Équipe INSERM dEndocrinologie (M.-P.V., J.O.,
M.P., J.F., J.-P.B.), Faculté de Pharmacie, 92296
Châtenay-Malabry, France,
Centre Nationale de la
Recherche Scientifique Unité de Recherche Associée 1967
(C.E., P.R.), Institut Gustave Roussy, 94805 Villejuif,
France,
Centre de Recherche Rhône-Poulenc Rorer
(A.B.-G.), 94403 Vitry-sur-Seine, France,
Service
dUrologie (P. B.), Centre Hospitals-Universitaire de
Bicêtre, 94270 Kremlin-Bicêtre, France
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ABSTRACT |
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INTRODUCTION |
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Human cells contain a 58-kDa cytosolic T3-binding protein (5). The sequence of its cDNA indicated that it is the monomer of the ubiquitous enzyme pyruvate kinase (6). Fructose 1,6-bisphosphate, a metabolite of the glycolytic pathway, regulates binding of T3 to the pyruvate kinase monomer. This cytosolic binding protein influences the regulation of the transcriptional responses mediated by T3 nuclear receptors, probably by helping to control the intracellular free T3 concentration (7). Another T3-binding protein has been identified in Xenopus laevis liver cytosol. This is a 59-kDa monomeric protein whose sequence is identical to that of a subunit of mammalian and avian aldehyde dehydrogenases (8).
Activation by NADPH and NADP+ of T3 binding in rat kidney cytosol was first reported by Hashizume et al. (9, 10). Purification of the binding activity indicated that it was due to a 58-kDa monomeric protein. This protein binds to isolated nuclei in vitro and regulates the access of T3 to the nuclear receptors (10). Yet another NADPH-activated T3-binding protein has been purified from rat liver cytosol by Kobayashi et al. (11) and found to be a dimer of a 38-kDa polypeptide.
We have analyzed the kinetics of the cytosolic T3-binding sites from rat cultured astrocytes (12) and from human kidney (13). Binding is strongly activated by a low concentration of NADPH, whereas NADP+ inhibits the activation by NADPH. The affinity of the cytosolic binding sites for T3 and T4 is similar to that of the thyroid hormone nuclear receptors, but there are 100 times more of them, so that they are responsible for most of the high-affinity T3 (and T4) binding in rat astrocytes and human kidney tissue. The NADP-regulated thyroid hormone-binding protein (THBP) was found to be a 35- to 38-kDa polypeptide by photoaffinity labeling (12, 13). Human NADP-regulated THBP is present in thyroid hormone target organs, such as kidney, heart, and liver, but not in the pancreas, fibroblasts, or red blood cells (13).
This study describes the purification of the human NADP-regulated THBP from kidney cytosol, peptide microsequencing of the purified protein, the cloning of its cDNA from a human brain cDNA library, and expression of recombinant THBP cDNA in E. coli. The cDNA of THBP was found to be nearly identical to previously cloned human cDNA (14) encoding a putative protein with no known function but with sequence homology to a lens crystallin produced in certain marsupial species (14).
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RESULTS |
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A search of the Genpept database showed that all four peptide sequences were identical to parts of the protein sequence translated from a 1,238-nucleotide human cDNA (14) (Genbank accession number L02950). This cDNA was cloned from a human retinal library because of its great homology to a kangaroo cDNA coding for µ-crystallin, a major protein component of the eye lens in this species (15). The putative protein encoded by the human cDNA had been therefore called human "µ-crystallin" (14), although it is not a lens structural protein in humans. Peptide 1 corresponded to the translation of nucleotides 514543 of the human cDNA L02950, peptide 2 corresponded to that of nucleotides 754786, peptide 3 corresponded to that of nucleotides 901933, and peptide 4 corresponded to that of nucleotides 940972.
Complementary DNA Cloning and Predicted Amino Acid Sequence of
Human THBP
A human brain library of adaptator-ligated double-strand cDNAs was
used for 5'- and 3'-rapid amplification of cDNA ends (RACE).
Gene-specific primers for PCR were synthesized according to the
sequence of the human cDNA L02950, which corresponds to the nucleotide
sequences of peptide 1 (sense primer:
5'-CCTTTAAGGAGGTGAGGATATGGAACCGCACC-AAAG-3') and peptide 3
(antisense primer: 5'-GGCTGCAACTGTGTCTTCCACTGCCATTCCC-3'). A single
PCR fragment of 1.1 kb was amplified using the antisense
primer (5'-RACE). Similarly, a single PCR fragment of
0.9 kb
was amplified using the sense primer (3'-RACE). The sequencing of 200
nucleotides at the 5'-end of the 5'-RACE PCR product indicated complete
identity with the 5'-end of cDNA L02950 (except for the lack of the
first five nucleotides in the 5'-untranslated region).
The two PCR products were cloned in pBluescript II KS. One clone was
selected in each case (clones TH5 and TH9) and sequenced. The sequences
corresponded to overlapping 5'- and 3'-fragments of the human
µ-crystallin cDNA (L02950), with the exceptions reported below. The
plasmids containing TH5 (3'-fragment) and TH9 (5'-fragment) inserts
were cut at a single restriction site in the overlapping region of the
inserts (BclI site, nucleotide 629 of the human
µ-crystallin clone) and in the multiple cloning site of pBluescript
(XhoI site). The full-length cDNA (TH5.9) was generated by
ligation of the 3'-fragment of the TH5 insert to the pBluescript
plasmid with the 3'-fragment of TH9 deleted. It was then cloned and
sequenced (Fig. 4).
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The putative THBP encoded by the open reading frame of TH5.9 cDNA is a 314- amino acid polypeptide whose sequence is identical to that deduced from L02950 cDNA. The protein has a theoretical mol wt of 33,775 and a theoretical pI of 5.06.
Expression of THBP cDNA in E. coli
A portion of the pBluescript plasmid containing the TH5.9 cDNA
insert was excised with EcoRI and ligated at the single
EcoRI site of pGEX-2T. The construct produced a glutathione
S-transferase (GST)-THBP fusion protein in E.
coli upon induction with isopropyl
ß-D-thiogalactopyranoside. The fusion protein was 569
residues long (227 amino acids of GST, 28 amino acids of a sequence
located 5' to the THBP coding region, and 314 amino acids of THBP),
with a theoretical mol wt of 63,134. It was purified by affinity
chromatography using a glutathione-agarose gel. The purified fusion
protein migrated on SDS-PAGE as one band with an apparent mol wt of
61,000 (Fig. 5A).
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DISCUSSION |
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The peptide sequencing data and the cloned cDNA sequence indicate that THBP is identical to a putative human protein related to kangaroo µ-crystallin (14). There is only one nucleotide difference within the coding region, which does not change the translated amino acid sequence. Using the first initiation codon (ATG) at nucleotide 26 and termination at nucleotide 968 (TAA) of the cDNA gave a protein with theoretical mol wt and pI close to those of the purified THBP.
Southern blots indicated that there is a single gene for human µ-crystallin (14), located on chromosome 16p (16). However, all four peptides sequenced from purified THBP were located in the C-terminal half, leaving open the possibility that there may be protein isoforms of similar mol wt but different thyroid hormone-binding properties produced by alternative splicing of the 5'-part of a single pre-mRNA. This seems unlikely because only one PCR product was amplified by 5'-RACE from the human brain library, and its sequence was identical to the 5'-sequence of the human µ-crystallin cDNA.
This conclusion was confirmed by the expression in E. coli of a vector containing the coding sequences of GST and of THBP cDNAs within the same reading frame. The purified fusion protein had the expected mol wt, as measured by SDS-PAGE, and bound T3 with high affinity in an NADPH-dependent manner. The binding characteristics of the fusion protein (T3 affinity, iodothyronine specificity, nucleotide dependence), as well as its photolabeling characteristics, were similar to those of the THBP in human kidney cytosol (13).
While µ-crystallin is a major lens protein in only a few marsupial
species (16), lower but significant concentrations are present in
several non-lens tissues in marsupials and humans (14), but until this
study identified it as a major NADP-regulated thyroid hormone-binding
protein, its biological function was unknown. The relationship between
human THBP and the major lens protein in kangaroos may appear
perplexing. In fact, most of the lens proteins that function as
crystallins are not specialized structural proteins, and their
synthesis is not restricted to the lens (17). The ubiquitous
-crystallins, which are major components of the lenses in all
vertebrate species, are related to small heat-shock proteins (17). Many
other crystallins, which are highly species-specific, are identical, or
at least closely related, to ubiquitous enzymes (18), such as
arginosuccinate lyase (
-crystallin in most birds and reptiles),
lactate dehydrogenase (
-crystallin in crocodiles and some birds),
NADPH:quinone oxidoreductase (
-crystallin in guinea-pigs and
camels), aldehyde dehydrogenase (
-crystallin in shrews), etc. Many
of these multifunctional species-specific crystallins are
NAD(P)H-binding proteins, which is believed to protect against
oxidation in the lens and/or to help filter UV radiation (18). It is
therefore remarkable that THBP has an unusually high affinity for NADPH
(12, 13), a property that could have contributed to the recruitment of
its gene to encode a crystallin in some marsupials. Finally, our
findings suggest that THBP has a regulatory or developmental role in
the lenses of higher mammals, although it does not act as a
crystallin.
The kinetic properties of the NADP-regulated THBP in crude human kidney cytosol (13) and its direct identification by photoaffinity labeling (13) suggested that it is different from the cytosolic T3-binding proteins previously characterized and/or identified in the cells or tissues of several species, such as the monomers of pyruvate kinase (6) and aldehyde dehydrogenase (8). These proteins are not regulated by NADPH and have much lower affinities for T3 and T4. The cloning of THBP cDNA confirms that the protein is not related to ubiquitous housekeeping enzymes.
Northern blot analysis of human tissues, using the kangaroo µ-crystallin cDNA as a probe, showed that the homolog human mRNA is abundant in the heart, brain, skeletal muscle, and kidney, that there is less in the lung and liver, and that there is none in the placenta or pancreas (14). This correlates well with our finding of high concentrations of NADP-regulated THBP in human heart, human and rat kidney (13), and rat brain (12), of less THBP in human liver, and of none in human pancreas (13). It also confirms that our photoaffinity labeling procedure (12, 13) has identified the main, if not the sole, NADP-regulated high-affinity thyroid hormone-binding protein in several human and rat tissues that respond to thyroid hormones.
A basic local alignment search tool (BLASTP) was used to screen the OWL data bank (SWISS-PROT, PIR 13, GenBank translations, Brookhaven). Significant homologies (4060%) were found between several amino acid stretches in THBP and two bacterial enzymes: lysine cyclodeaminase from Streptomyces hygroscopicus and the ornithine cyclodeaminases from Rhizobium meliloti and Agrobacterium tumefaciens (this converts ornithine to proline plus ammonia). The homology between the A. tumefaciens ornithine cyclodeaminase and kangaroo µ-crystallin was previously reported (14), but the kangaroo µ-crystallin had no ornithine-cyclodeaminase enzyme activity (14). The significance of these local homologies between bacterial enzymes and human THBP is hypothetical. The (iodo)thyronine binding site of THBP might be related to the amino acid (lysine, ornithine, arginine) binding sites of the cyclodeaminase enzymes. The pyridine nucleotide binding site of THBP might also be related to that of ornithine cyclodeaminase, since enzyme activity is stimulated by NAD+, which acts as a catalyst rather than a cosubstrate (19). This suggests that the mammalian THBP evolved from a very ancient family of amino acid- and pyridine nucleotide-binding proteins. The elucidation of the sequences of THBP and of its cDNA will allow further investigation of its pathophysiological functions and its regulation in thyroid hormone-responsive tissues.
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MATERIALS AND METHODS |
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Cytosol Preparation
Human kidney tissue was obtained at surgery from patients with
kidney carcinoma. The whole diseased kidney was removed for the benefit
of the patients. Normal-appearing tissue adjacent to neoplastic tissue
was collected on ice and frozen in liquid nitrogen. Cytosol was
prepared (13) and all the purification procedures were carried out at
04 C.
DEAE-Trisacryl Chromatography
The cytosol was applied to a DEAE-Trisacryl column (11 x
15 mm) equilibrated with buffer A at a flow rate of 1 ml/min. Bound
proteins were step-eluted with buffer A containing 50, 100, 200, 500,
and 1000 mM NaCl at a flow rate of 3 ml/min. Fractions
containing the NADPH-dependent T3 binding activity were
pooled and desalted by repeated (up to three times) ultrafiltration
(Vivaspin 4 Cut 10000, 7000 rpm for 30 min, 4 C) followed by dilution
in buffer A, until the NaCl concentration was 20 mM.
Blue-Sepharose Chromatography
The ultrafiltrate from DEAE-Trisacryl chromatography was loaded
at a flow rate of 1 ml/min onto a Blue-Sepharose CL-6B column (11
x 10 mm) previously equilibrated with buffer A. The proteins were
step-eluted (3 ml/min) with 0, 10-6, 10-5,
10-4 M NADPH in buffer A. The eluate fractions
were assayed for saturable T3 binding. Fractions
contain-ing binding activity were pooled and concentrated by
ultrafiltration.
Proteolytic Cleavage and Amino Acid Sequencing
The purified protein was transferred from a preparative
polyacrylamide gel to a Problot membrane (Perkin-Elmer, Norwalk, CT),
and the N-terminal amino acid sequence of the 36-kDa band was
determined by automatic Edman degradation on a protein microsequencer
(Perkin-Elmer), essentially as described in Ref. 20. Internal peptides
were obtained from the 36-kDa band ( 8 µg protein) excised from
the preparative acrylamide gel. The gel strip was incubated for 1
h in H2O, H2O-methanol (90:10),
H2O-methanol (80:20), H2O-methanol (50:50), cut
into small pieces, dried under vacuum, and incubated in 25
mM Tris (pH = 8.5), 1 mM EDTA, 0.05% SDS,
12.5 µg/ml proteinase Lys-C (overnight at 37 C). The supernatant was
lyophilized, solubilized in H2O, and injected onto an AX300
(2.1 x 30 mm) precolumn (Perkin-Elmer) followed by a 218TP52
Vydac HPLC reversed phase column (2.1 x 250 mm). The peptides
were eluted with 0.1% (vol/vol) trifluoroacetic acid in water for 10
min and with a 035% (vol/vol) linear gradient of 0.1%
trifluoroacetic acid in acetonitrile during 150 min at a flow rate of
200 µl/min. Fractions were collected each 0.5 min. The major peaks
were sequenced.
RACE by PCR
Both 5'- and 3'-RACE were performed using human brain
Marathon-Ready cDNA. PCR was performed as recommended by the
manufacturer. PCR products were cloned in pBluescript II KS
(Stratagene, La Jolla, CA). Complementary DNA fragments were ligated
using T4 DNA ligase. Nucleotide sequences were obtained using the
T3 primer (3'-end cDNA) and the M13 primer (5'-end and
full-sized cDNA).
Expression in E. coli
The full-sized cDNA sequence was subcloned into pGEX-2T
bacterial expression plasmid (Pharmacia-Biotech, Piscataway, NJ) which
contains the GST gene. The GST-fusion protein cDNA was expressed in the
TG1 strain of E. coli, after induction by isopropyl
ß-D-thiogalactopyranoside. Bacteria were lysed by
sonication in 10 mM EDTA, 1 mM
phenylmethylsulfonylfluoride, 50 mM Tris-HCl, pH 7.4, and
the GST-fusion protein was adsorbed onto glutathione-agarose beads. The
beads were washed three times with 50 mM Tris-HCl, pH 7.7,
and the GST-fusion protein was eluted with 10 mM reduced
glutathione in the same buffer. Purified GST was obtained by the same
procedure, using the empty pGEX-2T vector.
Miscellaneous Methods
The binding of [125I]T3 to soluble
proteins was assayed using dextran-coated charcoal to adsorb free
T3 (13). The methods used for photoaffinity labeling of
T3-binding proteins by UV-driven cross-linking of
underivatized [125I]T3, analysis by SDS-PAGE,
and autoradiography are described (13). Analytical SDS-PAGE (10%
acrylamide) was performed essentially as described by Laemmli (21). The
samples were boiled for 5 min in electrophoresis sample buffer. The
gels were run, fixed, and stained with 0.1% (wt/vol) Coomassie blue
R250, as described in Ref. 13. Apparent molecular weights were
calculated from the migration of standard proteins (High Molecular
Weight Markers, Sigma-Aldrich Chimie). Preparative gels ( 4 µg
THBP per lane) were stained with 0.2% (wt/vol) Coomassie blue R250 in
20% (vol/vol) methanol, 0.5% (vol/vol) acetic acid for 20 min,
destained in 30% (vol/vol) methanol, and extensively washed with
distilled water (22). The 36-kDa stained bands were cut out from the
gel for amino acid sequencing. Two-dimensional gel electrophoresis was
performed by the method of OFarrell (23): tube gel electrofocusing
was done in the presence of 9.2 M urea and 2% Nonidet P40,
and it was followed by 10% slab SDS-PAGE. Gels were silver-stained and
autoradiographed. Protein concentrations were usually measured by the
method of Bradford (24) using BSA as standard. Small amounts of
proteins were determined by comparing the intensity of Coomassie
blue-stained bands on SDS-PAGE to that of known amounts of BSA (240
µg/lane). The gels were scanned with a densitometer (BioImage
Electrophoresis System, Millipore Corp., Bedford, MA) to obtain the
integrated optical densities of stained bands. The equilibrium
dissociation constant (Kd) and inhibition constants
(Ki) were calculated by nonlinear least-square curve
fitting (mean ± SE of estimate), using an adaptation
(Biosoft, Cambridge, UK) for the Apple-Macintosh of the LIGAND program
of Munson and Rodbard (25).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the INSERM and the Fondation pour la Recherche Médicale.
1 Supported by a studentship from the Ligue Nationale contre le
Cancer.
Received for publication May 8, 1997. Accepted for publication July 14, 1997.
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REFERENCES |
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