Xenopus Cytosolic Thyroid Hormone-binding Protein
(xCTBP) Is Aldehyde Dehydrogenase Catalyzing the Formation of
Retinoic Acid*
Kiyoshi
Yamauchi
§,
Jun-ichiro
Nakajima
,
Hiroaki
Hayashi¶,
Ryuya
Horiuchi
, and
Jamshed R.
Tata**
From the
Department of Biology, Faculty of Science,
Shizuoka University, Shizuoka 422-8529, the ¶ Gunma Prefectural
College of Health Sciences, Maebashi 371-0052, the
Department
of Pharmacy, Gunma University School of Medicine,
Maebashi 371-8511, Japan, and the ** Laboratory of Developmental
Biochemistry, National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, United Kingdom
 |
ABSTRACT |
Amino acid sequencing of an internal peptide
fragment derived from purified Xenopus cytosolic thyroid
hormone-binding protein (xCTBP) demonstrates high similarity to the
corresponding sequence of mammalian aldehyde dehydrogenase 1 (ALDH1)
(Yamauchi, K., and Tata, J. R. (1994) Eur. J. Biochem. 225, 1105-1112). Here we show that xCTBP was
co-purified with ALDH and 3,3',5-triiodo-L-thyronine (T3) binding activities. By photoaffinity labeling with
[125I]T3, a T3-binding site in
the xCTBP was estimated to reside in amino acid residues 93-114, which
is distinct from the active site of the enzyme but present in the
NAD+ binding domain. The amino acid sequences deduced from
the two isolated xALDH1 cDNAs (xALDH1-I and xALDH1-II) were 94.6%
identical to each other and very similar to those of mammalian ALDH1
enzymes. The two recombinant xALDH1 proteins exhibit both
T3 binding activity and ALDH activity converting retinal to
retinoic acid (RA), which are similar to those of xCTBP. The mRNAs
were present abundantly in kidney and intestine of adult female
Xenopus. Interestingly, their T3 binding
activities were inhibited by NAD+ and NADH but not by
NADP+ and NADPH, whereas NAD+ was required for
their ALDH activities. Our results demonstrate that xCTBP is identical
to ALDH1 and suggest that this protein might modulate RA synthesis and
intracellular level of free T3.
 |
INTRODUCTION |
A major characteristic of 3,3',5-triiodo-L-thyronine
(T3),1 the active
form of thyroid hormone at the cellular level, is the multiplicity of
physiological processes. These include such diverse functions as
postembryonic and fetal development and postnatal growth in mammals,
amphibian metamorphosis, maturation of central nervous system, energy
metabolism in homeotherms, and environmental adaptation in
poikilotherms (1-4). It is now generally accepted that, at the
molecular level, most of these actions of thyroid hormone are initiated
by the interaction between thyroid hormone receptor and T3.
Thyroid hormone receptor is a member of a multigene family of nuclear
receptors that act as transcription factors in combination with
transcriptional co-activators and co-repressors and chromatin-modifying
factors (5-9). It is, however, not clear as to how T3
enters the cell and reaches the nucleus and what determines the
dynamics of cytoplasm-to-nucleus transfer of the hormone. A key
component of this intracellular process is most likely to be the
cytosolic thyroid hormone-binding protein (CTBP). Recently, CTBPs have
been detected in mammalian and amphibian cells (10-15), an interesting
feature of which is that these exhibit different biochemical
properties. We have earlier described a CTBP in adult
Xenopus liver (xCTBP) (11) which is a 59-kDa protein with a
higher affinity for T3 than L-thyroxine
(T4), T3 binding being neither
Ca2+- nor NADPH-dependent, as is the case for
Rana and rat CTBPs (12-15).
The physiological actions of retinoic acid (RA) and other retinoids are
also considered to be exerted through nuclear retinoic acid receptors
(7, 16). There is also good evidence that a large fraction of RA and
retinoids is present in the cell bound to cytoplasmic proteins,
identified as cytosolic retinoic acid (CRABP) and retinol-(CRBP)
binding proteins (16, 17). It has been suggested that these binding
proteins may not only determine the intracellular concentration of free
ligands but may also act as their transporters into the nucleus. A
similar suggestion, based on indirect evidence, has also been made for
a mammalian CTBP that has been identified as a monomer of pyruvate
kinase subtype M2 (18). Although there are many similarities between CTBP, CRABP, and CRBP, on the one hand, and retinoic acid receptors and
thyroid hormone receptors, on the other, a major difference is the
multiple types of CTBPs, unlike CRABP and CRBP (17). We have previously
reported three types of xCTBPs (19), each with a distinct pattern of
expression, which raises the possibility of a tissue-specific role for
CTBPs. It therefore became important to characterize CTBPs in greater detail.
A unique feature of xCTBP found in adult liver is that it has a region
similar to those of mammalian class 1 aldehyde dehydrogenase (ALDH1) (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) (11),
which is one of the enzymes catalyzing the oxidation of various
aliphatic and aromatic aldehydes to the corresponding acids. An
important and rather specific activity of ALDH1 is to act as an enzyme
catalyzing the synthesis of RA from retinal (20-23). It would be
highly possible that ALDH1 has a binding activity for hydrophobic
signaling molecules including T3 since human ALDH1 from
genital skin fibroblasts displays androgen binding activity (24).
Here we report studies carried out to unambiguously identify xCTBP as
ALDH1. Toward this aim, we have cloned two cDNAs encoding ALDH1
from a Xenopus hepatic cDNA library with human ALDH1
cDNA as the probe (25), determined their nucleotide sequences, and examined both the enzyme and T3 binding activities using
the recombinant proteins expressed in Escherichia coli.
Deduced amino acid sequences and studies of their activities clearly
showed that the two translated products are ALDH1 with T3
binding activity. The corresponding mRNAs were expressed
predominantly in the kidney and intestine in adult Xenopus.
T3 binding and ALDH activities of these proteins seem to be
expressed alternatively depending on NAD+ binding.
 |
EXPERIMENTAL PROCEDURES |
General--
Enzymes and chemicals were obtained from the
following sources: restriction enzymes from Life Technologies, Inc.,
New England Biolabs, Boehringer Mannheim, Takara Shuzo, and Toyobo; a
multiprime DNA labeling kit was from Amersham Pharmacia Biotech;
Moloney murine leukemia virus-reverse transcriptase, exonucleases III and VII, guanidinium isothiocyanate, and cesium chloride were from Life
Technologies, Inc.; Taq DNA polymerase was from Biotech International; a DNA sequencing kit was from Toyobo;
ethyl-3-aminobenzoate methanesulfonic acid salt was from Aldrich;
[
-32P]dCTP (110 TBq/mmol) was from ICN Biomedicals
Inc.; [125I]T3 (122 MBq/µg; carrier-free)
was from NEN Life Science Products; and unlabeled T3,
D-T3, T4,
3,3',5-triiodo-L-thyroacetic acid and
all-trans-retinal were from Sigma. Acetaldehyde was obtained from Merck and AG 1-X8 resin from Bio-Rad. Other reagents of molecular biology grade were purchased from Wako Pure Chemicals and ICN Biomedicals. Adult Xenopus hepatic cDNA library in ZAP
II
vector was kindly provided by Dr. A. Kawahara, Hiroshima
University, Japan. Human ALDH1 cDNA was a gift from Dr. A. Yoshida,
Beckman Research Institute of the City of Hope, CA. Protein was
determined by the dye binding method with bovine
-globulin as a
standard (26).
Preparation of Cytosol--
Adult female Xenopus
laevis were anesthetized by immersing in 0.2%
ethyl-3-aminobenzoate methanesulfonic acid salt. Animals were first
perfused with ice-cold Barth-X amphibian Ringer (27) containing 0.2 mg/ml heparin and then with ice-cold Barth-X amphibian Ringer alone.
Dissected tissue was minced with scissors in Barth-X amphibian Ringer,
followed by several washings in the same solution. The minced tissue
was homogenized in 4.5 volumes of 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, 1 mM
MgCl2, 1 mM dithiothreitol (DTT), 1 mM benzamidine hydrochloride, and 1 mM
phenylmethylsulfonyl fluoride, pH 7.5, as described previously (11).
After successive differential centrifugations at 3,000 × g for 10 min, 12,000 × g for 20 min, and
100,500 × g for 60 min, a clear supernatant was
obtained and stored in 10% glycerol at
85 °C until its use as a cytosol.
Purification of Native xCTBP from Liver Cytosol--
xCTBP was
purified as described with some modifications (11). All the following
procedures were carried out at 4 °C, unless otherwise noted. In
brief, solid ammonium sulfate at a final concentration of 1.4 M was added to 30 ml of cytosol, and the precipitate was removed by centrifugation at 12,000 × g for 15 min.
More solid ammonium sulfate, at a final concentration of 2.5 M, was added to the supernatant. The precipitate obtained
was collected by centrifugation in the same way and dissolved in 2-3
ml of 20 mM sodium phosphate, 0.5 mM DTT, pH
7.5. It was applied to a Cellulofine GCL-1000 column (3.0 × 76.5 cm, Seikagaku Co.), which had been equilibrated with the same buffer,
and eluted at a flow rate of 0.3 ml/min. The eluates with
T3 binding activity were applied to CM cation-exchange and
DEAE anion-exchange Sepharose columns (5 ml packed Fast Flow columns,
Amersham Pharmacia Biotech), which had been tandemly connected and
equilibrated with 20 mM sodium phosphate, 0.5 mM DTT, pH 7.5. Flow-through fractions were collected, and
the pH of the combined eluate was adjusted to 5.3 with 1 M acetic acid. The eluate was subjected to chromatography on Mono S
cation-exchange column (5 × 50 mm; Amersham Pharmacia Biotech), which had been equilibrated with 20 mM sodium acetate, 0.5 mM DTT, pH 5.3, and the proteins were eluted with a 60-min
gradient of this buffer to 20 mM sodium phosphate, 0.5 mM DTT, pH 7.5, at a flow rate of 0.5 ml/min in a fast
protein liquid chromatography apparatus (Amersham Pharmacia Biotech).
The proteins in the peak fractions exhibiting T3 binding
activity were further fractionated by chromatography on a
hydroxyapatite column (model Taps, Tonen) with a 20-min gradient of 20 mM sodium phosphate, 0.5 mM DTT, pH 7.5, to 300 mM sodium phosphate, 0.5 mM DTT, pH 7.5, at a
flow rate of 0.5 ml/min in a fast protein liquid chromatography
apparatus. Finally, the proteins with T3 binding activity
were further isolated by chromatography on a hydrophobic interaction
phenyl 5PW column (7.5 × 75 mm, Tosoh, Tokyo, Japan),
equilibrated with 0.5 M ammonium sulfate in 5 mM sodium phosphate and 0.5 mM DTT, pH 7.0. The
proteins were resolved with a 20-min linear gradient of the buffer to
60% ethylene glycol in 5 mM sodium phosphate, 0.5 mM DTT, pH 7.0, at a flow rate of 0.5 ml/min, using a high
performance liquid chromatography (HPLC) apparatus (Jusco 851-GI
system, Japan Spectroscopic Co.).
Screening of cDNA Library and Sequence Analysis--
An
hepatic cDNA library was screened with 32P-radiolabeled
human ALDH1 cDNA (25). The entire sequences of the two cDNAs
were determined for both strands by the method of Sanger et
al. (28). The computer program, Clustal W (1.60) in DNA Data Bank
of Japan was used on multiple sequence alignment and the construction
of unrooted tree by the Neighbor-joining method (29).
Northern Blot Analysis--
Total RNA was prepared from them by
the acid guanidinium isothiocyanate/phenol/chloroform method (30).
Total RNA (15 µg) was electrophoresed on a 1% agarose gel containing
2.6 M formaldehyde, and the separated RNAs were transferred
onto a nylon filter. Hybridization and washing were performed under
high stringency conditions as described (31). The probe for
Xenopus ALDH1 (xALDH1) cDNAs, a 0.3-kbp fragment that
contained nt 1708-2014 of xALDH1-I cDNA, was amplified by
polymerase chain reaction (PCR) and labeled with [
-32P]dCTP. To check the amount of total RNA loaded,
28 S ribosomal RNA hybridization signals on the same filter were
estimated as a loading control. Xenopus 28 S ribosomal
cDNA was amplified at the nt positions 1-346 by PCR, after the
reaction with Moloney murine leukemia virus-reverse transcriptase in
the presence of (dT)12-18 at 37 °C for 1 h.
Autoradiography was done with Kodak XAR5 film with intensifying screen
at
85 °C for 1-7 days.
Expression of Recombinant xALDHs in E. coli--
The coding
sequences of xALDH1-I and -II cDNAs, with a NdeI site
engineered into the start codon and a BglII site downstream from the stop codon, was prepared by PCR, subcloned into pET15b expression vector (Novagen, Madison, WI), and designated
pET15b/xALDH1-I and pET15b/xALDH1-II. These plasmids were transformed
into E. coli BL21. Bacteria were grown at 37 °C until the
absorbance at 600 nm reached 0.5. The temperature was lowered to
24 °C, 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside was added, and
incubation was continued for 24 h unless otherwise noted.
Purification of Recombinant xALDHs from E. coli--
Bacteria
were pelleted by centrifuging (1200 × g), resuspending
in 0.3 M NaCl, 50 mM Tris, pH 8.0, 10 mM imidazole, 1 mg/ml lysozyme, 1 mM
benzamidine hydrochloride, 1 mM phenylmethylsulfonyl fluoride, and 50 mM 2-mercaptoethanol, and then keeping on
ice for 30 min. The cells were disrupted by sonication for 10 s
three times on ice at the range 5 (UR200P type, Tomy) and subsequently by three cycles of freezing and thawing, and the lysate was centrifuged at 105,000 × g for 40 min at 4 °C. Recombinant
proteins with a histidine tag were isolated from the other proteins in
the supernatant by a nickel affinity chromatography (1 ml of the resin)
(ProBond Resin, Invitrogen, CA), with 0.3 M NaCl, 50 mM Tris, pH 8.0, 250 mM imidazole, after
washing the column with six times column volume of 0.3 M
NaCl, 50 mM Tris, pH 8.0, 80 mM imidazole. The
purified proteins were stored in 1 mM EDTA, 1 mM DTT, and 10% glycerol at
85 °C until use.
T3 Binding Activity--
Cytosolic or recombinant
proteins were incubated in 250 µl of 20 mM Tris-HCl, 1 mM DTT, pH 7.5, containing 0.1 nM
[125I]T3, in the presence of or absence of 5 µM unlabeled T3 at 0 °C for 30 min. The
[125I]T3 bound to proteins was separated from
free [125I]T3 by the Dowex method (11), and
these radioactivities were measured in a
counter (Auto Well Gamma
System ARC-2000, Aloka, Japan). The amount of
[125I]T3 bound nonspecifically was derived
from the radioactivity in the sample incubated with 5 µM
unlabeled T3 and subtracted from amount of the total bound
T3 to give the values for specific binding. The values for
the dissociation constant (Kd) and maximum binding
capacity were calculated from Scatchard plot (32).
Photoaffinity Labeling--
Photoaffinity labeling with
underivatized [125I]T3 was carried out as
described previously (11). The proteins were resolved by
SDS-polyacrylamide gel electrophoresis (PAGE) (33) and visualized using
Coomassie Brilliant Blue R-250 staining or silver staining. Phosphorylase b, bovine serum albumin, ovalbumin, and
carbonic anhydrase were used as molecular weight standards. The labeled proteins were detected by autoradiography exposed to x-ray XAR5 film
(Kodak) at
85 °C for 2-5 days.
ALDH Activity--
Assay was performed in duplicate or
triplicate (values within 10% of the mean) in 100 µl of 50 mM Tris, pH 8.0, 3.3 mM pyrazole, 100 mM KCl, 1 mM DTT, 0.33 mM
NAD+, and various concentrations of substrates by
monitoring for 1-2 min at 24 °C the formation of NADH (
at 340 nm = 6220) with aldehydes other than retinal. The reactions were
initiated by adding the enzyme. At least six concentrations were used
for determining kinetic constants of acetaldehyde, propionaldehyde, and
retinal, ranging 1-32 mM, 0.2-16 mM and 1-32
µM, respectively. For RA synthesis, the formation of NADH
and retinoic acid (
at 340 nm = 6220 + 39,200
22,800) were
monitored under dim light (34). In some cases, the formation of RA was
monitored at 340 nm by HPLC (35). There are few differences in the
values of kinetic parameters obtained from the two methods. Kinetic
constants were determined under initial velocity conditions linear with
time and protein.
Western Blot Analysis--
Cytosolic proteins from adult liver
and two recombinant xALDH1 proteins were separated by electrophoresis
on a SDS-10% polyacrylamide gel, transferred onto a nitrocellulose
membrane, and immunoblotted for 1 h at room temperature with a
primary polyclonal antibody to a peptide of xCTBP, which is identical
to amino acid residues 239-261 in xALDH1-II (see Fig. 2B).
Binding was detected by using the chemiluminescence kit (Boehringer
Mannheim) according to the manufacturer's directions.
Protein Sequencing--
The photoaffinity labeled xCTBP was
digested with lysyl endopeptidase, and the peptides were fractionated
by HPLC as described previously (36). The peptide with radioactivity
was hydrolyzed and analyzed by gas phase sequence analyzer (Applied
Biosystem, 470A).
 |
RESULTS |
Characteristics of xCTBP from Liver Cytosol--
The elution
profile of the final step of the chromatography of xCTBP, which was
purified on the basis of [125I]T3-binding on
a phenyl 5PW column, resembled that described earlier (11). The major
protein peak contained a single species of protein with 59 kDa that was
specifically photoaffinity labeled with
[125I]T3 and corresponded to the peak of
[125I]T3-binding activity (Fig.
1). To determine whether or not xCTBP has
ALDH activity, each fraction was assayed with acetaldehyde as a
substrate. The major protein peak also coincided with the peak of ALDH
activity. These results indicate that the 59-kDa protein has both
activities. The information about the sequence involved in the
[125I]T3-binding site was obtained by the
identification of the peptide with radioactivity after digestion of the
affinity labeled xCTBP with lysyl endopeptidase. Protein sequencing
revealed that the peptide (K)LADLVERDRLILSTM-, which corresponded well
to 91-106 residues of human ALDH1 enzyme (25) and to 92-107 residues
of xALDH1-I and -II enzymes (Fig.
2B), would constitute a part
of the [125I]T3-binding site.

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Fig. 1.
Hydrophobic interaction chromatography of
xCTBP. Fractions (0.5 ml) were collected as described under
"Experimental Procedures." The gradient of ammonium sulfate and
ethylene glycol was applied for 20 min (dotted line). The
solid line shows absorbance at 280 nm. T3
binding activity (open circles) in 25-µl aliquots of each
fraction and ALDH activity (closed circles) in 5-µl
aliquots of each fraction with acetaldehyde as a substrate were
examined. The inset depicts SDS-PAGE of the protein in the
peak fraction following photoaffinity labeling with
[125I]T3 in the absence (middle
lane) or presence (right lane) of 5 µM
unlabeled T3. A 10% gel was run and silver-stained
(left lane), followed by autoradiography (middle
and right lanes).
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Fig. 2.
Restriction maps, nucleotide sequences, and
deduced amino acid sequences of two xALDH1 cDNAs.
A, open boxes indicate the coding regions, and
solid lines indicate the non-coding regions. The
letters B, E, H, Ps, and Pv represent
BamHI, EcoRI, HindIII,
PstI, and PvuII restriction sites. B, first lines,
nucleotide sequences of xALDH1-I cDNA; second lines,
amino acid sequence deduced from xALDH1-I cDNA sequence;
third lines, amino acid sequence deduced from xALDH1-II
cDNA sequence; fourth lines, nucleotide sequences of
xALDH1-II cDNA. The AATAAA polyadenylation signal close to the 3'
end of the cDNAs is written in bold letters. The
two underlined peptides show the regions corresponding to
the sequences determined by a direct protein sequencing. The N-terminal
region was determined using the peptide derived from the liver xCTBP
after digestion with lysyl endopeptidase, and the C-terminal one was
determined using the peptide after treatment with CNBr (11). Residues
essential for catalytic activity (41-43), Trp170,
Asn171 Glu197, Glu270,
Cys304, and Gln489, are underlined.
Amino acid residues of xALDH1-I are represented by dots
where they are identical to the xALDH1-II sequence.
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Relative efficiencies of ALDH activity of xCTBP for two aldehyde
substrates, acetaldehyde and retinal, are shown in Table I. xCTBP catalyzed the conversion of
acetaldehyde into acetic acid or retinal into RA at a linear rate for
at least 1-2 min at 1.85 µg of the purified protein. The
Km value for acetaldehyde (380 ± 20 µM) was 220 times higher than that for retinal (1.7 ± 0.5 µM), although the Vmax for
acetaldehyde (0.54 ± 0.01 µmol/min/mg) was 7 times higher than
that for retinal (0.073 ± 0.002 µmol/min/mg). xCTBP exhibited
allosteric characteristics for retinal with the Hill coefficient
of 2.5 ± 0.2 but not for acetaldehyde as the substrate (Fig.
5A).
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Table I
Kinetic constants of ALDH activities of xCTBP and ALDH1 enzymes
Substrate Km values are millimolar for acetaldehyde
and propionaldehyde, and micromolar for retinal. Coenzyme
Km values are micromolar. Km
values for the three substrates were determined at 0.33 mM
NAD+ and that for NAD+ at 10 mM
acetaldehyde. Vmax values are µmol/min/mg purified
protein. -, not determined because of hyperbolic kinetic
characteristics for acetaldehyde, propoinaldehyde, and NAD+.
The number of determinations is shown in parentheses. Each value is
shown as mean ± S.E. for Km,
Vmax, and the Hill coefficient.
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Cloning and Analysis of xALDH1 cDNAs--
We selected adult
Xenopus liver cDNA library for cloning ALDH cDNA
because T3 binding activity is higher in liver cytosol than
in other tissues of adult Xenopus (36). Human ALDH1 cDNA (25) was used as a screening probe from the fact that amino acid
sequence of xCTBP showed high identity to that of human ALDH1 in the
two regions selected (see Fig. 2B). Two cDNAs with 2.4 kbp, xALDH1-I and xALDH1-II, which were quite similar to but slightly different from each other, were thus isolated. xALDH1-I was
distinguished from xALDH1-II in one of the two internal PstI
restriction sites (Fig. 2A). When amino acid sequences were
deduced from the two cDNAs and aligned with human ALDH1 sequence
(25), it is likely that xALDH1-I cDNA starts at the third nt of the
putative ATG codon and ends at nt position 2301 and that xALDH1-II
cDNA starts at 21 nt upstream from possible start codon and ends at
nt position 2341 (Fig. 2B). For xALDH1-II cDNA, the
flanking sequence of the possible start site conformed partially to the
Kozak criteria (37). A putative polyadenylation signal was present
beginning at nt position 2276 for xALDH1-I cDNA and 2317 for
xALDH1-II cDNA, in their 3'-untranslated regions. Deduced amino
acid sequences of xALDH1-I and -II consisted of both 502 residues
including the start site Met and whose molecular weights were
calculated to be 55,020 and 55,215, respectively, which agreed well
with 59 kDa for xCTBP estimated by SDS-PAGE. Estimated pI values of
xALDH1-I (7.08) and -II proteins (7.44) also agreed well with the
measured pI value of xCTBP, 7.0 ± 0.1 (11). The amino acid
compositions of the xALDH1-I and -II were highly similar to that of the
purified xCTBP (11). The amino acid sequence of xALDH1-I showed 94.6% identity with that of xALDH1-II through 502 residues. The two sequences
exhibited the highest identity (74-80%) with ALDH1 sequences from
various species, as well as the subclass of ALDH1 reported as a retinal
dehydrogenase type II, RalDH(II) (38, 39), and the second (71%), third
(67-68%), and fourth (65-66%) highest identities with human ALDH6,
mammalian ALDH2, and human ALDH5, respectively. The cladogram derived
from multiple alignment of amino acid sequences of several classes of
ALDHs clearly suggests that the two Xenopus proteins belong
to the class of ALDH1 (Fig. 3).

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Fig. 3.
Cladogram derived from the amino acid
sequences of animal ALDHs. These sequence data were collected
from PIR protein data base and EMBL/GenBankTM DNA data
base. The tree was derived using the neighbor-joining method (29) after
aligning these sequences by a multiple sequence alignment program,
Clustal W (1.60) over residues 10-498 of xALDH1 sequences. In this
alignment, 97 invariant and 88 conserved residues were found through
the 22 sequences. Bootstrap values (%) from 1000 replications are
indicated. This analysis supports the specific branching orders for the
major groups, which are ((ALDH6, ALDH1) and (ALDH5, ALDH2)), ALDH9) as
shown in the tree by Yoshida et al. (62). Proteins with
catalytic activity converting retinal to RA are indicated by
asterisks (20, 22, 23, 38, 39, 45, 52-55, 57).
1, human ALDH6; 2, mouse RalDH(II); 3,
rat RalDH(II); 4, xALDH1-I; 5, xALDH1-II;
6, chicken ALDH1; 7, human ALDH1; 8,
horse ALDH1; 9, rat phenobarbital-induced ALDH;
10, rat RalDH(I); 11, rat kidney ALDH1;
12, mouse ALDH1 (AHD-2); 13, elephant
shrew(ee) crystallin; 14, elephant shrew
(mpe) crystallin; 15, human ALDH5;
16, horse ALDH2; 17, human ALDH2; 18,
bovine ALDH2; 19, mouse ALDH2 (AHD-1); 20, giant
octopus crystallin; 21, earthworm ALDH; and 22,
human ALDH9.
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As regards functional significance of the above comparisons, these
Xenopus proteins had all 23 of the strictly conserved
residues of the aldehyde dehydrogenase superfamily, expressed in
phylogenetically diverse organisms (40). By analogy to the human ALDH1
sequence, Cys304 (41) and Glu270 (42) might
play catalytically essential roles in Xenopus proteins, whereas human ALDH2 enzyme also require the Gln489 (43).
All three residues were conserved in the two xALDH1 sequences. Three of
the amino acids interacted with NAD+ in the
NAD+ binding domain of rat ALDH3 (44) and bovine ALDH2
(45), corresponding to Trp170, Asn171, and
Glu197 in the Xenopus sequences, and were
conserved in xALDH1 as well as mammalian ALDH1 sequences.
To identify whether or not the xCTBP purified from liver cytosol is
xALDH1, the amino acid sequences of the two regions in xCTBP determined
by direct protein sequencing were compared with those deduced from the
two xALDH1 cDNAs. The sequences (K)LADLVERDRLILSTM and
(M)DIDKVAFTGSTEVGKLIKEAAG were identified to the amino acid positions
92-107 and 239-261 of xALDH1-II, but both were distinct from the
corresponding sequences of xALDH1-I at amino acid positions 92 and 256. Thus xCTBP is more likely to be xALDH1-II than xALDH1-I.
Characteristics of Recombinant xALDH1 Expressed in E. coli--
The xALDH1-I and -II proteins expressed in E. coli contain an additional 20 residues of a histidine tag. We
purified them to almost single band by a nickel affinity chromatography
(Fig. 4A). Approximately 7 mg
of the purified proteins were obtained from a 250-ml culture. In
SDS-PAGE, the apparent molecular weights of the recombinant xALDH1-I
and -II proteins with a histidine tag were estimated to be 60 × 103, which was a bit bigger than that of xCTBP in liver
cytosol, 59 × 103 (Fig. 4, B and
C). The two purified recombinant proteins were specifically
photoaffinity labeled with [125I]T3 (Fig.
4B), like the xCTBP purified from liver cytosol (see inset in Fig. 1). The photoaffinity labeling of xALDH1-II
was more strongly inhibited by 5 µM unlabeled
T3 than that of xALDH1-I. Polyclonal antibody to the
peptide of xCTBP recognized both recombinant proteins as well as xCTBP
in adult liver cytosol (Fig. 4C).

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Fig. 4.
Purification of two recombinant xALDH1
proteins and comparison of their T3 binding activities and
immunoreactivities with those of xCTBP in liver cytosol.
A, purification of two recombinant xALDH1 proteins. Bacteria
harboring pET15b/xALDH1-I (lanes 1-4) or pET15b/xALDH1-II
(lanes 5-8) were cultured in the presence of 0.2 mM isopropyl-1-thio- -D-galactopyranoside for
24 h at 24 °C. These extracts were mixed with a nickel-bound
resin, and the mixtures were packed into a disposable mini-column
(7 × 23 mm). After washing the column, xALDH1-I and -II were
eluted as described under "Experimental Procedures." Lanes
1 and 5, bacterial extract; lanes 2 and
6, flow-through fraction; lanes 3 and
7, eluates with 80 mM imidazole buffer;
lanes 4 and 8, eluates with 250 mM
imidazole buffer. B, photoaffinity labeling of xCTBP and
xALDH1 with [125I]T3. Cytosolic proteins (65 µg) from adult liver (lane 1) and the purified recombinant
proteins (each 5.6 µg), xALDH1-I (lanes 2 and
3) and xALDH1-II (lanes 4 and 5), were
photoaffinity labeled with [125I]T3 in the
absence (lanes 1, 2, and 4) or presence
(lanes 3 and 5) of 5 µM unlabeled
T3. A 10% gel was run, followed by autoradiography.
C, immunoreactivity of two recombinant xALDH1 proteins with
a polyclonal antibody against a peptide of xCTBP. Cytosolic proteins
(93 µg) from adult liver (lane 1) and the purified
recombinant proteins (each 8 µg), xALDH1-I (lane 2) and
xALDH1-II (lane 3), were immunoblotted after SDS-PAGE as
described under "Experimental Procedures." Arrowheads
indicate recombinant xALDH1 proteins with a histidine tag.
|
|
ALDH activity was found in the two recombinant proteins expressed from
the xALDH1-I and -II cDNAs with all the substrates examined. The
Km and Vmax values for
acetaldehyde, propionaldehyde, retinal, and NAD+ are
summarized in Table I. Both proteins showed the lowest
Km values for retinal (6.9 ± 0.5 µM in xALDH1-I and 4.2 ± 0.2 µM in
xALDH1-II) (Fig. 5, B and
C); the Km values for propionaldehyde (0.45 ± 0.13 mM in xALDH1-I and 0.32 ± 0.14 mM in xALDH1-II) and acetaldehyde (3.2 ± 0.4 mM in xALDH1-I and 1.7 ± 0.2 mM in
xALDH1-II) were 2 and 3 orders of magnitude higher than those for
retinal. Vmax values of the two proteins for
acetaldehyde (0.40 ± 0.12 in xALDH1-I and 0.14 ± 0.02 µmol/min/mg in xALDH1-II) were similar to those for propionaldehyde
(0.31 ± 0.07 in xALDH1-I and 0.12 ± 0.01 µmol/min/mg in
xALDH1-II) and were 1 order of magnitude higher than those for retinal
(0.062 ± 0.005 in xALDH1-I and 0.045 ± 0.003 µmol/min/mg
in xALDH1-II). The Vmax/Km
values indicate that substrate preference of the two xALDH1 proteins is
the following order: retinal > propionaldehyde > acetaldehyde. The kinetics for NAD+ were characterized by
Km of 38 ± 3 and 9.2 ± 1.8 µM, and Vmax of 0.44 ± 0.10 and 0.18 ± 0.03 µmol/min/mg, for xALDH1-I and -II,
respectively, at the concentration of NAD+ ranging from 6 to 120 µM. Compared with the kinetics of the purified xCTBP, the Km of Xenopus recombinant
proteins exhibited values 3-8 times higher, although their
Vmax value was very similar. The order of
substrate specificity of the Xenopus enzymes was in
agreement with that of mammalian ALDH1 enzymes (38, 46, 47) but quite
distinct from other classes of mammalian ALDHs (48-51). Positive
cooperativity showing allosteric kinetics could only be detected when
the ALDH activities of the xALDH1-I and -II proteins were examined with
various concentration of retinal (Hill coefficients, n = 1.8 ± 0.4 and 2.7 ± 0.6, respectively) (Fig. 5,
B and C) but were undetectable with acetaldehyde
and propionaldehyde as substrates.

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Fig. 5.
Rates of RA synthesis from retinal catalyzed
by xCTBP purified from adult liver and by recombinant xALDH1 enzymes
versus substrate concentrations. The rate of RA
synthesis was measured in the presence of the xCTBP (A),
xALDH1-I (B), or -II (C) enzymes. These reactions
were performed at 24 °C with 1.85 µg of xCTBP or 5 µg of
xALDH1-I and -II enzymes. The insets depict Hill plots. Each
value is the mean of triplicate determinations.
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Relationship between T3 Binding and ALDH Activities on
xCTBP/xALDH1--
To confirm whether or not the two recombinant xALDH1
proteins are dual-functional proteins, T3 binding activity
was also examined. The two recombinant proteins bound specifically
T3 (insets in Fig.
6). Their binding specificities were
T3 > D-T3 > T4 > 3,3',5-triiodo-L-thyroacetic acid, which was very similar
to that of the xCTBP in adult liver cytosol (11).
[125I]T3 binding to xALDH1-II was more
strongly inhibited by 320 nM unlabeled T3 than
that to xALDH1-I, which was in good agreement with the results of the
photoaffinity labeling (Fig. 4B). The results of Scatchard
analysis of the xALDH1-I and -II proteins shown in Fig. 6 and their
Kd values for T3 binding are compared
with those of the xCTBP from liver cytosol determined previously (11)
(Table II). The Kd
values, 142 ± 0 and 48.0 ± 7.2 nM for the
recombinant xALDH1-I and -II proteins, were 16 and 5 times higher than
that for the xCTBP purified from liver cytosol (9 nM),
respectively.

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Fig. 6.
Scatchard analysis of T3 binding
to the xALDH1 proteins. The purified recombinant xALDH1-I (24.8 µg, A) and -II (12.8 µg, B) were incubated
with 0.1 nM [125I]T3 at various
concentrations of unlabeled T3 for 30 min at 0 °C in a
final volume of 250 µl of 20 mM Tris-HCl, 1 mM DTT, pH 7.5. B/F, bound/free ratio. Each
value indicates the mean of triplicate determinations. The
insets illustrate the effect of four competitors (320 nM) on [125I]T3 binding to the
purified recombinant xALDH1-I (A) and xALDH1-II
(B). Triac,
3,3',5-triiodo-L-thyroacetic acid. *, p < 0.01; **, p < 0.001, compare
[125I]T3 binding in the presence of the
competitor with that in the absence of the competitor. Nonspecific
binding was subtracted from total binding. Each value indicates the
mean ± S.E. of triplicate determinations.
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Table II
T3 binding to xCTBP and xALDH enzymes
The Kd and maximum binding capacity (MBC) values for
xCTBP are cited (4). The number of determinations is shown in
parentheses.
|
|
As the recombinant xALDH1 proteins required 10
5 to
10
4 M NAD+ to display ALDH
activity (Table I), the effects of NAD+ on T3
binding by the two recombinant proteins were examined. NAD+
can inhibit the T3 binding to the two xALDH1 proteins in a
dose-dependent manner. The concentrations of
NAD+ necessary to inhibit 50% of specific T3
binding were about 100 and 43 µM for xALDH1-I and
xALDH1-II, respectively (not shown). These findings suggest that
xCTBP/xALDH1 expresses alternatively T3 binding and ALDH
activities dependent on NAD+ binding. Therefore, we next
examined the effect of four coenzymes, including NAD+, on
the T3 binding to the recombinant xALDH1 proteins.
NAD+ and NADH both inhibited T3 binding
activity by 47 and 27% for xALDH1-I and 23 and 18% for xALDH1-II,
respectively, at the concentration of 0.2 mM, but
NADP+ and NADPH failed to exert the effect at the same
concentration (Fig. 7).

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Fig. 7.
Effect of coenzymes on T3 binding
activity of the recombinant xALDH1 proteins. The recombinant
proteins were incubated with 0.1 nM
[125I]T3 in the presence or absence of 5 µM unlabeled T3 for 30 min at 0 °C in a
final volume of 250 µl of the same buffer with or without 0.2 mM each coenzyme. *, p < 0.05; **,
p < 0.001, compare [125I]T3
binding in the presence of the coenzyme with that in the absence of the
coenzyme. Nonspecific binding was subtracted from total binding. Each
value indicates the mean ± S.E. of triplicate
determinations.
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|
Expression of xCTBP/xALDH1 in Different Tissues--
In all
tissues examined, a single band of mRNA was detected in a size
between 28 S and 18 S ribosomal RNAs. The accumulation of
xCTBP/xALDH1 transcripts was particularly strong in kidney and
intestine, with smaller amounts found in liver and stomach. These
transcripts were expressed at very low levels in heart and skeletal
muscles (Fig. 8).

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Fig. 8.
Tissue distribution of xALDH1 mRNAs.
Lane 1, liver; lane 2, kidney; lane 3,
stomach; lane 4, intestine; lane 5, heart;
lane 6, skeletal muscle. Fifteen micrograms of total
cellular RNAs obtained from adult female Xenopus tissues
were electrophoresed in 1% agarose gel, blotted onto a nylon filter,
and hybridized with the 0.3-kbp fragment of the 3' non-coding region of
xALDH1-I cDNA (upper) or the 0.35-kbp fragment of the 5'
region of 28 S ribosomal cDNA (lower).
|
|
 |
DISCUSSION |
From the following five lines of evidence, we conclude that xCTBP
is xALDH1. First, the xCTBP purified from liver cytosol displayed ALDH
activity (Fig. 1 and Table I), and its enzymatic properties agreed with
those of mammalian ALDH1. Second, the recombinant proteins expressed
from two xALDH1 cDNAs in E. coli also showed both ALDH
and T3 binding activities (Figs. 4-7), all of which were similar to those of the liver xCTBP (Tables I and II). Third, in the
two internal regions, the primary sequence of xCTBP coincided exactly
with the corresponding sequences in xALDH1-II and in xALDH1-I except
for two residues. Fourth, the apparent molecular weights estimated by
SDS-PAGE and pI values and amino acid compositions of xCTBP, described
previously (11), were consistent with the molecular weights, pI values,
and amino acid compositions calculated from the sequence data of the
xALDH1 cDNAs. Fifth, polyclonal antibody to a peptide of xCTBP
reacted to both xCTBP and the two recombinant xALDH1 proteins. Since
retinal is a preferred substrate for xALDH1, our conclusion raises the
possibility that xCTBP/xALDH1 can modulate actions of RA and
T3 via their nuclear receptors by regulating RA synthesis
and intracellular levels of free T3.
As regards specifically the Xenopus proteins, the
Km and Kd values of the
recombinant xALDH1 proteins were several times higher than those of
xCTBP from liver cytosol. The lower affinities of the recombinant
xALDH1 proteins for the substrates and T3 might be due to
the presence of the histidine tag at their N termini, which may be a
contributory factor for the variation. A comparison of xALDH1-I with
xALDH1-II reveals some differences between their molecular, enzymatic,
and T3 binding properties. However, it is not possible to
ascertain from these results that the two proteins have distinct
functions. Sequencing data of both the xCTBP (11) and the two cDNAs
strongly indicate that the xCTBP we purified from liver cytosol is
xALDH1-II, rather than xALDH1-I. Even if xALDH1-I was contained in the
fractions with T3 binding activity of the final
purification step, by phenyl 5PW column chromatography, the ratio of
xALDH1-I to xALDH1-II might be low.
It is well known that ALDH isozymes form a superfamily (40). The
determination of the nucleotide sequences of the isolated cDNAs
allowed us to compare their amino acid sequences with those of the
known ALDHs from various species and to construct the cladogram shown
in Fig. 3. First, this tree clearly illustrates that translated products of the two Xenopus cDNAs and the mammalian and
chicken ALDH1 proteins group together. This relationship among the
ALDH1 sequences reflects the evolutional distances among vertebrates. The second interesting point about this tree is the position of the two
rodent RalDHs(II) and the rat retinal dehydrogenase type I, RalDH(I).
The RalDH(II) proteins were outside of the vertebrate ALDH1 group with
the bootstrap value of 91.4%, whereas RalDH(I) protein fell within the
vertebrate ALDH1 group, suggesting that RalDH(II) is a sister group of
ALDH1 and that it is hard to distinguish RalDH(I) from ALDH1 on the
basis of the data of primary sequences alone. We could not exclude the
possibility that a Xenopus homolog of the rodent RalDH(II),
which has not yet been found so far, exists and also plays a role in
the synthesis of RA from retinal. It is worth noticing that the ability
to convert to RA is found in many ALDH1 proteins, including RalDH(II)
as a subtype, from various vertebrate species (indicated by
asterisks in Fig. 3). In view of the involvement of RA in
many developmental processes, it would be valuable to survey the
presence of subtypes of ALDH1 and to examine the expression patterns of
a group of xALDH1 proteins during early embryogenesis and limb
formation in Xenopus.
Sequence comparison of ALDHs depicted in the cladogram (Fig. 3) shows
that the residues Glu270, Cys304, and
Gln489, which are thought to be involved in the catalytic
role of ALDH enzymes (41-43), were highly conserved in
Xenopus sequences. The region participating in
T3 binding by xCTBP/xALDH1 is located at amino acid
positions 92-107. The corresponding region is present away from the
catalytic domain but in the NAD+ binding domain in bovine
ALDH2 (45). Although this region does not seem to interact directly
with NAD+, the structural basis for the effects of
NAD+ on T3 binding will probably require a
structure for ALDH1.
Both recombinant xALDH1 enzymes can catalyze the dehydrogenation of
acetaldehyde, propionaldehyde, and retinal (Table I). The substrate
specificity estimated as a
Vmax/Km is the highest for
retinal, this value being 7-12- and 50-90-fold higher than those for
propionaldehyde and acetaldehyde, respectively, whereas for xCTBP, the
Vmax/Km for retinal is
30-fold higher than that for acetaldehyde. Interestingly, allosteric
kinetics showing positive cooperativity was observed for the
recombinant xALDH1 enzymes and the xCTBP with the Hill coefficient of
1.8-2.7 and 2.5, respectively, when retinal was used as the substrate (Table I). A similar observation was made for a rat RalDH(I), with Hill
coefficient of 1.4-1.8, by Napoli's group (52-55). Napoli's group
also reported that retinal associated with cytosolic retinoid-binding proteins (CRABP and CRBP) could be important for its recognition as a
substrate by the enzyme, since CRABP stimulated the production of RA
and CRBP suppressed it (53). This interesting finding suggests that it
would be useful in future studies to determine a similar effect of
retinoid-binding proteins on the enzyme activities of xCTBP/xALDH1.
The two xALDH1 enzymes reported here are quite similar to each other as
regards their the primary sequences, substrate specificities, and
T3 binding properties (Tables I and II, and Fig. 2).
Probably the presence of two genes is most likely due to the
tetraploidy of the Xenopus genome (56). For this reason we
used the 3' non-coding region of the xALDH1-I cDNA as a probe for
Northern analysis, since it would recognize both xALDH1 transcripts but
not those of the other members of ALDH superfamily. The relatively high amounts of xALDH1 mRNAs, migrating as a single band with a mobility intermediate to those of ribosomal 18 S and 28 S RNAs in adult Xenopus kidney and intestine (Fig. 8), are compatible with
the finding for rat kidney ALDH1 (57). The level in liver was not so
high, although T3 binding activity was found almost
exclusively in liver (19). This discrepancy suggests the possibility of post-transcriptional or -translational regulation, or some other form
of modulation of T3 binding activity of xCTBP. Dual
functional properties of ALDH1 have also been reported for other
species, such as for elephant shrew
(23) and giant octopus
crystallins as major component of lens proteins (see Fig. 3) and human
56-kDa androgen-binding protein in genital skin fibroblast (24).
Interestingly, the formation of RA from retinal via rabbit ALDH
(probably ALDH1) was stimulated by diethylstilbestrol,
dehydroisoandrosterone, estrone, and cortisone but inhibited by
progesterone, deoxycorticosterone, testosterone, and androsterone (34),
thus suggesting that compounds with steroid hormonal activities could
bind to rabbit ALDH. Although very recent study indicated that human
liver ALDH1 and ALDH2 both could bind T3 and
3,3',5-triiodo-L-thyroacetic acid, their
Kd values were micromolar ranges (58). It would
therefore be interesting to determine whether or not the same or other
hydrophobic signaling molecules bind to xCTBP/xALDH1.
Among other studies on CTBPs, there are those reporting an
enzyme-linked CTBP, which is a monomeric form of pyruvate kinase subtype M2 in human cell lines and whose conversion to the tetramer is
regulated by an intermediate of the glycolytic pathway, fructose 1,6-bisphosphate (10, 18). Recently, Shi et al. (59)
isolated the cDNA for a Xenopus homolog of the human
pyruvate kinase subtype M2 and described high levels of its transcript
in Xenopus tadpole tail just before metamorphic climax, in
hindlimb during the progression of metamorphosis, whereas relatively
low levels were detected in the intestine during metamorphosis.
However, it is uncertain whether or not the monomer of pyruvate kinase
subtype M2 functions as a CTBP in Xenopus as in the human
cell lines. Our earlier studies have shown that xALDH1 is the
predominant CTBP in adult Xenopus (11, 19). It is worth
pointing out that there is no similarity of primary sequence between
the monomer of the pyruvate kinase subtype M2 and xCTBP/xALDH1. Another
type of NADPH-activated CTBPs has been reported in rat kidney (13),
liver (14), and brain (15), comprising molecular species with different
molecular weight values. For example, kidney CTBP is a monomer of 58 kDa, liver CTBP is a homodimer with 76 kDa, and rat brain CTBP is a 58-kDa protein. T3 binding activity of all of these CTBPs
is strongly activated by NADPH and slightly by NADH, but not by
NADP+, whereas NAD+ has no effect on the
T3 binding activity of the rat CTBPs (13, 14). A very
recent study indicated that human kidney CTBP is a 38-kDa protein,
which is homologous to kangaroo µ crystallin (60). On the other hand,
T3 binding activity of xCTBP is inhibited by
NAD+ and NADH, and neither NADPH nor NADP+ is
effective. If the total concentration of cytoplasmic NAD+
plus NADH, which ranges 10
4 M in mammalian
cells (61), changes in similar ranges in Xenopus cells, it
would be one of the important factors modulating T3 binding
to xCTBP/xALDH1. These observations and our present results show that
xCTBP might be a novel type of CTBP.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB016717 and AB016718.
§
To whom correspondence should be addressed: Dept. of Biology,
Faculty of Science, Shizuoka University, 836 Oya, Shizuoka 422-8529, Japan. Tel.: 81-54-238-4447; Fax: 81-54-238-0986; E-mail: sbkyama{at}ipc.shizuoka.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
T3, 3,3',5-triiodo-L-thyronine;
CTBP, cytosolic
thyroid-hormone-binding protein;
xCTBP, Xenopus CTBP;
T4, L-thyroxine;
RA, retinoic acid;
CRABP, cytosolic retinoic acid-binding protein;
CRBP, cytosolic
retinol-binding protein;
ALDH, aldehyde dehydrogenase;
DTT, dithiothreitol;
HPLC, high performance liquid chromatography;
xALDH, Xenopus ALDH;
kbp, kilobase pair(s);
nt, nucleotide(s);
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
RalDH(II), retinal dehydrogenase type II;
RalDH(I), retinal
dehydrogenase type I.
 |
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