(Received for publication, April 29, 1997)
From the Division of Clinical Chemistry, Karolinska
Institute, Huddinge University Hospital, 14186 Huddinge, Sweden, the
Institute for Nutrition Research, University of Oslo,
0316 Oslo, Norway, and the ** Ludwig Institute for Cancer Research,
Biomedicum, Uppsala University, 75124 Uppsala, Sweden
The steroid side chain cleavage in bile acid
formation is catalyzed by liver peroxisomal enzymes (Pedersen, J. I. and Gustafsson, J. (1980) FEBS Lett. 121, 345-348; Kase,
F., Björkhem, I., and Pedersen, J. I. (1983) J. Lipid
Res. 24, 1560-1567). We here describe the cloning and sequencing
of a cDNA coding the first of these enzymes, a
3,7
,12
-trihydroxy-5
-cholestanoyl-CoA oxidase
(THCA-CoA oxidase) from rabbit liver peroxisomes. After tryptic
digestion of purified protein in a polyacrylamide gel, five peptides
were isolated and sequenced. Using two oligonucleotides deduced from the amino acid sequence data, two overlappping clones were isolated from a rabbit liver cDNA library, which together made up a unique cDNA sequence of 2139 base pairs. It contained an open reading frame of 2046 base pairs encoding a protein of 681 amino acids with a
molecular mass of 76,209 daltons. All five peptides could be localized
within the sequence. Transfection of COS cells with the coding part of
the cDNA resulted in a significant expression of THCA-CoA oxidase
activity. We were not able to demonstrate 3
,7
-dihydroxy-5
-cholestanoyl-CoA oxidase activity under the same conditions. The obtained sequence showed 73.6% similarity with a
proposed rat THCA-CoA oxidase and 81% similarity with a recently
reported human branched chain acyl-CoA oxidase, indicating that these
three proteins represent the same enzyme. The similarity with rat
palmitoyl-CoA oxidase was 41.8%. The C-terminal tripeptide of the
protein was SNL, a previously undescribed variant of the main class of
peroxisomal targeting signals. Northern blot analysis revealed that the
gene is transcribed in liver and kidney, and the major mRNA
fraction had a size of approximately 2.6 kilobase pairs.
Formation of bile acids from cholesterol takes place exclusively
in the liver. The sequence of reactions involves a large number of
enzymes localized to several compartments of the cell (1). The final
steps start with activation to CoA esters of 3,7
,12
-trihydroxy-5
-cholestanoic acid (THCA)1
and of 3
,7
-dihydroxy-5
-cholestanoic acid
(DHCA) in the endoplasmic reticulum (2, 3) followed by oxidative
cleavage of the steroid side chain in peroxisomes with formation of
cholic acid and chenodeoxycholic acid, respectively. The reaction
mechanism of this oxidative cleavage is similar to that of
-oxidation of fatty acids (4-6), but a different oxidase seems to
be involved (7, 8). Recently, it was shown that rat liver peroxisomes
contain a noninducible THCA-CoA oxidase that could be separated from
palmitoyl-CoA oxidase inducible by clofibrate and another noninducible
acyl-CoA oxidase (9). This last enzyme was found to have the highest
activity toward branched chain fatty acids such as the synthetic
2-methylpalmitic acid and the natural pristanic acid and has therefore
been termed pristanoyl-CoA oxidase (10). In human liver, only two
acyl-CoA oxidases have been detected, palmitoyl-CoA oxidase and a
branched chain acyl-CoA oxidase active also on bile acid intermediates (11, 12).
THCA-CoA oxidase has been difficult to purify both because of the small amounts of protein available and because of the instability of the enzyme. Recently, Van Veldhoven et al. reported on the successful purification (13) and (during preparation of this manuscript) also on the cloning of this enzyme from rat liver (14) as well as of a similar enzyme from human liver (15). We have noted that a THCA-CoA oxidase from rabbit liver was more stable than its rat counterpart, and this source also provided more material. In the present work we report on the partial purification of THCA-CoA oxidase from rabbit liver, cloning and sequencing of a cDNA coding for the enzyme, and expression of enzyme activity in COS cells after transfection with this cDNA.
3,7
,12
-Trihydroxy-5
-[7-3H]cholestan-27-oic
acid as well as 3
,7
,-dihydroxy-5
-cholestan-27-oic acid were
prepared as described (16) using bile from Alligator
mississipiensis as starting material. The natural form of THCA in
bile of A. mississipiensis has been identified as the
25R-diastereoisomer (17), but the strong alkaline hydrolysis
generally used during isolation causes isomerization at C-25 and the
appearance of the 25S-isomer (18). The isolated material was
separated by HPLC on a 5-µm C-18 Nucleosil column (0.5 × 25 cm)
with 24% 30 mM trifluoroacetic acid (pH 2.9 with triethylamine) in methanol as eluting solvent. The 25R- and
the 25S-isomers of THCA were almost completely separated in
this system (19). The ratio between the 25R and the
25S isomers in the material primarily isolated was about
7:3. The combined fractions of tritium-labeled (25R)- and
(25S)-THCA as well as unlabeled DHCA were converted into CoA
esters using the same method as used for the synthesis of choloyl-CoA
(20). Purified palmitoyl-CoA oxidase was a gift from Prof. T. Hashimoto
(Shinshu University, Matsumoto, Japan). 2
,7
-Dichlorofluorescein
diacetate was from Eastman Kodak Co. (Rochester, NY). The sources of
special reagents and kits are given below. Other chemicals were
commercial high purity material.
Liver peroxisomes were prepared from a male rabbit fasted for 24 h essentially as described for rat liver (21). The light mitochondrial fraction generated by centrifugation of the postmitochondrial supernatant at 16,200 rpm (24,000 × gav) for 10 min in the Kontron T324 centrifuge (A8.24 rotor) was layered on a linear Nycodenz gradient ranging from 15% (w/v) in 0.25 M sucrose, 1 mM EDTA, and 1 mM Hepes at pH 7.4 to 45% in 1 mM EDTA, 1 mM Hepes at pH 7.4. The gradient tubes contained a prelayered 2-ml Maxidenz cushion and were centrifuged at 30.000 rpm for 30 min at 4 °C in the Sorvall TV 850 vertical rotor. Fractions of 1.7 ml were collected and assayed for catalase activity (22, 23). The five fractions with the highest activity were combined, diluted with 0.25 M sucrose, and centrifuged at 33,000 rpm (100,000 × g) for 45 min. The pellet (14 mg of protein from two gradients) was resuspended in 10 ml of solubilization buffer consisting of 10 mM sodium pyrophosphate, pH 9, 10% ethylene glycol, 0.1% Triton X-100, 10 µM FAD, 0.5 mM dithiothreitol. After stirring on ice for 1 h and centrifugation at 33,000 rpm for 45 min, the supernatant was brought to pH 7.4 and applied to a hydroxylapatite (Bio-Gel HTP, Bio-Rad) column (volume, 5 ml; diameter, 1 cm). The column was equilibrated in 10 mM phosphate buffer pH 7.4, 10 µM FAD, 0.5 mM dithiothreitol, 10% ethylene glycol and eluted with the same buffer to remove unbound protein. The flow rate was 0.35 ml/min, and 1.75-ml fractions were collected. Protein containing THCA-CoA oxidase (and devoid of palmitoyl-CoA oxidase activity) was eluted with 60 mM potassium phosphate buffer, pH 7.4, 10 µM FAD, 0.5 mM dithiothreitol, and 10% ethylene glycol. The active fractions (total volume, 9.5 ml) were concentrated to 2.2 ml in a dialysis bag (Spectra-Por 2 membrane) covered with polyethylene glycol (molecular weight, 15,000-20,000; Sigma). Aliquots (0.45 ml) of the concentrated protein solution were subjected to gel filtration on a SuperdexTM 200 HR 10/30 column connected to a SmartTM chromatography system (Pharmacia Biotech, Inc., Uppsala, Sweden). Elution was performed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM KPi buffer, pH 7.4) containing 0.5 mM dithiothreitol and 20% ethylene glycol at a flow rate of 0.25 ml/min, and fractions of 0.25 ml were collected.
Generation of Peptides by Digestion in GelProtein in the peak fractions from the gel filtration above was concentrated by precipitation (24), and the dried sample was dissolved in 25 µl of sample buffer containing 8 mM dithiothreitol. After heating to 95 °C for 4 min, the reduced sample was alkylated by incubation at room temperature for 20 min with 16 mM iodoacetamide. The sample was subjected to polyacrylamide gel electrophoresis in SDS (25) on a 10% gel of 1-mm thickness using the Mini-Protean II Slab Cell (Bio-Rad). After Coomassie Blue staining, the band corresponding to a molecular mass of 72 kDa, using the 72-kDa band of the palmitoyl-CoA oxidase as a reference (26), was excised, transferred to an Eppendorf tube, and subjected to in-gel digestion essentially according to Hellman et al. (27). In brief, the gel pieces were washed with 0.5 M Tris-Cl, pH 9.2, and 50% acetonitrile and then completely dried. To introduce the protease into the gel piece, 10 µl of 0.1 M Tris-Cl, pH 9.2, containing 0.5 µg of endo-LysC (protease source Achromobacter lyticus, WAKO Chemicals GmbH, Neuss, Germany) was added. Rehydration was continued by adding 0.1 M Tris-Cl, pH 9.2, in small aliquots until the gel piece was immersed. After overnight incubation at 30 °C, the supernatant was saved and combined with extractions from the gel piece. Generated peptides were isolated by reversed phase liquid chromatography on a µRPC C2/C18 SC 2.1/10 column in a SMARTTM chromatography system. Peptides were sequenced on a Perkin-Elmer Applied Biosystems model 494 instrument, following the manufacturer's instructions.
cDNA CloningOligonucleotides were synthesized on a
380B DNA synthesizer (Applied Biosystems, CA), using the
-cyanoethylphosphoamidite chemistry. A cDNA library made from
New Zealand White rabbit liver in the phage
gt10 was obtained from
CLONTECH (Palo Alto, CA). The library (5
-STRETCH
PLUS cDNA library) was constructed with oligo(dT) and random
priming utilizing EcoRI linkers and was found to contain
2.5 × 1010 plaque-forming units/ml. The library was
propagated in Escherichia coli C600 Hfl according to the
manufacturer's recommendations. Screening of the library was performed
with oligonucleotide probes, end-labeled with 32P. Positive
clones were isolated, and the
DNA was purified with the
WizardTM Lambda Preps DNA purification system (Promega,
Madison, WI).The inserts were excised from the vector by digestion with
EcoRI, extracted from the agarose gel
(GenEluteTM agarose spin columns, Supelco, Bellefonte, PA),
and subcloned into pUC18 using the Ready-To-GoTM DNA Ligase
(Pharmacia). Plasmid DNA was purified with the Jetstar Mini Plasmid
Purification system (Genomed, Research Triangle Park, NC). Sequencing
of inserts were done automatically on a 373 DNA sequencer stretch
(Applied Biosystems), using the ABI PRISMTM dye terminator
cycle sequencing kit with AmpliTaqR polymerase FS
(Perkin-Elmer).
Stock cultures of the cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 100 units/ml penicillin, 100 µg/ml streptomycin. Transfection was carried out by electroporation in 0.4-cm cuvettes (Gene Pulser II without the pulse controller unit; Bio-Rad), using a single pulse of 0.40 kV and 25 microfarads. After transfection, the cells were grown for 48 h, harvested by low speed centrifugation, and homogenized (sonication for 6-10 s) in the incubation medium.
Enzyme Assays and Analytical ProceduresThe conversion of
THCA-CoA into
3,7
,12
-trihydroxy-5
-cholest-24(E)-enoic acid
(
24-THCA) was measured at 30 °C in a volume of 0.1 ml
in an incubation medium that contained 0.1 M Tris-HCl, pH
8, 75 µM FAD, 1 mg/ml bovine serum albumin, 0.01% Triton
X-100, and 42 µM (25R)- and (25S)-[3H]THCA-CoA (60.000 cpm). The
incubations were stopped with 2.5 µl of 6 M KOH and
hydrolyzed at 60 °C for 30 min to remove bound CoA. After
acidification with HCl and extraction with ethyl acetate, the samples
were analyzed by reversed phase HPLC on a 5-µm YMC-Pack ODS-A column
(250 × 4.6-mm inner diameter) (YMC Co., Ltd. Kyoto, Japan). The
eluting solvent was 20% 30 mM trifluoroacetic acid (pH 2.9 with triethylamine) in methanol. Radioactivity in the eluent was
monitored by a Flo-One Beta radiochromatography detector (Radiomatic
Instruments & Chemical Co., Inc. Tampa, FL).
THCA-CoA racemase activity was assayed in the same incubation buffer by following the conversion of (25R)-THCA-CoA into (25S)-THCA after hydrolysis and extraction as above. Protein was determined by the method of Bradford (28) using bovine serum albumin as a standard. Protein in the eluent from the gel filtration column was estimated based on the UV absorbance in the eluent compared with that of a fixed amount of injected bovine serum albumin.
Analysis by Gas Chromatography-Mass SpectrometryExtracts
of incubations were dried under N2, and the material was
methylated by treatment with methanol, 2,2-dimethoxypropane, concentrated HCl (1:0.7:0.011, v/v/v) for 30 min at 55 °C. The mixture was dried under N2 and sialylated by treatment with
300 µl of pyridine, hexamethyldisilazane, trimethylchlorosilane
(3:2:1, v/v/v) for 30 min at 60 °C. The solvent was removed by
N2, and the material was dissolved in isooctane. Gas
chromatography-mass spectrometry of the isooctane solution was
performed on a Hewlett Packard 5890 series II Plusgas chromatograph
equipped with an HP-ultra capillary column (25 m × 0.30 mm,
0.33-µm thickness), connected to an HP 5972 mass selective detector
and an HP 7673A automatic sample injector. The oven temperature program
was as follows: 180 °C for 1 min, 35 °C/min to 270 °C, and
then 20 °C/min to 300 °C, where the temperature was kept for 19 min. Helium was used as carrier gas. The gas chromatograph was operated
in the constant flow mode, with the flow rate set to 0.8 ml/min. The injector was operated in the splitless mode and kept at 270 °C, and
the detector transfer line was kept at 280 °C. The mass spectrometer was operated in the selected ion monitoring mode, and two ions were
detected simultaneously. The ions used for analysis were as follows:
3,7
-dihydroxy-5
-cholestanoic-acid and
3
,7
-dihydroxy-5
-cholest-24-enoic acid,
m/z 412 and m/z 410;
3
,7
,12
-dihydroxy-5
-cholestanoic acid and
3
,7
,12
-dihydroxy-5
-cholest-24-enoic-acid,
m/z 500 and m/z 498. The
retention times were as follows: 3
,7
-dihydroxy-5
-cholestanoic acid, 17.54 min; 3
,7
-dihydroxy-5
-cholest-24-enoic acid, 20.47 min; 3
,7
,12
-trihydroxy-5
-cholestanoic acid, 17.82 min;
3
7
12
-dihydroxy-5
-cholest-24-enoic acid, 20.83 min.
During our attempts to purify the enzyme, we experienced, as did others (13), considerable problems with the stability of the enzyme. During the different purification steps, we noticed, however, that activity was always associated with a band on SDS-polyacrylamide gel electrophoresis of about 72 kDa and a migration distance identical to that of the 72-kDa fragment of purified palmitoyl-CoA oxidase (26). Even if it was possible after several purification steps to obtain a preparation that showed only this band on SDS-polyacrylamide gel electrophoresis, the total amount of protein was too small for further characterization (29). We therefore decided to reduce the number of purification steps and base our cloning strategy on in-gel digestion and peptide analysis of this protein band. The steps used are shown in Table I. Solubilization of the peroxisomes in hypotonic pyrophosphate buffer in the presence of detergent on ice was found to be efficient. Heat treatment was avoided, since it was found that, unlike palmitoyl-CoA oxidase (26), THCA-CoA oxidase was very heat-labile. The acyl-CoA oxidase activity and the THCA-CoA activity were separated on a hydroxylapatite column. THCA-CoA oxidase eluted at 60 mM phosphate buffer, while acyl-CoA oxidase was eluted at 200 mM phosphate buffer. No acyl-CoA oxidase was detected in the 60 mM fraction eluate, while a small amount of THCA-CoA oxidase eluted in the 200 mM fraction. The 60 mM fraction eluate was concentrated by dialysis against polyethylene glycol and further fractionated by gel filtration (Fig. 1). Maximum THCA-CoA oxidase activity was detected in fraction 14 that eluted between 13.00 and 13.25 ml, which was immediately in front of the elution volume corresponding to bovine serum albumin (13.5 ml), suggesting a molecular mass of the oxidase of about 70 kDa. Fractions between 12.75 and 13.50 ml were used as a source of the oxidase. Activity measurement in the active fractions was complicated by the presence of a THCA-CoA racemase activity (interconversion of (25R)- and (25S)-THCA-CoA) (19) that started to elute immediately after the peak fraction of THCA-CoA oxidase activity. Maximum THCA-CoA racemase was found to be in a fraction between 14.75 and 15.00 ml, corresponding to a molecular mass of about 40 kDa. This compares with the values given by Schmitz et al. of 45 kDa for a similar rat liver mitochondrial enzyme (30) and 47 kDa for a human liver peroxisomal enzyme (31). Fractions between 14.5 and 15.5 ml (fractions 20-23) were pooled and used as a source of racemase in incubations when required (see legend to Fig. 1). The racemase preparation was found to be contaminated with some THCA-CoA oxidase activity, corresponding to about 10% of that in the peak fraction of the oxidase. The substrate used for the THCA-CoA oxidase assay contained the 25R- and 25S-isomers in a ratio of 7:3, and since the S-isomer appears to be the physiological one (19), the amount of this substrate was not at saturation level. The presence of the racemase activity therefore resulted in an erroneously high activity in some of the fractions on the low molecular side of the eluate. For this reason a fixed amount of racemase was included in all incubations of fractions from the gel filtration step to be assayed for the oxidase activity.
|
Isolation and Sequencing of Tryptic Peptides and Preparation of Oligonucleotides for Screening
Polyacrylamide gel electrophoresis
of the active fractions from the gel filtration showed the appearance
of a band at aout 72 kDa that varied in intensity corresponding to the
activity (Fig. 2). The migration of this band was
identical to that of the 72-kDa band of palmitoyl-CoA oxidase. In-gel
digestion of the protein contained in this band and separation by HPLC
generated a large number of peptides (Fig. 3), five of
which were sequenced and found to contain 6-17 amino acids (not
including the initial lysine required by the protease endo-LysC for
cleavage). The sequence of peptide 3 was used to design two
oligonucleotides intended for screening for the THCA-CoA oxidase
cDNA (Fig. 4). In accordance with the cleavage
pattern of the enzyme endo-LysC, it was assumed that a lysine residue
would precede the amino-terminal aspartic residue of the peptide. Two
oligonucleotides were synthesized, denoted Ox-1 and Ox-2, which
differed only in the codon for serine, where different triplets were
utilized (Fig. 4). The most prevailing codons for the amino acids found
in the rabbit genome were used (32), and in two positions with a high
degree of degeneracy inosine was inserted (Fig. 4).
Isolation of Rabbit THCA-CoA Oxidase Clones and Sequence of a Full-length Clone
Approximately 500,000 plaque-forming units were
screened, and only those plaque-forming units were picked that
generated a signal with both oligonucleotides. In this way, several
cDNA-clones were isolated. One of these, denoted 10, numbered
2092 bp and contained an open reading frame of 2046 bp (Fig.
5). Another shorter clone (
69) overlapped with
10
in the 5
-end with 47 bp, making the total cDNA sequence obtained
2139 bp. Since the cDNA sequence contained an EcoRI site
(in position 1173 of the complete sequence), the insert of
10 was
cleaved in two fragments, which were subcloned separately in plasmid
vectors. The sequence around the EcoRI site was verified by
sequence analysis of a polymerase chain reaction fragment from the
phage clone, spanning this region. All of the five peptides could be
localized within the sequence (Fig. 6). The shortest of
the peptides (peptide 2) represented the C-terminal end of the
polypeptide chain. However, this peptide partly overlapped with another
of the peptides (peptide 4), and therefore the enzyme cleavage must
have occurred after a glutamine and not, as expected, after a lysine
residue. Whether this indicates the presence of microheterogeneity in
this position or possibly some protease contaminant in the endo-LysC
preparation is presently not known.
The open reading frame encodes a protein of 681 amino acids with a molecular mass of 76,209 daltons and a theoretical isoelectric point of 7.74.
The obtained peptide sequence showed a homology with rat THCA-CoA
oxidase of 73.6% and a homology with rat acyl-CoA oxidase of 41.8%
(Fig. 7). Interestingly, there are two additional
methionine residues in the amino-terminal part of the deduced sequence
(in positions 18 and 22). The most proximal of these corresponds to the
initiation methionine residue of rat acyl-CoA oxidase, the sequence of
which is 17 amino acid residues shorter in the amino-terminal end than
the other two enzymes. Very recently, the sequence for a branched chain
acyl-CoA from human liver was published (15) that has a homology of
81% with our enzyme and contains the same number of amino acid
residues.
The C-terminal tripeptide in our protein was found to be SNL (Ser-Asn-Leu), which is different from previously described peroxisomal targeting signals (33). In particular, the sequence is different from HKM (His-Lys-Met) found in rat THCA-CoA oxidase (14) and SKL (Ser-Lys-Leu) found in human branched chain acyl-CoA oxidase (15).
Expression Pattern of THCA-CoA OxidaseTo visualize the
mRNA for THCA-CoA oxidase, a cDNA probe of 1135 bp
(corresponding to positions 47-1172 in the cDNA sequence) was
used. A strong signal was obtained with rabbit liver total RNA
corresponding to a band at 2.6 kilobase pairs (Fig. 8).
A much weaker signal was obtained from rabbit kidney, while no signal was detected from other organs tested (not shown). Signals were also
obtained with mouse and rat liver (Fig. 8). Fasting had no significant
effect on mRNA level in any of the three species tested.
Expression of THCA-CoA Oxidase Activity in COS Cells
To
express THCA-CoA activity in eukaryotic cells, a plasmid vector,
pCAGGS, carrying a modified chicken -actin promoter (35) was used. A
cDNA fragment including the coding sequence of the oxidase was
prepared from the
clone 10 by polymerase chain reaction (Gene Amp
XL polymerase chain reaction kit; Perkin-Elmer). The primers contained
a restriction site for XbaI, and the fragment obtained
numbered 2081 bp, corresponding to positions 46-2126 in the cDNA.
This fragment was ligated into the XbaI site of the expression vector pCAGGS, and the construct was propagated in Epicurian
Coli XLI-Blue MRF
Kan supercompetent cells (Stratagene, La Jolla, CA).
The insert could be cleaved out of the vector by digestion with
XbaI, and the orientation was established by DNA sequencing.
An estimated quantity of 20 µg of plasmid DNA with the cDNA
inserted the correct way was used to transfect approximately 2 × 106 cells. In a control experiment, a similar quantity of
plasmid DNA with the cDNA inserted in the opposite direction was
allowed to transfect the same number of cells.
When incubating a homogenate of the transfected cells with
tritium-labeled THCA-CoA as described under "Experimental
Procedures," a low but significant THCA-CoA oxidase activity was
detected with the assay based on radio-HPLC (Fig. 9).
Two independent experiments resulted in 3 and 2.3% total conversion of
(25R)- and (25S)-THCA-CoA into
24-THCA. A homogenate of cells transfected with a
construct with the coding region inversely inserted had no detectable
activity. It should be noted that it is only (25S)-THCA-CoA
that is a substrate of the enzyme (19). It could be calculated that the
conversion of this stereoisomer into
24-THCA was 13 and
16%, respectively, in the above two experiments with cells transfected
with the cDNA inserted the correct way. (cf. Fig.
9).
Combined gas chromatography-mass spectrometry allows a considerably
more sensitive and accurate assay than the above HPLC method. Fig.
10 shows analysis by this method of hydrolyzed extracts of incubations of (25R)- and (25S)-THCA-CoA with
the same amount of cells transfected by either a complete or a
truncated insert. The ion at m/z 500, corresponding to the molecular ion of the methyl ester trimethylsilyl
derivative of THCA, and the ion at m/z 498, corresponding to the molecular ion of the same derivative of
24-THCA, were followed through the gas chromatography.
There was no separation of the R- and
S-stereoisomers of THCA on the column used. In the analysis
of the incubation with cells transfected with the complete insert (Fig.
10A), a conversion of 1.2% of the substrate
(25R)- and (25S)-THCA-CoA, was obtained. In
addition, there was another product peak, corresponding to a conversion of 0.3%, occurring in the tracing of the ion at
m/z 498 (Fig. 10A). This peak had a
retention time identical to that of the methyl ester trimethylsilyl
ether of 24-OH-THCA. This compound has a prominent ion at
m/z 498 in its mass spectrum, corresponding to the M-90 ion. Formation of 24-OH-THCA is most probably due to the
presence of hydratase activity in the COS cells, converting the newly
synthesized
24-THCA-CoA into the corresponding
24-hydroxylated product. A cDNA fragment devoid of the N-terminal
sequence corresponding to the region up to the second methionine (in
position 18) was also used in a separate expression experiment. A very
small product peak calculated to represent a conversion of about 0.3%
of the substrate was observed (Fig. 10B). Most probably,
this represents a small endogenous THCA-CoA oxidase activity in the COS
cells.
When using unlabeled DHCA-CoA as a substrate and analyzing the product
by gas chromatography-mass spectrometry as above, no conversion into
24-DHCA could be detected in incubations with the
transfected cells. In these analyses, the ion at
m/z 412, corresponding to the molecular ion of
the methyl ester trimethylsilyl derivative of DHCA, and the ion at
m/z 410, corresponding to the molecular ion of
the same derivative of
24-DHCA, were used. A significant
conversion was obtained, however, in incubations with either purified
rabbit liver peroxisomes or with the crude enzyme from the gel
filtration step. The rates of these reactions were about half of those
obtained with THCA-CoA as substrate.
In the present work, we have cloned a THCA-CoA oxidase from rabbit liver peroxisomes. That the cloned protein truly represents this enzyme was demonstrated by the expression of THCA-CoA oxidase activity after transfection in COS cells. Even if the activity was low, it exceeded severalfold that in mock-transfected cells. Furthermore, the product of the oxidase reaction was verified both by HPLC and by gas chromatography-mass spectrometry. During the preparation of this manuscript, the cloning of two proteins closely related to the one described here was published, a rat peroxisomal THCA-CoA oxidase (14) and a human peroxisomal branched chain acyl-CoA oxidase (15). Expression of the branched chain acyl-CoA oxidase cDNA was demonstrated by the use of antibody against the protein. For neither of the two has enzyme activity been demonstrated in an expression system, however. Formal proof of the correct protein being cloned was thus not presented. The high degree of homology between the protein described here and both rat peroxisomal THCA-CoA oxidase and human peroxisomal branched chain acyl-CoA oxidase (73.6 and 81%, respectively) makes it clear that these three proteins represent the same enzyme from the three different species.
When compared with peroxisomal rat acyl-CoA oxidase, the similarity was only 41.8%. Of special interest was the presence of two methionines at the N-terminal part of rabbit THCA-CoA oxidase, the second of which (at position 18) corresponds to the start of rat acyl-CoA oxidase. Obviously, the initial 17 amino acid residues are essential for catalytic activity, since a clone lacking this portion was unable to elicit a significant THCA-CoA activity when transfected into COS cells. The more specific function of this part of the protein is unknown; possibly, it is required for correct binding of the substrate.
The C-terminal tripeptide of the protein was found to be SNL (Ser-Asn-Leu). This is different from the previously reported C-terminal tripeptides considered to be peroxisomal targeting signals. From site-directed mutagenesis experiments it is known that certain variations of the tripeptide motif are tolerated for it to function as a peroxisomal targeting signal (36). A substitution of Asn in SNL for Lys in SKL (Ser-Lys-Leu), the most common peroxisomal targeting signal (33), is compatible with the rule of a basic amino acid in the second position of the tripeptide (36). It thus appears evident that SNL represents another variant of the major class of peroxisomal targeting signals found in eukaryotic cells (33). It should be noted that SNL differs from the C-terminal tripeptide of human peroxisomal branched chain acyl-CoA oxidase only by Asn being altered to Lys, while it differs from that of rat THCA-CoA oxidase (HKM) in all three positions. From this point of view, rabbit THCA-CoA oxidase is closer to the human protein than to its rat counterpart. Baumgart et al. (14) could not confirm the interaction of the C terminus of rat THCA-CoA oxidase with the human peroxisomal targeting signal receptor, in contrast to the situation with rat palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, and suggested that THCA-CoA oxidase was targeted to peroxisomes by a hitherto unknown mechanism. In view of the finding that SKL and SNL are the C termini of the counterparts of this enzyme in human and rabbit liver, respectively, this suggestion does not appear to be true for THCA-CoA oxidases of other species.
It has generally been considered that both THCA and DHCA are chain-shortened by the same peroxisomal enzymes. The crude enzyme preparation that eluted from the gel filtration column (Fig. 1) converted both THCA-CoA and DHCA-CoA to the respective unsaturated derivatives, although DHCA-CoA was converted at a considerably lower rate than THCA-CoA. This may not be surprising in view of the fact that chenodeoxycholic acid is normally found only in trace amounts in rabbit bile (37). After transfection of COS cells with the appropriate clone, we were not able to detect any activity toward DHCA-CoA. Whether this is due to the activity being too low for detection by the method used or to the existence of a separate oxidase for DHCA-CoA can only be speculated on.
As expected, the enzyme was expressed in livers from both rat and mouse in addition to rabbit. As previously demonstrated (14), kidney also gave a weak signal. Formation of bile acids is considered to take place only in the liver. The function of the enzyme in the kidney is not known. The possibility cannot be excluded that small amounts of cholesterol hydroxylated in the 27-position in other organs may eventually be further oxidized and chain-shortened in the kidneys.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y13279.
We are grateful to Turid Veggan, Manfred Held, and Maria Olin for expert technical assistance and to Dr. Jun-ichi Miyazaki (Institute of Development, Aging and Cancer, Tohoku University, Japan) for a generous gift of the pCAGGS expression vector.