(Received for publication, August 29, 1994; and in revised form, December 15, 1994)
From the
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 HO
. The
inducibility of this H
O
-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
10
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
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.
In animal cells, mitochondria as well as peroxisomes oxidize
fatty acids via -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
-oxidation system, whereas
the long and very long chain fatty acids are processed predominantly in
peroxisomes(1) . The peroxisomal
-oxidation system
consists of three proteins: H
O
-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), (
)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.
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.
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.
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 10
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) .
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, 8,600; B,
14,000; and C,
13,000.
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.
Figure 6:
Co-infection of Sf9 insect cells with
recombinant baculovirus expressing components B and C. Sf9 insect cells
(5 10
) 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.
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 10
) 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.
Human ACOX, a dimeric protein with a molecular mass of 140
kDa, contains FAD as the prosthetic group and catalyzes a
HO
-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 -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
-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.