(Received for publication, January 8, 1997, and in revised form, March 10, 1997)
From the Departments of Biochemistry and
Anatomy, Queen's University, Kingston,
Ontario, K7L 3N6 Canada
Treatment of Chinese hamster ovary (CHO) cells by the aldehyde containing calpain inhibitor I resulted in the induction of a 35-kDa protein that was partially sequenced and shown to be a member of the aldo-keto reductase superfamily (Inoue, S., Sharma, R. C., Schimke, R. T., and Simoni, R. D. (1993) J. Biol. Chem. 268, 5894-5898). Using rapid amplification of cDNA ends polymerase chain reaction, we have sequenced the cDNA for this protein (CHO reductase). This enzyme is a new member of the aldo-keto reductase superfamily and shows greatest amino acid sequence identity to mouse fibroblast growth factor-regulated protein and mouse vas deferens protein (92 and 80% sequence identity, respectively). The enzyme exhibits about 70% sequence identity with the aldose reductases (ALR2; EC 1.1.1.21) and about 47% with the aldehyde reductases (ALR1; EC 1.1.1.2). Northern analysis showed that it is induced in preference to either ALR1 or ALR2 and RNase protection assays showed gene expression in bladder, testis, jejunum, and ovary in descending order of expression. The cDNA for this inducible reductase was cloned into the pET16b vector and expressed in BL21(DE3) cells. Expressed CHO reductase showed kinetic properties distinct from either ALR1 or ALR2 including the ability to metabolize ketones. This protein joins a growing number of inducible aldo-keto reductases that may play a role in cellular regulation and protection.
To characterize the calpain inhibitor I-sensitive protease(s) involved in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, Simoni's group (1) attempted to isolate Chinese hamster ovary (CHO)1 cells resistant to this peptide aldehyde (N-acetyl-leucyl-leucyl-norleucinal (ALLN)). Instead of inducing a protease, a 35-kDa protein was overexpressed which gave tryptic peptide fragments with a high degree of sequence identity to members of the aldo-keto reductase superfamily.2 This superfamily is a rapidly growing group of monomeric oxidoreductases containing at least 40 members at present (2). This group includes the aldehyde and aldose reductases, a number of hydroxysteroid dehydrogenases, Shaker channels, and plant chalcone reductases. They are characterized by a TIM-barrel structure (3) and the preferential use of NADPH over NADH. Substrates include aliphatic and aromatic aldehydes, monosaccharides, steroids, prostaglandins, polycyclic aromatic hydrocarbons, and isoflavinoid phytoalexins.
Several members of this family have been shown to be induced in response to hormonal or chemical factors. These include fibroblast growth factor-regulated protein (FR-1) (4), mouse vas deferens protein (MVDP) (5), aldose reductase (ALR2) (6-8), and dihydrodiol dehydrogenase (9). To determine the relationship of this new reductase to the other members of the family we have used RACE PCR to isolate and sequence its mRNA from CHO cells. It shows highest sequence identity to FR-1 and MVDP at 92 and 80%, respectively. FR-1 and MVDP have not been extensively characterized kinetically but CHO reductase showed a greater ability to reduce ketones than the aldehyde or aldose reductases and a strong preference for aromatic aldehydes.
Calpain inhibitor I was purchased from Calbiochem (La Jolla, CA). Daunomycin was purchased from Rhône-Poulenc Rorer (Montreal, Quebec, Canada). NADPH and NADH were from Boehringer Mannheim (Laval, Quebec). All steroids were from Steraloids (Wilton, NH). p-Nitroacetophenone was from Aldrich while all other enzyme substrates were from Sigma. T7 RNA polymerase, Moloney murine leukemia virus reverse transcriptase, and all restriction enzymes were from Promega (Madison, WI), while RNase A, RNase T1, and DNase I were from U. S. Biochemical Corp. (Cleveland, OH).
Cell CultureInduced CHO-K1 cells, kindly provided by Dr.
Simoni, were grown essentially as described by Inoue et al.
(1) to a final ALLN concentration of 70 µM in the
presence of 20 µM verapamil. Cells were collected and
stored at 70 °C until assayed. Control CHO-K1 cells were obtained
from the American Type Culture Collection and grown without ALLN or
verapamil.
Total RNA was isolated
from induced cells by the TRIzol method (10). First strand synthesis
was primed with the RACE adapter T17 primer using Moloney
murine leukemia virus reverse transcriptase (11). An initial cDNA
fragment for CHO reductase was generated using 3-RACE PCR with a
primer based on the human aldose reductase sequence
(5
-CTCAACAACGGCGCCAAGATGCCCA corresponding to position 19 to 43 of the
coding region). Subsequently, gene specific primers were used with
5
-RACE PCR to obtain the 5
-end cDNA. The 5
-RACE procedure was
improved by titrating the amount of gene-specific primer over the range
0.001 to 10 pmol per reaction (12). The entire cDNA was sequenced
in both directions using overlapping gene specific primers. Sequencing
was performed by the dye terminator method using an Applied Biosystems
model 377A sequencer.
Total RNA from noninduced cells was used with 3-RACE PCR to generate
cDNA fragments for hamster aldehyde reductase (ALR1; EC 1.1.1.2)
and aldose reductase (ALR2; EC 1.1.1.21). A coding region fragment for
aldehyde reductase of 735 bp was generated using the primers
5
-TGTCCTCCTGCACACTG (sense) (position 15 to 31 of the rat coding
region) and 5
-GGACCTGCCACCTGAGC (antisense) (position 734 to 750 of
the pig coding region). A 3
-end fragment of hamster aldose reductase
was generated using the RACE adapter primer and the primer
5
-GCCTTGATGAGCTGTG (sense) (position 898 to 913 of the mouse coding
region). These fragments were sequenced and aligned with known members
of the AKR family to ensure their correct assignment for use as probes
in Northern analysis. 3
-RACE cDNA from noninduced total RNA was
used to generate a Hamster glyceraldehyde-3-phosphate dehydrogenase
probe using a sense (5
-GTTGCCATCAACGACCCCTT) and antisense
(5
-AGCATCAAAGGTGGAAGAATG) primer derived from the rat sequence.
The
entire CHO reductase cDNA was amplified using primers in the 5-
and 3
-untranslated region containing restriction sites for
EcoRI and XhoI, respectively. This fragment was
digested with EcoRI and XhoI and ligated into
pCRII (Invitrogen) similarly digested. The plasmid CHOpCRII was
transformed into CJ236 cells. Introduction of a NdeI site at
the N-terminal methionine was performed using the Kunkel method (13).
Successful mutagenesis was screened by NdeI digestion and
positive clones were resequenced to ensure introduction of no other
mutations. The NdeI-XhoI fragment was subcloned
into the pET16b vector (Novagen) to generate an in-frame histidine
leader sequence for HisTag purification. The CHOpET16b plasmid was
transformed into BL21(DE3) cells containing pLysS. One-half liter
cultures were grown in LB supplemented with 50 µg/ml ampicillin and
34 µg/ml chloramphenicol at 30 °C to an absorbance of 0.5 at 600 nm. Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM and the cultures were grown for a further 3 h. The cells were harvested by centrifugation at
1000 × g for 15 min. The pellets were resuspended at
30 ml/liter of culture in bind buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9) and stored at
20 °C. Cells were thawed and phenylmethylsulfonyl fluoride added
to a final concentration of 1 mM. Cells were disrupted by
sonication and the insoluble fraction separated by centrifugation at
12,000 × g for 20 min at 4 °C. The supernatant was
applied to a 5-ml chelating Sepharose column (Pharmacia) previously
charged with nickel sulfate and washed with bind buffer. The loaded
column was washed with 60 mM imidazole, 0.5 M
NaCl, 20 mM Tris, pH 7.9 (wash buffer), and CHO reductase
was eluted using a linear gradient from wash buffer to elute buffer (1 M imidazole, 0.5 M NaCl, 20 mM
Tris, pH 7.9). The pooled protein was dialyzed against 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA and stored at 4 °C or
20 °C. Protein
samples from the purification of CHO reductase were run on 12%
SDS-polyacrylamide minigels and stained with Coomassie Brilliant Blue
R.
Total RNA was isolated by the TRIzol method as described above. Total RNA was glyoxalated (13) and separated on a 1% agarose gel in 10 mM sodium phosphate, pH 7.0, and transferred to a nylon membrane (Zeta-Probe, Bio-Rad). Prehybridization was done at 37 °C for at least 1 h in 6 × SSC, 5 × Denhardt's, 0.5% SDS, and 100 µg/ml salmon sperm DNA. Hybridization was performed overnight at 42 °C in the same solution containing at least 1 × 107 cpm/ml of each probe at 1 × 108 cpm/µg. The probes were either an 800-bp hamster glyceraldehyde-3-phosphate dehydrogenase cDNA, a 735-bp hamster ALR1 cDNA, a 450-bp hamster ALR2 cDNA, or the full-length CHO cDNA prepared by nick-translation (Nick Translation System, Life Technologies, Inc.). Final membrane washes were at 0.1 × SSC, 0.5% SDS, 60 min, 70 °C with exposure for 16 h at 25 °C with DuPont Reflection NEF film and one intensifying screen.
Ribonuclease Protection Assay of Chinese Hamster TissuesA
radiolabeled riboprobe was prepared by in vitro
transcription of the 3 portion of the hamster CHO reductase gene with
T7 RNA polymerase in the presence of [32P]CTP. The
plasmid CHOpCRII was linearized at the unique AccII site to
generate a 200-bp cRNA transcript. The polyacrylamide gel-purified
riboprobe (0.5 × 106 cpm) and tissue RNA (20 µg) or
CHO-K1 cell-derived RNA (2-5 µg) were denatured at 85 °C for 10 min and annealed overnight at 55 °C in hybridization buffer (80%
formamide, 40 mM PIPES, pH 6.5, 0.4 M NaCl, 1 mM EDTA). Sample processing and display were as described previously except that the ribonuclease digestion buffer contained 50 µg/ml RNase A in addition to 2 µg/ml RNase T1 (14).
All substrates were assayed at 25 °C in
1 ml of 0.1 M sodium phosphate, pH 7.0, using a
Hewlett-Packard 8452A diode array spectrophotometer, except for
daunomycin which was assayed in 0.1 M HEPPS, pH 8.5. Apparent Km values for the substrates were assayed
in the presence of 200 µM NADPH. Apparent
Km values for the cofactors were assayed in the
presence of 1 mM pyridine-3-aldehyde. NADH was
assayed in a 5-mm path-length cell at 370 nm with correction for the
different at that wavelength. All steroids were assayed in the
presence of 2.5% acetonitrile. Kinetic constants were determined using
the Marquardt algorithm (15) supplied with the spectrophotometer.
Sequence
information obtained from the initial 3-RACE reaction performed on
induced CHO-K1 total RNA using the primer to human ALR2 was different
from known ALR2 sequences. Specific primers were used to sequence the
sense and antisense strands and to obtain the 5
-untranslated region
using 5
-RACE. The full-length cDNA sequence generated by RACE
PCR consisted of 1268 nucleotides with a 5
-noncoding region of
43 nucleotides, an open reading frame of 951 nucleotides, and a
3
-untranslated region of 274 nucleotides (Fig. 1). The
open reading frame coded for a protein of 316 amino acids. The original
peptide fragments sequenced by Inoue et al. (1)
matched exactly with the sequence determined from cDNA sequencing.
The deduced protein sequence of CHO reductase showed high identity to
members of the aldo-keto reductase superfamily. The highest identity
was to the mouse fibroblast growth factor regulated (FR-1) protein at
92% (4) (Fig. 2). CHO reductase was 80% identical to
the MVDP (5). The aldose reductases were approximately 70% identical
while the aldehyde reductases were approximately 47% identical.
Multiple sequence alignment analysis showed that CHO reductase, FR-1,
and MVDP form a distinct subgroup within the superfamily and should be
classified in the AKR1B group using the nomenclature system proposed by
Jez et al. (2). CHO reductase contained the same residues
implicated in the catalytic mechanism as other members of the aldo-keto
reductase family. These included Asp-44, Tyr-49, Lys-78, and
His-111.
Induction of CHO Reductase mRNA in CHO-K1 Cells
RNA gel
blot hybridization analysis from cells treated with ALLN at a
concentration of 70 µM confirmed the induction of CHO reductase (Fig. 3). This induction was instead of aldose
reductase or aldehyde reductase. The selectivity of the aldose
reductase probe was maximized by using the 3-end of its cDNA,
which contained the greatest differences between the hamster aldose
reductase and CHO reductase sequences.
CHO Reductase mRNA Levels in Chinese Hamster Tissues
Analysis of a range of CHO tissues revealed no detectable
levels of CHO reductase mRNA when probed by Northern blot using the
full-length CHO cDNA as probe. These blots did confirm the integrity of the RNA. RNase protection assay analysis of the same RNA
stocks showed transcript in the bladder, testis, jejunum, and to a
lesser extent in the ovary (Fig. 4). The basal level in
CHO-K1 cells was higher than in all tissues tested comparing the signal
from 5 µg of noninduced cells total RNA with 20 µg of tissue RNA.
As indicated by the Northern analysis, cells induced by ALLN showed a
markedly elevated transcript level.
Expression and Kinetic Analysis
The recombinant protein yield
was 45-50 mg/liter of cells after chromatography on the
nickel-Sepharose column. The protein was greater than 98% pure as
judged by Coomassie staining of SDS-polyacrylamide gels. The expressed
CHO reductase was active when tested with a variety of common
substrates for the aldo-keto reductases. The Km
values for the cofactors NADPH and NADH were very similar to those
obtained for aldehyde and aldose reductase (Table I).
ALLN, which was used to induce the protein, was a substrate although it
bound poorly as indicated by its millimolar Km. Overall aliphatic substrates such as the commonly used
DL-glyceraldehyde were poor substrates. The only kinetic
values for FR1 or MVDP are a Km of 0.92 mM for FR1 with DL-glyceraldehyde (16). This
value falls between that obtained for CHO reductase and what has been
observed with ALR2 (Table I). The poor ability to catalyze aliphatic
substrates also included glucose which was only converted at a level of
2-3 times background up to a concentration of 1.2 M. Small
aromatic aldehydes were better substrates (Table II), with micromolar Km values and
kcat values 2-10-fold higher than for the
aliphatic substrates. A number of ketones were substrates for the CHO
reductase (Table III). They exhibited low
kcat values (<1 s1) and tighter
binding for aromatic or heterocyclic substrates. The diagnostic ketone
substrate for the carbonyl reductases, menadione (Km
of 0.045 mM (17)), was not a substrate for CHO reductase.
CHO reductase was also able to catalyze the reduction of a number of
steroid aldehydes and ketones (Table IV). The affinities were high (low µM) while the turnover rates were again
less than 1 s
1. The enzyme showed selectivity for
aldehydes or ketones on ring D (e.g.
17
-hydroxyprogesterone) and no reactivity toward ketones on ring A
(e.g. testosterone). The enzyme showed a pH optimum of 6 to
6.5 with all substrates except daunomycin which was maximally reduced
at pH 8.5 (data not shown). CHO reductase was inhibited 35% by 1 mM sodium barbitone and 31% by 1 mM sodium
valproate.
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We have isolated and sequenced a full-length cDNA from Chinese hamster ovary cells that encodes CHO reductase, a new member of the aldo-keto reductase superfamily. Initial characterization of this protein was described by Inoue et al. (1) and the sequence presented here confirms their assignment to the aldo-keto reductase superfamily. The enzyme showed highest protein sequence identity to the fibroblast growth factor-regulated protein (FR-1) and to MVDP. FR-1, MVDP, and now CHO reductase comprise a subgroup of the aldo-keto reductases that are inducible. FR-1 is induced in cells by exposure to acidic fibroblast growth factor-1 (4) and thus may play a role in the mitogenic action of fibroblast growth factor-1. MVDP expression in the vas deferens was dependent on the presence of testosterone (5) and androgen response elements have been isolated in the MVDP gene (18). Both these murine aldo-keto reductases appear to be expressed instead of the more common aldehyde or aldose reductases. CHO reductase induction was also instead of the Chinese hamster aldehyde or aldose reductases.
Besides FR-1 ((AKR1B8); nomenclature proposed by Jez et al.
(2)), MVDP (AKR1B7) and now CHO reductase (AKR1B9), many other members
of the AKR superfamily have been shown to be induced by chemical,
hormonal, or extracellular factors. AKR inducing agents have included
ethacrynic acid (9), ethoxyquin (19),
3-methyl-4-dimethyl-aminoazobenzene (20), and
N-methyl-N-nitrosourea (21), while spontaneous
induction has been observed during the development of hereditary
hepatitis leading to hepatocarcinogenesis (6). Hormonal regulation of AKR members has been observed during the estrous cycle (7), in the cell
cycle of Leishmania major (22) and due to the effects of
basic fibroblast growth factor on astrocytes (8). Androgen response
elements have been found for ALR2 (23) in addition to that mentioned
for MVDP. ALR2 was also regulated by extracellular osmolytes (24, 25)
and an osmotic response element has been found in the rabbit ALR2 gene
(26). The different members of the aldo-keto reductase superfamily are
clearly important in a number of cellular defense and developmental
mechanisms.
RNase protection assay analysis showed that the CHO reductase was variably expressed in the organs of the Chinese hamster. The highest levels were seen in the bladder with transcript also present in the jejunum, testis, and to a lesser extent, ovary. This is in contrast to the FR-1 and MVDP genes that were expressed in testis, heart, ovary (FR-1), and adrenal (both) (27). The low level of CHO reductase transcript in the ovary is puzzling since a high basal level was clearly evident by RNase protection assay analysis of the CHO-K1 cells. This may indicate that the protein is developmentally regulated or that transcriptional regulation in the CHO-K1 cells line has been altered. These cells may thus be primed for the tremendous induction that occurred when the aldehyde ALLN was introduced.
Expressed CHO reductase was functional and displayed kinetics distinct
from other members of the aldo-keto reductase family. It is interesting
that despite its close sequence identity to the aldose reductases
(approximately 70%) it showed an affinity for aliphatic substrates
more closely comparable to the aldehyde reductases (Table I).
Conversely, for aromatic substrates, its affinity was generally
comparable with aldose reductase (Table II). In contrast to the work of
Vander Jagt et al. (28), on human aldose reductase, which
reported no substituent effects for aromatic aldehydes, CHO reductase
showed a 10-fold lower Km for
p-nitrobenzaldehyde versus benzaldehyde. The
preference for aromatic substrates likely accounts for the ability of
CHO reductase to reduce the anthracycline-based ketone daunomycin. CHO
reductase showed similar kinetic constants to the previously reported
daunorubicin reductase (29) (Km, 0.043 versus 0.08 mM; kcat,
0.47 versus 1.5 s1, respectively at pH 8.5).
However, it is not daunorubicin reductase since CHO reductase, as with
other aldose reductase members (30), was only slightly inhibited by 1 mM sodium barbitone. Daunorubicin reductase and other
aldehyde reductases are almost completely inhibited at this
concentration (31, 32). Daunomycin also bound tighter to CHO reductase
than it did to the ketone preferring carbonyl reductase (0.043 versus 0.13 mM) (17). However, CHO reductase was
not able to reduce menadione. The observed preference for aromatic and
other heterocyclic substrates could also explain why CHO reductase was
able to metabolize ketone steroids, even though aldehyde containing
steroids were preferred. This ability to use ketones makes this enzyme
kinetically distinct from the aldehyde and aldose reductases.
The underlying structural reasons for these subtle kinetic differences
between CHO reductase and the aldose reductases can be explained by
analysis of the crystal structures of both these proteins. CHO
reductase has not been crystallized. However, the FR-1 crystal
structure can be used in this comparison because of the high degree of
sequence identity to CHO reductase, particularly around the substrate
site. Comparison of the ternary complexes of FR-1 (16) and human aldose
reductase (33) complexed with NADP and the inhibitor zopolrestat show
that there are many aromatic residues involved in the binding of
zopolrestat. These include Tyr-49, Trp-80, Trp-112, Phe-123, and
Tyr-310. These residues would provide the primary binding energy for
aromatic substrates such as pyridine-3-aldehyde or steroids through
-bond stacking interactions. This would account for the similar
Km values using aromatic substrates for CHO
reductase and aldose reductase. However, the substrate site is bounded
by two regions of considerable sequence difference between FR-1 (and
thus CHO reductase) and aldose reductase (Fig. 5). These
differences result in the active site pocket in FR-1 being wider due to
the more remote position of the loop containing Cys-299. In addition,
more space is seen around the trifluoromethyl group of zopolrestat in
FR-1. This larger substrate pocket in CHO reductase may not clamp small
aliphatic substrates as firmly as does aldose reductase, resulting in
the lower observed affinity. This larger substrate pocket may also allow for more movement of larger substrates such as daunomycin or the
ketone containing steroids thus allowing for the correct orientation
and reduction of these ketones.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81045[GenBank].
We thank Dr. John Watson for assistance with cell culture, Dr. Florante Quiocho and Dr. David Wilson for access to the crystal coordinates for zopolrestat bound FR-1 and ALR2, and Dr. Zongchao Jia for help with interpreting the crystal structures.