acid residue, due to a missing histidine at amino acid position 651. This could result from either a deletion of a codon in the human or an insertion of a codon into rat ACOX. To exclude the possibility of an amplification or sequencing error, five individual cDNA clones spanning exons 10-14 (7) were checked by sequencing. It was confirmed that the missing codon is not an artifact since all the cDNA clones upon sequencing were identical to the corresponding exonic sequences (Fig. 2).


Figure 2: The nucleotide sequence of portions of genomic and cDNA clone of the 14th exon of human ACOX. Panel A, genomic sequence and Panel B cDNA sequence. Arrowheads indicate the location of the missing His codon.



Expression and Purification of the Full-length Human ACOX in Sf9 Insect Cells

A major aim of this study was to establish a system that would produce large quantities of recombinant human ACOX for determining its enzymatic properties. Human ACOX encoded by the full-length cDNA is composed of 660 amino acid residues and has a predicted molecular mass of 72 kDa. The final construct had 6 histidines at the N-terminal end. Expression of the full-length cDNA in insect cells revealed a major 72-kDa protein band on SDS-PAGE. This 72-kDa protein increased in a time-dependent manner following infection with recombinant virus (Fig. 3A) and became prominent by 48 h postinfection. Densitometric scanning of the gels showed that this 72-kDa protein accounted for 15% of the total cellular protein in Sf9 insect cells 72 h postinfection. Immunoblot analysis with antibodies raised against rat ACOX revealed that the 72-kDa protein is the predominant immunoactive protein in insect cell lysates. Besides the 72-kDa band, two other bands with lower molecular masses of 51 and 21 kDa were also recognized (Fig. 3B). The 72-, 51-, and 21-kDa immunoactive bands correspond to components A, B, and C of human liver ACOX, respectively. Thus, human ACOX expressed in insect cells exhibited a pattern similar to that of rat ACOX(17) . Fig. 3C shows the time course of the ACOX activity in Sf9 insect cells infected with recombinant virus. ACOX activity increased from 24 h postinfection until peak levels of enzyme activity were found at 72 h postinfection. The time course of increase in ACOX activity correlated well with the increase in the amount of protein detected on SDS-PAGE. The specific activity was 0.9 unit/mg of protein at the peak level of expression.


Figure 3: Analysis of human ACOX expressed in Sf9 insect cells. Panel A, SDS-PAGE analysis of whole cell lysates of Sf9 insect cells infected with the recombinant baculovirus containing full-length coding sequence of human ACOX cDNA. Cells, grown in monolayer culture, at 80% confluence were infected with the recombinant virus and harvested at the indicated time (hr) after infection. Cell lysates (75 µg of protein) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Panel B, immunoblot analysis of recombinant human ACOX expressed in Sf9 insect cells. The cell extracts were prepared (as in panel A) and processed for immunoblotting using antibodies raised against rat liver ACOX. Arrowheads indicate the position of components A (72 kDa), B (51 kDa), and C (21 kDa). Panel C, time course of expression of human ACOX activity in Sf9 insect cells infected with the recombinant virus. Cells were infected and harvested as described for Panel A and assayed for ACOX activity. The values are expressed as the average of two assays. Panel D, the recombinant human ACOX purified from Sf9 insect cell lysates infected for 72 h. The histidine-tagged human ACOX expressed in Sf9 insect cells was purified on a Ni-NTA affinity column. The purified protein (5 µg) was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Lane M represents molecular mass standards (values in kilodaltons) in Panels A and D.



Recombinant human ACOX was purified by a single-step procedure as described under ``Experimental Procedures.'' As shown in Fig. 3D, a major 72-kDa band was eluted at 500 mM imidazole. The bands of lower molecular masses of 51 and 21 kDa could be visualized only by silver staining (data not shown). The low ratio of the component B and C cleaved from component A in insect cells limited the yield of lower molecular mass components B and C of ACOX. Using this purification procedure, about 20 mg of pure human ACOX was obtained from 1 liter of Sf9 insect cell suspension culture (1 times 10^9 cells) which is two times greater than the yield of recombinant rat ACOX purified from Sf9 insect cells by the ``heating method'' described elsewhere(3) .

Estimation of Apparent K(m) Value for the Human ACOX

Samples (5 µg) of the purified human recombinant ACOX were incubated with increasing concentrations of palmitoyl-CoA as described under ``Experimental Procedures.'' At a low concentration of palmitoyl-CoA (2-10 µM), the enzymatic activity increased linearly with an increasing substrate concentration. The highest specific activity was 0.95 unit/mg of protein at 15 µM palmitoyl-CoA. Nevertheless, at a high concentration of palmitoyl-CoA, there was substrate inhibition. The specific activity decreased to 0.25 unit/mg of protein when the concentration of palmitoyl-CoA increased to 50 µM. From the linear parts of the Lineweaver-Burk plots (not illustrated) an apparent K(m) of 10 µM and V(max) of 1.4 units/mg of protein were calculated for palmitoyl-CoA. The K(m) of purified human ACOX is similar to that of the purified rat enzyme, but much lower than that reported (62 µM) for the crude enzyme of human liver (10) . Although it has been reported that ACOX contains FAD as a prosthetic group(4) , omission of FAD from the reaction mixture did not affect the enzyme activity and kinetic properties (data not shown).

Chain Length Specificity

The substrate specificity of human ACOX was examined with 50 µg of crude Sf9 insect cell extracts infected with hisA-NPV and 5 µg of protein of the purified enzyme. 10 µM octanoyl CoA (C(8)), decanoyl-CoA (C), lauroyl-CoA (C), myristoyl-CoA (C(14)), palmitoyl-CoA (C), and stearoyl-CoA (C(18)) were used as substrates for the assay. Maximal activities for saturated fatty acids were observed with C substrates. The specific activities toward C-C(18) were 0.07-0.1 unit/mg of protein for crude extract and 0.85-0.95 unit/mg of protein for the purified enzyme. The activity toward C(8) and C was less than 40% of that toward C acyl-CoA. Since ACOX is the first enzyme of the peroxisomal beta-oxidation pathway, the lack of short chain acyl-CoA oxidase activity of ACOX led to the conclusion that human peroxisomes predominantly, if not exclusively, oxidize very long chain acyl-CoAs.

Localization of Recombinant Human ACOX in Sf9 Insect Cells

In normal human liver, ACOX is localized exclusively within the peroxisomal matrix(24) . A tripeptide peroxisomal targeting signal (Ser-Lye-Leu, or a conservative variant such as Ser-Arg-Leu) located at the C terminus of a majority of peroxisomal proteins, including ACOX, that targets proteins to peroxisomes has been identified(25) . The conserved peroxisomal targeting signal Ser-Lye-Leu is also present at the C terminus of human ACOX as reported earlier(7) . In peroxisomal deficiency disorders, peroxisomal proteins are synthesized at their normal rate. However, they are not found in the particulate fraction of the cell but in the cytosol, which indicates that they were not incorporated into peroxisomes(12) . In this study, as shown in Fig. 4, the human ACOX expressed in insect Sf9 cells was distributed predominantly in the cytosol as visualized at the electron microscopic level by the protein A-gold immunocytochemical procedure. It is of interest to note that the electron density of the protein in Sf9 insect cells and the peroxisomal matrix of induced rat liver recognized by the protein A-gold method is similar and tends to be higher than that of other cytosolic proteins.


Figure 4: Immunocytochemical localization of human ACOX by protein A-gold labeling procedure. Recombinant human ACOX is expressed abundantly, as indicated by the density of gold particles, and it is distributed predominantly in the cytosol. Panel A, an infected Sf9 insect cell showing aggregates of electron dense material which on higher magnification (Panel B) reveals gold particles indicating the cytosolic localization of human ACOX. Panel C, a portion of a hepatic parenchymal cell from a rat treated with ciprofibrate, a peroxisome proliferator, which shows the presence of ACOX in the matrix of peroxisomes. N, nucleus; C, cytoplasm; P, peroxisome; M, mitochondria. A, times8,600; B, times14,000; and C, times13,000.



Expression of Truncated ACOX

It has been reported that rat ACOX is originally composed of two identical 72-kDa subunits. Following translocation into peroxisomes, the polypeptide is cleaved proteolytically between Val and Ala, into two components (B and C) at a frequency of 80%(4) . Although the molecular composition of mature human ACOX subunits is not available, three components similar to rat with molecular masses of 72, 51, and 21 kDa were also recognized by immunoblot analysis(12) . To ascertain whether components B and C perform independent functions in human ACOX, they were expressed separately in Sf9 insect cells by insertion of their respective coding sequences in the baculovirus genome as described under ``Experimental Procedures.'' As shown in Fig. 5, Sf9 insect cells infected with the recombinant baculovirus harboring component B or C (hisB-NPV, hisC-NPV) exhibit a major band of 51 kDa corresponding to B or a smaller band of 21 kDa representing C. However, enzyme activity assay revealed that cell lysates infected with hisB-NPV or hisC-NPV alone had barely detectable activity. The mixture of the two lysates also had no activity. Furthermore, component B and C protein were purified from the infected cell lysates with an Ni-NTA affinity column. Incubation of the purified B and C either separately or together with substrate did not show any catalytic activity, suggesting that either component A is necessary or dimerization of B and C is essential to form an active complex.


Figure 5: Expression of truncated human ACOX in Sf9 insect cells. Panel A, SDS-PAGE analysis of Sf9 insect cells expressing truncated human ACOX. Whole cell lysates of insect cells infected with recombinant baculovirus containing full-length or truncated human ACOX cDNA were subjected to SDS-PAGE. Approximately 50 µg of protein of Sf9 insect cell lysate was loaded in each lane. Panel B, Western blot analysis of the truncated human ACOX expressed in Sf9 insect cells. Samples were prepared (same as Panel A) and processed as described in Fig. 3. Arrowheads indicate the bands corresponding to component A (full-length, 72 kDa), component B (N-terminal 468 amino acids, 51 kDa), and component C (C-terminal 192 amino acids, 21 kDa). The constructs encoding N-terminal 468 amino acids(1-468) and C-terminal 192 amino acids(469-660) were created as described in Fig. 1. Lanes 1, 2, and 3 in both Panels A and B represent Sf9 insect cells infected with recombinant baculoviruses coding for components A, B, and C, respectively.



Co-expression of Component B and C in Sf9 Insect Cells Forms Active Enzyme

Sf9 insect cells were further co-infected with hisB-NPV and hisC-NPV at different ratios. Surprisingly, all of the co-infected cells exhibited ratio-dependent ACOX activity. As shown in Fig. 6, the highest activity obtained was with the Sf9 insect cells infected with m.o.i. of 10 hisB-NPV + 10 hisC-NPV (Fig. 6). It was about 50% of the control cells producing full-length ACOX. With a total m.o.i. of 10, the co-infection of 5 hisB-NPV + 5 hisC-NPV exhibited 25% activity. When Sf9 insect cells were infected with same amount of hisB-NPV (10 m.o.i.), the activity increased with an increase in hisC-NPV (2-10 m.o.i.). Conversely, in infection of Sf9 cells with 10 m.o.i. of the hisC-NPV, the activity was increased with the increase in hisB-NPV.


Figure 6: Co-infection of Sf9 insect cells with recombinant baculovirus expressing components B and C. Sf9 insect cells (5 times 10^6) were infected with various amount of recombinant baculovirus containing truncated human ACOX cDNA. The m.o.i. of each infection is indicated at the top of each lane. Three days after infection, the cells were harvested, washed with PBS, and processed either for SDS-PAGE analysis (A) or for ACOX enzyme activity assay. The activity was expressed as percent of the full-length protein (component A). Values represent the mean of four assays from two independent infections.



Interestingly, when 0.05% Triton X-100 was present in the protein extraction, which involved three cycles of freeze/thaw, all the co-infected cell lysates lost activity, suggesting that active complexes of component B and C can be formed in the absence of component A with an equal molarity of B and C proteins.

Heterodimerization of Components B and C in Sf9 Insect Cells

To examine whether components B and C form an active heterodimeric complex, the Sf9 insect cells were infected either independently with hisA-NPV, hisB-NPV, hisC-NPV or coinfected with hisB-NPV + hisC-NPV, hisB-NPV + C-NPV and B-NPV + hisC-NPV. As shown in Fig. 7, after Ni-NTA affinity column purification, the co-infection with hisB-NPV + hisC-NPV revealed equal amounts of component B and C protein. Co-infection with hisB-NPV + C-NPV, in which the component C protein has no histidine tag, exhibited more component B and less C; while co-infection with B-NPV + hisC-NPV showed less component B and more C. These data reveal that components B and C form a heterodimeric complex and can be co-purified with its partner using a histidine tag for either one. The relatively low enzymatic activity after co-infection indicates that only part of the co-expressed protein forms catalytically active heterodimeric complexes.


Figure 7: Co-purification of component B and C from Sf9 insect cells infected with recombinant baculovirus expressing 6 histidine-tagged component B or C. A linker sequence encoding 6 histidines was added in front of the coding sequence of component B or C as described under ``Experimental Procedures.'' Sf9 insect cells (2 times 10^8) were infected appropriately with recombinant baculovirus with a total m.o.i. of 20. Approximately 72 h postinfection, infected cells were harvested, lysed, and passed onto an Ni-NTA affinity column. The column was extensively washed and eluted as described under ``Experimental Procedures.'' The peak fractions were pooled together, concentrated with Centricon-10 (Amicon), loaded onto an SDS-PAGE gel, and stained with Coomassie Blue. The protein of each lane were purified from the infection of: lane 1, hisA-NPV; lane 2, hisB-NPV; lane 3, hisC-NPV; lane 4, hisB-NPV + hisC-NPV; lane 5, hisB-NPV + C-NPV; lane 6, B-NPV + hisC-NPV. Note the presence of heterodimeric partner in lanes 5 and 6.




DISCUSSION

Human ACOX, a dimeric protein with a molecular mass of 140 kDa, contains FAD as the prosthetic group and catalyzes a H(2)O(2)-generating dehydrogenation of fatty acyl-CoA to a 2-trans-enoyl-CoA(4) . The activity of ACOX in human liver is 5-fold lower than that found in rat liver(9) . The low abundance of ACOX in human liver and other tissues has precluded structural studies requiring large quantities of enzymes. Since it is difficult and cumbersome to obtain sufficient quantities of purified protein for biochemical and physicochemical characterization, we constructed a cDNA encoding a 660-residue human ACOX and expressed it as a catalytically active recombinant protein in Sf9 insect cells using the baculovirus. Our strategy for engineering a cDNA construct encoding the 1980-nucleotide open reading frame of human ACOX involved the usage of human liver RNA for reverse transcription-PCR to obtain four cDNA fragments for ligation so as to yield the entire coding sequence. The cDNA we had engineered matched perfectly with the amino acid sequence deduced from sequencing the human ACOX gene(7) . It should be noted that the human ACOX cDNA encodes 660 amino acids, whereas the rat ACOX cDNA encodes 661 amino acids due to a single codon deletion/insertion event; the one missing amino acid in human ACOX is a histidine at position 651. Recently, Aoyama et al.(8) reported that human ACOX cDNA encodes 661 amino acid residues, i.e. similar in length to that of rat ACOX. The reason for this discrepancy of an entire codon is not clear and casts doubt on the authenticity of this sequence as that of human in origin. Furthermore, their human ACOX cDNA sequence shows 7 other amino acid differences when compared to our human ACOX cDNA (data not presented). The authenticity of our human cDNA sequence is confirmed by its identity with the human ACOX genomic sequences(7) .

Rat ACOX is originally composed of two identical subunits (component A), but after translocation into the peroxisome the polypeptide is cleaved proteolytically between Val and Ala into two components (51-kDa component B and 21-kDa component C)(3) . The Sf9 insect cells infected with recombinant baculovirus produced human ACOX as 15-20% of the total cellular protein. The ACOX expressed in Sf9 insect cells exhibited three polypeptide components A, B, and C (with relative molecular masses of 72, 51, and 21 kDa) which are identical to those present in human liver (12) . However, components B and C, which are proteolytically derived from component A, are present at a lower ratio. It was reported earlier that the lower ratio of components B and C of rat ACOX generated by baculovirus expression was due to incomplete processing of the overexpressed protein(17) . Our previous studies with uninfected Sf9 insect cells revealed that these cells lack recognizable peroxisomes and do not possess immunorecognizable ACOX(17) . Accordingly, the lack or paucity of peroxisomes in Sf9 insect cells accounts for the presence of overexpressed human ACOX as electron dense aggregates in the cytoplasm of these cells and for the existence predominantly as component A. Thus, it appears that posttranslational packaging and processing within the peroxisome is not a prerequisite for the preservation of ACOX catalytic activity.

Additionally, the baculovirus Sf9 insect cell expression system has enabled us, for the first time, to examine the relationship between ACOX components B and C. Expression of components B and C alone showed no catalytic activity for either of the separate components. Nevertheless, co-expression of components B and C in Sf9 insect cells exhibited ACOX activity in the absence of component A. It is proposed that the co-expression of components B and C in insect cells could form an active B + C complex. The mechanism by which these two components form a catalytically active heterodimeric complex in a cell that has no visualizable peroxisomes remains unclear. In this study, we presented evidence to demonstrate that components B and C indeed form heterodimeric complexes within the insect cell cytosol to yield a catalytically active complex. Dissociation of this complex by addition of detergent during protein extraction resulted in loss of enzymatic activity.

In Zellweger syndrome patients, no morphologically distinct peroxisomes are present in the parenchymal cells of liver and kidney and in skin fibroblasts(24) . Pulse-chase experiments using fibroblasts derived from these patients demonstrated that ACOX could not be processed into components B and C; the failure of this proteolytic cleavage was attributed to the lack of peroxisomes in these cells(12) . Nevertheless, palmitic acid was reported to be oxidized at an efficient rate by the homogenates of Zellweger fibroblasts(26) . Our previous data revealed that, although recombinant rat ACOX expressed in insect cells had reduced amounts of components B and C (with an estimated molecular ratio of A, B, and C = 5:1:1 in Sf9 insect cells versus 1:5:5 in rat liver), the specific activity of the expressed protein was similar to the endogenous rat liver enzyme. The present study also has demonstrated a high activity of the purified recombinant human ACOX that is predominantly composed of the 72-kDa component A. These results imply that component A, as well as, the heterodimeric complex of components B and C display catalytic activity, whereas when expressed individually neither component B nor component C exhibit such activity. The presence of ACOX components B and C in insect cells by 72 h postinfection suggests that some processing of component A into components B and C can occur in the cytosol, since these cells appear to lack morphologically recognizable peroxisomes. In addition, proteolytic degradation may account for some cleavage as a result of cell lysis which is evident 48 h postinfection.

Of evolutionary interest is that the initial reaction in the mitochondrial and peroxisomal beta-oxidation cycle is catalyzed by MCAD and ACOX, respectively(1) . It was suggested that the MCAD and peroxisomal ACOX families evolved from a common primordial gene belonging to a superfamily(27) . The short, medium, and long chain MCAD (28) contain 388-400 amino acids in the mature form of the proteins, as compared to peroxisomal ACOX which has 661 (rat) or 660 (human) amino acids. Although the sequence similarity between MCAD and peroxisomal ACOX is low, it has been clearly shown that 70% of the rat and human ACOX sequence on the N-terminal portion could be aligned to the entirety of individual rat MCAD sequences (Fig. 8). The percent identity between rat MCAD and rat or human ACOX ranges from 13.9 to 20.4%(27) . Of particular interest is that the most recently cloned rat very long chain MCAD(29) , like the peroxisomal ACOX, contains a 22-25-kDa extra polypeptide as compared with the other shorter chain fatty acid metabolizing MCAD (Fig. 8). In this context it is of particular interest to note that extra residues of the ACOX sequence are similar to those located downstream in the C terminus of very long chain MCAD. When the human ACOX component C and the rat very long chain MCAD terminal extra sequences are compared, a surprising 15.2% identity and 38.7% similarity was noted (Fig. 8). The identity was higher than the 15% which is regarded as borderline; whether these identical residues are shared by chance or signify a distant evolutionary relationship remains a conjuncture. Tanaka and Indo (27) proposed that the primordial MCAD/ACOX gene was similar to the ancestral MCAD gene, and that the ACOX family diverged from it as a result of a fusion of the C-subunit (component C) domain from another gene, and that the fusion of the C-domain was closely linked to the genesis of the peroxisomes. Our results suggest that ACOX component B alone lacks enzymatic activity and that the presence of component C is crucial for the oxidative activity. Thus, in peroxisomal ACOX, the N-terminal 1-468 amino acid sequence of component B shared homology with short, middle, and long chain MCAD, while the C-terminal 469-660 amino acid sequence (component C) shared homology with the C-terminal extra sequence of very long chain MCAD. Since beta-oxidation toward long chain fatty acids is known to be catalyzed both by very long chain MCAD and the peroxisomal ACOX, the shared C-terminal sequence must, therefore, be responsible for the common substrate which is very long chain acyl-CoA. ACOX and very long chain MCAD should be closer on the phylogenetic tree to peroxisomal ACOX than the other shorter MCAD.


Figure 8: Comparison of the C-terminal sequence of human ACOX and rat very long chain MCAD. Deduced amino acid sequences of human ACOX 469-660 (component C) and rat very long chain MACD 486-655 are aligned to maximize identity. Human ACOX component C residues identical with the rat very long chain MCAD C-terminal residues are shown as vertical bars, + indicates similar amino acids, and gaps indicated by dashes were introduced into the sequence to facilitate their alignment.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant R37 GM23750, Joesph L. Mayberry Sr. Endowment Fund, and Adrian Mayer Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8144; Fax: 312-503-8240; jkreddy{at}merle.acns.nwu.edu.

(^1)
The abbreviations used are: ACOX, peroxisomal acyl-CoA oxidase(s); PCR, polymerase chain reaction; bp, base pair(s); PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; AcNPV, Autographa californica nuclear polyhedrosis virus; Sf9, Spodoptera frugiperda; m.o.i., mutiplicity of infection; Ni-NTA, nickelnitrilo-triacetic acid; MCAD, mitochondrial acyl-CoA dehydrogenase(s).


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