Purification, Molecular Cloning, and Functional Expression of the Human Nicodinamide-Adenine Dinucleotide Phosphate-Regulated Thyroid Hormone-Binding Protein

Marie-Pierre Vié1, Claudine Evrard, Jeannine Osty, Aline Breton-Gilet, Pascal Blanchet, Martine Pomérance, Pierre Rouget, Jacques Francon and Jean-Paul Blondeau

Équipe INSERM d’Endocrinologie (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 d’Urologie (P. B.), Centre Hospitals-Universitaire de Bicêtre, 94270 Kremlin-Bicêtre, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo µ-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [125I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (Kact) = 10-8 M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The major effects of thyroid hormones occur via the binding of T3 to its nuclear receptors. These receptors belong to a superfamily of ligand-dependent transcription factors that includes the receptors for steroid hormones, retinoids, and vitamin D. Steroid receptors shuttle continuously between the nucleus and cytoplasm (1), and they are believed to interact with their appropriate hormones in the cytoplasm. In contrast, the retinoid and T3 receptors remain bound to their target genes, even in the absence of ligand (2, 3). The intracellular transport of retinoids and thyroid hormones and the control of their intracellular concentrations are undertaken by cytosolic binding proteins. Complementary DNA cloning of the retinol- and retinoic acid-binding proteins showed that they belong to a family of lipid-binding proteins (4). The binding of T3 to cytosolic proteins in several types of cells and tissues has recently been shown to be regulated, indicating that these proteins are involved in the control of intracellular T3 homeostasis.

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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Purification of Human THBP
Various purification steps were tested on an analytical scale. Fractionation of human kidney cytosol by ammonium sulfate precipitation, affinity chromatography on T3-Sepharose, and hydrophobic chromatography on Octyl-Sepharose were unable to achieve significant purification of THBP. Human kidney cytosol was applied to a column of diethylaminoethyl (DEAE) Trisacryl and eluted stepwise with increasing concentrations of NaCl. Most of the proteins were eluted in the flow-through and in the 50- and 100-mM NaCl fractions. The NADPH-dependent saturable T3 binding activity was eluted by 200 mM NaCl with a yield of {approx} 50% (Fig. 1AGo). A small amount of T3 binding activity was eluted by 100 mM NaCl, but it was not NADPH-dependent and was probably due to serum albumin contaminating the cytosol preparations (as revealed by Coomassie blue staining after SDS-PAGE of the fraction; not shown). The 200-mM NaCl fraction was desalted and adsorbed onto Blue-Sepharose gel. The column was eluted stepwise with increasing concentrations of NADPH (Fig. 1BGo). Most of the proteins were not retained (Table 1Go), but the T3 binding activity was eluted with 10-6 M NADPH (Fig. 1BGo).



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Figure 1. Chromatography of THBP from Kidney Cytosol

THBP-containing preparations were applied to a DEAE-Trisacryl column (panel A) and a Blue-Sepharose column (panel B). Protein ({circ}) was determined by the method of Bradford. [125I]T3 binding activity (bound/unbound ratio, mean of duplicates) was measured by the dextran-coated charcoal method without additives ({blacksquare}), or with NADPH (3 x 10-7 M in panel A or 10-6-10-4 M in panel B) (•), or with NADPH and 10-7 M unlabeled T3 ({blacktriangleup}). The concentrations (M) of NaCl (panel A) and NADPH (panel B) in the elution buffers are indicated by arrows.

 

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Table 1. Purification of Thyroid Hormone Binding-Protein from Human Kidney Cytosol

 
The purification scheme adopted for large-scale partial purification of THBP was: 1) DEAE-Trisacryl chromatography, 2) desalting of the 200-mM NaCl fraction, and 3) Blue-Sepharose chromatography. The purity and recovery of the THBP at each step is shown in Table 1Go (this particular preparation was used for sequencing the proteolytic peptides). The results of two large-scale purifications indicated that the specific activity of binding was increased {approx} 6-fold by DEAE-Trisacryl chromatography and {approx} 1000-fold by Blue-Sepharose chromatography over the specific activity in the cytosol. Figure 2AGo illustates the purification of the 36-kDa band after the two chromatographic steps. The photolabeling of this protein after Blue-Sepharose chromatography was strongly reduced by oxidation of NADPH to NADP+ and suppressed by excess unlabeled T3 (Fig. 2BGo). The resulting THBP was estimated to be 45–60% pure, based on the specific activity of the purified fraction (Table 1Go and another independent experiment). These data are in agreement with the SDS-PAGE and silver staining analyses (Fig. 2AGo): the 36-kDa silver-stained band represented {approx} 50% of the proteins stained in the lane. The yield of THBP was {approx} 5%.



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Figure 2. SDS-PAGE of THBP-Containing Fractions

Panel A, Proteins from crude cytosol (lane 1), 200-mM NaCl fraction from DEAE-Trisacryl chromatography (lane 2), and 10-6 M NADPH fraction from Blue-Sepharose chromatography (lane 3) were silver-stained. Panel B, The 10-6 M NADPH fraction from Blue-Sepharose chromatography was incubated with [125I]T3, UV-irradiated, and analyzed by SDS-PAGE and autoradiography. Incubation and photolabeling were perfomed with 5 x 10-5 M oxidized glutathione plus 0.033 U/ml glutathione reductase (lane 1), without additives (lane 2), or with 10-7 M unlabeled T3 (lane 3).

 
Two-dimensional electrophoresis of the purified fraction showed that only one 36-kDa spot with an isoelectric point (pI) = 5.3 was present, and it corresponded to the spot labeled by photoaffinity (Fig. 3Go). Preparative SDS-PAGE was therefore used as the final purification step. The Coomassie blue-stained 36-kDa band gave pure denatured THBP, which was suitable for amino acid sequencing.



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Figure 3. Two-Dimensional PAGE of Blue-Sepharose-Purified THBP

The fraction eluted from the Blue-Sepharose column with 10-6 M NADPH was incubated with [125I]T3, UV-irradiated, and analyzed by two-dimensional PAGE and autoradiography. The 36- to 97-kDa (vertical scale) regions of the silver-stained gel (panel A) and of the autoradiogram (panel B) are shown. The complete pH range is shown (horizontal scale).

 
Amino Acid Microsequencing of Purified THBP
No sequence information was obtained by direct NH2-terminal sequencing of purified THBP, suggesting that the NH2 terminus of the protein was modified either as the result of a posttranslational event or due to protein handling. The protein was cleaved with endoproteinase Lys-C, and the resulting peptides were separated by reverse-phase HPLC. Three peaks eluting at 81.9, 93.2, and 97.9 min were selected for NH2-terminal sequencing. One of them gave the sequence [K]EVRIWNRTK (peptide 1), another gave [K]EAVLYVDSQE (peptide 2), and the third one gave two sequences: [K]XXGMAVEDTV (peptide 3) and [K]LIYDSWSSGK (peptide 4) (the lysine residue preceding the specific cleavage site by endoproteinase Lys-C is shown in parentheses; X indicates unidentified amino acids).

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 514–543 of the human cDNA L02950, peptide 2 corresponded to that of nucleotides 754–786, peptide 3 corresponded to that of nucleotides 901–933, and peptide 4 corresponded to that of nucleotides 940–972.

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 {approx} 1.1 kb was amplified using the antisense primer (5'-RACE). Similarly, a single PCR fragment of {approx} 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. 4Go).



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Figure 4. Nucleotide and Predicted Amino Acid Sequence of Cloned Human THBP

The deduced amino acid sequence of the encoded protein (single-letter code) is shown in boldface. The underlined regions represent amino acids determined by microsequencing of proteolytic peptides 1 to 4. The asterisk denotes the 3'-stop codon. Nucleotide variations with human µ-crystallin cDNA are indicated above the appropriate THBP nucleotides. The gene-specific primers used for 3'- and 5'-RACE are indicated above the nucleotide sequence as dotted arrows. The nucleotide sequences of human THBP have been deposited with GenBank under accession no. U85772.

 
The TH5.9 is 1,228 nucleotides long, and its sequence is nearly identical to that of the human µ-crystallin cDNA (L02950). The only differences are: 1) the absence of the first five nucleotides at the begining of the 5'-untranslated region (AGACT) of the L02950 sequence and of the last five nucleotides (CAGTG) at the end of the 3'-untranslated region, just upstream of the poly-A tail of TH5.9; 2) substitution of G for A at position 1013 in the 3'-untranslated region of the THBP sequence; 3) substitution of G for C at position 889 in the open reading frame of the THBP sequence. This substitution does not alter the sequence of the translated protein since ACG and ACC both code for threonine. TH5.9 and L02950 cDNAs are strongly homologous ({approx} 80%) to the cDNA coding for the kangaroo µ-crystallin.

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. 5AGo).



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Figure 5. SDS-PAGE of GST and GST-THBP Fusion Protein

Panel A, GST-THBP fusion protein (lane 1) and GST (lane 2), produced in E. coli, were purified on glutathione-agarose beads, resolved by SDS-PAGE (3.6 µg/lane), and stained with Coomassie blue. Panel B, 0.4 µg purified GST-THBP fusion protein (lanes 1–4) or 0.16 µg purified GST (lanes 5 and 6) were incubated with [125I]T3, UV-irradiated, and analyzed by SDS-PAGE and autoradiography. Incubation and photolabeling were perfomed without additives (lanes 1 and 5), with 10-7 M NADPH (lanes 2 and 6), with 10-7 M NADH (lane 3), or with both 10-7 M NADPH and 10-7 M unlabeled T3 (lane 4).

 
Purified GST-THBP bound T3 in a NADPH-dependent manner, whereas the GST produced in E. coli (using the empty vector) did not (Fig. 6AGo). Half-maximal activation was obtained with {approx} 10-8 M NADPH, whereas NADH was without effect up to 10-6 M (Fig. 6BGo). The binding of T3 to GST-THBP promoted by 3 x 10-7 M NADPH was abolished by an exogenous NADPH-oxidizing enzyme system (5 x 10-5 M oxidized glutathione plus 0.033 U/ml glutathione reductase), indicating that NADP+ is not an activator (not shown). Iodothyronine binding to GST-THBP was studied in the presence of an optimal concentration of NADPH (Fig. 6CGo). The T3 dissociation constant (Kd) is 0.48 ± 0.15 nM (as determined by nonlinear least-squares curve fitting). The affinity of T4 [inhibition constant (Ki) = 2.3 ± 0.2 nM] and rT3 (Ki = 84 ± 8 nM) are {approx} 5 and {approx} 200 times lower, respectively, than that of T3.



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Figure 6. Thyroid Hormone Binding to GST-THBP Fusion Protein

Purified GST-THBP fusion protein was incubated with [125I]T3 and additives, and the ratio of bound to unbound T3 was determined by the dextran-coated charcoal method (mean of duplicates). Panel A, Increasing amounts of GST-THBP were incubated without additives ({square}), with 10-5 M NADPH ({circ}), or with both 10-5 M NADPH and 10-7 M unlabeled T3 ({triangleup}), and increasing amounts of GST were incubated under the same conditions as GST-THBP (points superimposed as {triangleup}). Panel B, 0.2 µg GST-THBP was incubated with increasing concentrations of NADPH (•) or NADH ({blacksquare}). Panel C, 0.2 µg GST-THBP was incubated with 3 x 10-7 M NADPH and increasing concentrations of unlabeled T3 ({circ}), T4 ({square}), rT3 ({triangleup}).

 
NADPH stimulated the photolabeling of the 61-kDa purified GST-THBP with [125I]T3 but NADH did not, and the NADPH-activated photolabeling was blocked by excess unlabeled T3 (Fig. 5BGo). In contrast, recombinant GST was not photolabeled, whether or not NADPH was present.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This report describes the purification and sequencing of a human THBP and the molecular cloning of its cDNA. THBP was purified to homogeneity by two chromatographic steps and preparative SDS-PAGE. Approximately 8 µg pure THBP was obtained from 8 g kidney tissue. The specific activity of binding of the partially purified THBP (before preparative SDS-PAGE) was {approx} 1000-fold greater than that of the cytosol. The THBP was {approx} 50% pure. The THBP retained its T3 binding properties and was activated by NADPH. It could be photolabeled with radioactive underivatized T3 and behaved on two-dimensional electrophoresis as a polypeptide with a mol wt and pI similar to that of the THBP in kidney cytosol (13).

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 {alpha}-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 ({delta}-crystallin in most birds and reptiles), lactate dehydrogenase ({epsilon}-crystallin in crocodiles and some birds), NADPH:quinone oxidoreductase ({zeta}-crystallin in guinea-pigs and camels), aldehyde dehydrogenase ({pi}-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 1–3, GenBank translations, Brookhaven). Significant homologies (40–60%) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals
[3'-125I]T3 (specific activity: 3 mCi/µg) was purchased from Amersham-France (Les Ulis, France), rT3 was from Calbiochem (Meudon, France). Pyridine nucleotides, endoproteinase Lys-C, and isopropyl ß-D-thiogalactopyranoside were from Boehringer-Mannheim-France (Meylan, France). T3, T4, oxidized and reduced glutathione, glutathione reductase, glutathione-agarose, and HCl-washed charcoal were from Sigma-Aldrich Chimie (St-Quentin-Fallavier, France). DEAE-Trisacryl was from Sepracor (Villeneuve-la-Garenne, France). Dextran T-70, Octyl-Sepharose, activated CH-Sepharose 4B, and Blue-Sepharose CL-6B were from Pharmacia-Biotech (St-Quentin-Yvelines, France). Oligonucleotides were synthesized by Genset (Paris, France). Human brain Marathon-Ready cDNA and Advantage KlenTaq Polymerase Mix were from CLONTECH (Palo Alto, CA). Vivaspin 4 Cut 10000 membranes were from Vivascience (Barjouville, France). Restriction enzymes and T4 DNA ligase were from Ozyme (Montigny-le-Bretonneux, France). Other chemicals were pur-chased from Merck-Clévenot (Nogent-sur-Marne, France).

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 0–4 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 ({approx} 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 0–35% (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 ({approx} 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 O’Farrell (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 (2–40 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).


    ACKNOWLEDGMENTS
 
We thank Dr. D. Faucher (Rhône-Poulenc Rorer, Vitry-sur-Seine, France) for helpful discussions about the microsequencing of THBP.


    FOOTNOTES
 
Address requests for reprints to: Dr J. P. Blondeau, INSERM Endocrinologie, Tour D1, Faculté de Pharmacie, 5 Rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France.

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. Back

Received for publication May 8, 1997. Accepted for publication July 14, 1997.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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