INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THYROID HORMONES INFLUENCE GROWTH, differentiation, and metabolism by modulating gene transcription rates after binding to receptors (for reviews, see Refs. 42 and 59). A nongenomic mechanism is also discussed as a possible mechanism of action for some of the effects of thyroid hormones (7, 12, 40, 65). Like many other hormones, thyroid hormones are distributed by the bloodstream from the site of synthesis to their targets. However, during their way from the site of synthesis to the target, the hormonal signals are subjected to various modulations. There are two main ways of modulating the signals. The first concerns the distribution of the hormones in the body; the second is the generation of a more efficient form from a less-efficient form of the hormone.

Thyroid hormones readily partition into cell membranes because of their high solubility in lipids (14, 24, 56). Therefore, no complex signal-transduction system is needed for the transfer of information across cell membranes. The hormone can directly interact with the intracellular receptors after permeating through cell membranes (68).

Recent research suggests that the evolution of an increasing complexity of regulation by thyroid hormones in vertebrates is paralleled by the evolution of an increasing complexity of thyroid hormone distribution (see Ref. 55). Plasma proteins binding thyroid hormones establish an appropriate distribution of these hormones between extracellular and intracellular compartments. In larger mammals, including humans, there are three thyroid hormone-binding plasma proteins: thyroxine-binding globulin, transthyretin (TTR), and albumin. Smaller placental mammals, diprotodont marsupials, and birds possess only albumin and TTR. In polyprotodont marsupials, monotremes, and reptilians, albumin, but not TTR, is synthesized during adult life by the liver and secreted into the blood (45). In adult individuals of these classes and subclasses of vertebrates, TTR is made only in the choroid plexus of the brain and in the retina. Thus TTR is essentially an extracellular brain protein in most vertebrate species. In adult animals of species with efficient endothermic regulation of body temperature, which appeared later in evolution, TTR is also a blood protein synthesized and secreted by the liver. Recently, the transient synthesis of TTR was observed during early development in the liver of bullfrog tadpoles (67) and seabream (53).

During evolution of mammalian TTRs from TTR in reptile-like ancestors, the NH₂-termini of the TTR subunits became shorter and less hydrophobic (see Refs. 54 and 55). It was suggested that the molecular mechanism of this was a stepwise shift in the 3' direction of the splice site at the 5' end of exon 2 of the precursor mRNA for the TTR subunit (3). However, the NH₂-termini of the TTR in bullfrog tadpoles do not seem to fit into this general scheme for TTR evolution (67). Also, the binding properties of bullfrog TTR for L-thyroxine (T₄) and 3,5,3'-triiodothyronine (T₃) (66) drastically differ from those of the TTRs in other species (10). In this paper, we report the cloning and sequencing of the cDNA for TTR in Xenopus laevis (xTTR; the African clawed toad) and the analysis of the TTR gene in genomic DNA by Southern blotting. We also analyze the timing and tissue pattern of TTR gene expression in X. laevis. For physicochemical studies of the properties of xTTR, its cDNA was incorporated into two protein expression vectors for the yeast Pichia pastoris. The cleavage recognition site of the -factor (using the pPIC9 vector) of yeast and the xTTR presegment (using the pPIC3.5 vector) allowed normal processing of the recombinant xTTR in the yeast. The obtained protein product was characterized as TTR by sequencing of the NH₂-terminus, determination of the association of subunits into a tetramer, binding to human retinol-binding protein, and quantitation of the binding of T₄ and T₃. The nucleotide sequence of the genomic DNA in the 5' area of the xTTR gene was determined. Comparison of the nucleotide sequence of the genomic DNA with the cDNA sequence derived from mature RNA gave the position of the splice site between exons 1 and 2. Comparison with splice-site positions in other vertebrate TTR mRNAs allowed conclusions about the molecular mechanism of the evolution of the NH₂-terminal region of TTR in vertebrates.

Comparisons of the amino acid sequences of TTRs from different species, with TTR genelike segments in the genomes of Caenorhabditis elegans, Schizosaccharomyces pombe, Salmonella dublin, and Escherichia coli were used to identify the position of xTTR in the evolution of TTR from bacteria to humans. A most parsimonious phylogenetic tree was constructed based on the amino acid sequences of the TTR subunits derived from the nucleotide sequences of TTR cDNAs.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials, sources of animals, cDNA library, cDNA probe, and yeast expression vectors. T₃, D-T₃, T₄, and 3,5,3'-triiodothyroacetic acid were purchased from Sigma. X. laevis tadpoles were from Hassei Biological (Hamamastu, Japan). Developmental stages were assessed according to the criteria of Nieuwkoop and Faber (41). A cDNA library, from the middle regions of the bodies of X. laevis tadpoles at stages 54-57, in ZAP II -vector was kindly provided by Dr. J. R. Tata, National Institute for Medical Research. A 0.35-kb fragment of X. laevis elongation factor 1-cDNA (32) was a gift from Dr. T. Amano (Yoshizato MorphoMatrix Project, Japan). Yeast expression vectors were purchased from Invitrogen (Adelaide, Australia).

Screening of cDNA library and sequence analysis. The X. laevis cDNA library was screened with ³²P-labeled Rana catesbeiana TTR cDNA (67). Six positive clones were obtained. The entire sequence of the longest cDNA was determined for both strands by the method of Sanger et al. (51). The other five were partially sequenced on one strand only.

Southern analysis of X. laevis genomic DNA. Genomic DNA from liver of adult X. laevis was isolated using pancreatic RNase and proteinase K, as described by Sambrook et al. (49). Aliquots (30 µg) were digested with various restriction enzymes that do not have sites within the DNA to which the probes were designed. The digested DNA was subjected to electrophoresis in a 1% agarose gel, and the DNA was transferred onto a Hybond-N+ nylon membrane (Amersham, Australia) by capillary blotting (49). Southern hybridization was performed using Rapid-hyb buffer (Amersham) with ³²P-radiolabeled exon-1 fragment (nucleotide position 1-103) or exon-2 fragment (nucleotide position 122-202) of xTTR cDNA, with prehybridization for 2 h and hybridization for 5 h at 60°C. The filters were washed twice with 2× standard sodium citrate (SSC), 0.1% SDS at room temperature for 20 min each, followed by washing twice with 1× SSC, 0.1% SDS at 60°C for 20 min each. The filters were then exposed to Kodak BioMax MS film with an intensifying screen, at 70°C, for 2 days.

Detection of TTR in serum by Western analysis. Serum samples from adult human, adult koala, adult Tasmanian devil, and adult X. laevis were analyzed for the presence of TTR by Western analysis using antiserum from a rabbit raised against a mixture of purified TTRs from human, tammar wallaby (Macropus eugenii), and chicken (Gallus gallus) serum, as described previously (47).

Northern blot analysis: timing of gene expression during development. Livers were collected from 10 to 20 metamorphosing tadpoles at stages 53-54, 58-59, 60, 62-63, and 66, deposited in liquid nitrogen, and stored at 80°C until use. Total RNA was extracted by the acid guanidinium isothiocyanate-phenol-chloroform method (11). Total RNA (15 µg/lane) was analyzed by electrophoresis in 1% agarose gel containing 2.6 M formaldehyde. Hybridization with ³²P-labeled xTTR cDNA and washing were performed under high-stringency conditions, as described in Ref. 27. Autoradiography was carried out at 80°C for 1 to 7 days. Hybridization signals of elongation factor 1-mRNA on the same filter were used as loading controls. The amount of radioactive cDNA probe hybridizing with mRNA was quantitated by computer-assisted image analysis (software Photoshop from Adobe). The amount of the TTR mRNA is shown as the ratio of the density for the TTR mRNA bands to that for the elongation factor 1-mRNA bands.

Northern blot analysis: sites of gene expression. Total RNA was extracted from heads, livers, and carcasses (heads and livers removed) of X. laevis tadpoles at stages 57-59. An amount of 10 µg of RNA per lane was analyzed by electrophoresis in 1% agarose gel (37). Hybridization was performed at 65°C using digoxygenin (DIG)-labeled xTTR cRNA or at 60°C using ³²P-labeled cane toad (Bufo marinus) choroid plexus lipocalin cDNA (2) as probes. Filters were washed twice for 15 min in 0.3 M NaCl, 0.03 M trisodium citrate (2× SSC), 0.1% SDS at room temperature, once for 15 min in 1× SSC and 0.1% SDS at hybridization temperature and once for 15 min in 0.5× SSC and 0.1% SDS or 0.1× SSC and 0.1% SDS at hybridization temperature. Color detection of the hybridized DIG probe was performed with anti-DIG-alkaline phosphatase-conjugated antibody followed by the reaction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, essentially according to the instruction of Roche Diagnostics.

Construction of expression vectors. BamH I and EcoR I (pPIC3.5 vector) or Xho I and EcoR I (pPIC9 vector) sites were introduced by polymerase chain reaction into xTTR cDNA such that cleavage by BamH I occurred immediately before nucleotide 39 of xTTR cDNA or by Xho I such that cleavage occurred immediately before nucleotide 96 of xTTR cDNA or by EcoR I to cleave immediately after the stop codon TAA (498) of the xTTR cDNA. The PCR product was ligated with the Pichia expression vectors pPIC3.5 or pPIC9. The inserted vector was linearized by digestion with Sal I and transformed into P. pastoris strain GS115 by electroporation.

Affinity chromatography of recombinant xTTR on human retinol-binding protein. Recombinant xTTR was purified by affinity-column chromatography on human retinol-binding protein coupled to Sepharose 4B, as described by Larsson et al. (34).

Determination of the mass of the TTR monomer by mass spectrometry. The mass of the recombinant xTTR subunit was determined by a Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (Finnigan MAT), using -cyano-4-hydroxycinnaminic acid (10 mg/ml) in 70% acetonitrile with 0.1% trifluoroacetic acid as the matrix. The spectrum was detected with laser power 100 µJ and laser aim 4.

Determination of the mass of the TTR tetramer by gel filtration. The molecular mass of recombinant TTR was estimated by fast-performance liquid chromatography gel filtration on Superose-12 (HR 10/30, Pharmacia) equilibrated in 50 mM potassium phosphate buffer saline (pH 7.4). Column and sample volumes were 24 ml and 50 µl, respectively, the flow rate was 0.5 ml/min, and the standards were bovine serum albumin (68 kDa), ovalbumin (45 kDa), horse heart myosin (16.7 kDa), and horse heart cytochrome c (12 kDa).

Analysis of NH₂-terminal amino acid sequence. The NH₂-terminal amino acid sequences of the recombinant TTRs were determined on a commercial basis by the Australian Proteome Analysis Facility (Macquarie Univ., Sydney, New South Wales, Australia).

Analysis of T₄ and T₃ binding to recombinant xTTR. Purified xTTR (100 nM) was incubated with T₄ or T₃ concentrations from 0 to 1,000 nM in the presence of tracer [¹²⁵I]T₄ or [¹²⁵I]T₃ at 4°C, and free T₄ or T₃ was separated from the TTR-bound hormones according to the method described by Chang et al. (10). The radioactivity was determined using an LKB 1270 Rackgamma II counter with an efficiency of 70%. Nonspecific binding was estimated by extrapolation of the saturation curve, and other corrections were performed (as described in Ref. 10) before analysis by Scatchard plotting (52).

Amplification of the TTR gene from genomic DNA. Genomic DNA from adult X. laevis liver was prepared by phenol extraction, as described by Sambrook et al. (49). Oligonucleotide primers consisting of xTTR cDNA segments near the 3' end of exon 1 (5'-ACACCTTCTGAACAACAATGGC-3') and complementary to sequences near the 5' end of exon 2 (5'-TTCACTAGCAGATTGGCAGCGG-3') were used in polymerase chain reactions to amplify TTR genomic DNA fragments containing the exon 1/intron 1 and intron 1/exon 2 regions, as previously described (3). Polymerase chain reactions were carried out in 0.02-ml reaction mixture for 25 cycles at a denaturation temperature of 94°C for 5 min, an annealing temperature of 55°C for 30 s, and an extension temperature for 1 min. The product from the polymerase chain reaction mixture was then purified and subcloned into pCR2.1 vector.

Phylogenetic analysis of TTR sequences. TTR cDNA sequences were obtained from databases: human (Homo sapiens X59498), pig (Sus scrofa X82258), sheep (Ovis aries X15576), cow (Bos taurus AB009591), rat (Rattus norvegicus X14876) mouse (Mus musculus X03351), shrew (Sorex araneus AJ223149), hedgehog (Erinaceus europaeus AJ277290), wallaby (Macropus eugenii X79113), kangaroo (Macropus giganteus X97689), sugar glider (Petaurus breviceps X80999), dunnart (Sminthopsis macroura X80998), opossum (Monodelphis domestica X80193), chicken (Gallus gallus X60471), lizard (Tiliqua rugosa M97509), bullfrog (Rana catesbeiana AB006134), Xenopus (Xenopus laevis AB026996), sea bream (Sparus aurata AF059193) and imported into an Australian National Genomic Information Service (ANGIS) project. Bases before the first ATG were deleted, and amino acid sequences were derived using the Etranslate program. Derived sequences were checked against published derived sequences, and additional residues in the COOH-terminal region were deleted. For species in which the NH₂-terminal amino acid residues had been determined by Edman degradation, residues comprising the presegment were deleted. For species in which the NH₂-terminal residue had not been determined (pig, shrew, mouse, sugar glider, X. laevis, sea bream, dunnart, and lizard), it was designated as the residue corresponding to residue 1 in human TTR. These "mature TTR sequences," together with those from rabbit (Oryctolagus cuniculus P07489), were put into multiple-sequence format using the Pileup program. Bootstrap resampling (500 replicates) was carried out using the Eseqboot program. Eprotpars was used for calculation of maximum parsimony, and Econsense was used to generate the consensus tree. All programs were accessed via the ANGIS. Specific programs used were from the Genetics Computer Group (21).

Database searches for sequences similar to those of TTRs. Database searches for protein sequences and open reading frames (ORF) were performed using the BCM Search Launcher General Protein Sequence/Pattern Searches with BLASTP+BEAUTY postprocessing. The same sequences were suggested by the program as being most closely related to TTR, regardless of which TTR amino acid sequence (from different species) was entered as the query sequence. When the xTTR sequence was entered, the number of matches with the non-TTR sequences was higher than when a reptilian, avian, marsupial, or eutherian TTR sequence was entered. Hydrophobicity profiles were generated by the method of Kyte and Doolittle (33), using a scanning window of seven amino acid residues.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Molecular cloning and sequencing of xTTR cDNA. In preliminary experiments, TTR cDNA from R. catesbeiana (67) was found to hybridize with RNA extracted from X. laevis tadpole livers but not with RNA from the livers of adult X. laevis frogs. Therefore, a cDNA library from the livers of X. laevis tadpoles in middevelopment (stages 54-57) was screened with a Rana TTR cDNA probe. Six independent cDNA clones with the lengths of cDNA in the range of 0.4 to 0.65 kb were isolated. The longest was sequenced on both strands (Fig. 1). The alignment of the deduced amino acid sequence with those of other TTRs from various species makes it likely that the coding sequence for xTTR starts with nucleotide 39 and ends with nucleotide 497. The partial matching of the flanking region of the possible start site satisfied Kozak's rules (31). The deduced amino acid sequence of xTTR was 153 residues long and included a signal peptide. The molecular weight was calculated to be 16,683. A putative polyadenylation signal was present at nucleotide positions 639 to 644. 

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence of the cDNA for tadpole [Xenopus transthyretin (xTTR)] and its deduced amino acid sequence. A: sequencing strategy. xTTR cDNA is represented as a small checkered bar. Arrows indicate direction and extent of sequencing. B: nucleotide sequence and derived amino acid sequence of xTTR cDNA. Nucleotide numbers at the right, beginning with the first nucleotide of cDNA insert. Signal peptide amino acids are numbered 19 to 1. Amino acid numbers are indicated underneath the nucleotide sequence. The NH2-terminal residue of the mature transthyretin (TTR), obtained from NH2-terminal amino acid sequencing of recombinant xTTR, is designated as +1. The first in-frame stop codon is represented by 3 asterisks. The putative polyadenylation signal in the 3' untranslated region of cDNA is underlined.

Southern analysis of genomic DNA from X. laevis. The number of chromosomes (61) and the DNA content per nucleus (58) suggest that a duplication of the genome occurred in the ancestor of the X. laevis group. Immunochemical analysis of albumins allowed dating of that duplication event to ~10 million years ago, after the branching of TTR evolution into the lines leading to X. tropicalis and to the X. laevis group ~30 million years ago (8). Furthermore, Southern analysis of the genomic DNA from R. catesbeiana was consistent with the presence of a single copy of the albumin gene per haploid R. catesbeiana genome (4). There is only one TTR gene copy per haploid genome in the rat (18). To clarify the situation for X. laevis, a Southern analysis was carried out for genomic DNA from X. laevis, which had been digested with the restriction enzymes EcoR I, BamH I, Pst I, or Sac I, and either xTTR exon 1 cDNA or xTTR exon 2 cDNA as probes. The nucleotide sequences of exons 1 and 2 do not contain a cleavage site for any of the restriction endonucleases used in the Southern analysis. More than one band was obtained in the analysis for all of the used restriction enzymes (Fig. 2), suggesting that more than one copy of the TTR gene is present per haploid genome of X. laevis.

View larger version (75K): [in this window] [in a new window]   Fig. 2.   Southern blot hybridization. Thirty micrograms of adult Xenopus laevis liver genomic DNA was digested with EcoR I, BamH I, Pst I, or Sac I and subjected to electrophoresis in a 1% agarose gel. Southern blot analysis was performed using exon 1 fragment (A) or exon 2 fragment (B) of xTTR cDNA as probes, as described in MATERIALS AND METHODS. The positions of the DNA molecular size markers are indicated on the left.

Western analysis of serum of adult X. laevis for TTR. Western analysis of the serum from adult X. laevis was performed using antiserum against a mixture of TTRs from a human, tammar wallabies, and chickens (see MATERIALS AND METHODS). Human serum was used as a positive control, koala serum was used to demonstrate cross-species recognition of the TTRs by the antiserum, and serum from Tasmanian devil [which does not contain TTR (45)] was used as a negative control. Background nonspecific binding in the globulin region was present for all four species, a strong signal corresponding to the TTR subunit was observed in the lanes containing serum from human and koala but not in the lanes containing serum from Tasmanian devil or adult X. laevis (Fig. 3A, left).

View larger version (61K): [in this window] [in a new window]   Fig. 3.   A, left: Western analysis for detection of TTR in adult X. laevis serum. Aliquots of 6 µl serum from adult X. laevis and, for comparison, aliquots of 2 µl serum from Tasmanian devil (S. harrisii), koala (P. cinerius), and human (H. sapiens) were separated in SDS-PAGE, then transferred onto a nitrocellulose membrane. Triple the volume of amphibian serum was analyzed (lower protein concentration compared with mammalian serum). Antiserum (1:5,000) had been generated against a mixture of purified TTRs from human, wallaby, and chicken. The secondary antibody (1:10,000) was horseradish peroxidase-linked anti-rabbit immunoglobulin, raised in sheep. Detection used an enhanced chemiluminescence kit (Amersham) and X-ray film. The positions of TTRs are indicated by a bracket. A, right: TTR mRNA expression in X. laevis tadpole liver during development. Total RNAs were prepared from tadpole liver at stages 53-54, 58-59, 60, 62-63, and 66 (lanes 1-5). Electrophoretically separated RNA (15 µg) was transferred to a nylon membrane and probed with a 32P-labeled 0.65-kb EcoR I fragment of xTTR cDNA (top) and with a 32P-labeled 0.35-kb fragment of the X. laevis elongation factor 1 (EF-1)-cDNA (middle). Hybridization was in 50% and 30% formamide hybridization buffer for TTR mRNA and EF-1-mRNA, respectively, at 42°C. The amounts of xTTR mRNA relative to those of EF-1-mRNA are shown below the autoradiograms (bottom). Three analyses were carried out for each stage. SE are indicated by vertical bars. MW, molecular weight. B: sites of TTR gene expression. Total RNA was prepared from livers (lane 1), heads (lane 2), and the rest of the body (lane 3) of X. laevis tadpoles, stage 57 to 59. An amount of 10 µg of RNA per track was subjected to Northern analysis. Digoxygenin (DIG)-labeled cRNAs for xTTR and X. laevis EF-1 were synthesized with T3 or T7 RNA polymerase after linearizing the plasmids with appropriate restriction enzymes. Hybridization was in 50% formamide hybridization buffer for TTR and EF-1 messages, respectively, at 65°C.

Levels of TTR mRNA in X. laevis tadpole liver during development. RNA was extracted from the livers of X. laevis tadpoles from the premetamorphic stage 53 to the end of the metamorphic climax stage 66 and hybridized in Northern analysis to xTTR cDNA (Fig. 3A, right top). For standardization, the filter was simultaneously analyzed with a cDNA probe for elongation factor 1 (Fig. 3A, right middle). Hybridization signals were quantitated by densitometry. The levels of TTR mRNA were found to reach a maximum at prometamorphic stages 58 to 59. Thereafter, they declined and became 1.1% of the maximum level at stage 66 at the end of metamorphic climax (Fig. 3A, right bottom). No signal was obtained for the RNA from the livers of adult X. laevis frogs (data not shown).

Sites of TTR gene expression. Total RNAs were isolated from livers, whole heads, and carcasses (bodies without livers and heads) of X. laevis tadpoles at prometamorphic stages 57-59. RNAs were separated by electrophoresis and transferred to a Hybond N-nylon membrane. Northern analysis was performed using ³²P-labeled xTTR cDNA (Fig. 3B) as a probe. A strong signal for TTR mRNA was detected in RNA isolated from tadpole liver (48-h exposure). In contrast, only an extremely faint TTR mRNA signal was detected in RNA isolated from tadpole heads, and no signal at all from RNA from carcasses without liver and head but including skin.

Synthesis of xTTR in Pichia pastoris. To obtain sufficient amounts of xTTR for characterization, a recombinant xTTR gene was constructed and cloned using two different signal sequences: the xTTR presegment and the -factor signal sequence. The two different plasmids constructed for the expression of xTTR are shown in Fig. 4. In plasmid pPIC3.5-xTTR, the xTTR cDNA had been placed under the control of the native AOX1 promoter at BamH I and EcoR I sites, and the xTTR presegment sequence was used in expression. The second plasmid was constructed by ligating the 5' end of xTTR cDNA to the 3' end of the -factor signal sequence in the vector pPIC9. [The -factor signal sequence synthesized in the yeast is removed within the Golgi by the KEX2 protease specific for dibasic amino acid sequences (28, 29). This is known to occur efficiently in P. pastoris (48).] The plasmids were linearized with SalI before transformation into the P. pastoris strain GS115. Twenty putative Pichia recombinants of each expression vector were selected and incubated for protein synthesis for 72 h. The analysis of the culture supernatant by SDS-PAGE and silver staining showed the efficient synthesis and secretion of a protein with a subunit molecular mass of ~16 kDa (data not shown). This value is consistent with the molecular mass of the TTR subunits from other species. The recombinant xTTRs from both plasmid constructs were purified by affinity chromatography on a human retinol-binding protein-Sepharose column. The purified TTR-like proteins were sequenced for the first five amino acids from the NH₂-terminus. They had the same sequence, namely alanine, proline, proline, glycine, and histidine. Apparently, the cleavage of the -factor signal sequence by KEX2 protease occurred at exactly the same site as that of the xTTR presegment by the signal peptidase in Pichia. Because the protein was expressed with similar efficiency in all of the 40 screened clones, one recombinant clone of each vector was selected for further characterization. Similar to TTRs from other vertebrates, the recombinant xTTR was found to bind specifically to retinol-binding protein (Fig. 5A). This feature was used for the purification of recombinant xTTR from the yeast culture. Approximately 4 mg of the recombinant xTTR were obtained from 1 liter of Pichia culture.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Construction of vectors for xTTR expression. Expression plasmids were constructed from pPIC3.5 using an xTTR presegment (A) and from pPIC9 using -factor signal sequence (B) for expression of the xTTR gene and secretion of xTTR. The figures show the sequence at the 5' end and the 3' end of xTTR after restriction sites were introduced by polymerase chain reaction. 5AOX1, promotor of P. pastoris alcohol oxidase 1 gene; 3(TT), native transcription termination and polyadenylation signal of alcohol oxidase 1 gene; 3AOX1, sequence from the alcohol oxidase 1 gene, 3' to the TT sequences; HIS4, histidinol dehydrogenase gene; Amp, ampicillin resistance gene; ColE1, E.coli origin of replication; S, -factor secretion signal; Sal I, Sal I restriction site for linearization of the vector.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Physicochemical properties of the X. laevis recombinant TTR. Physicochemical properties were analyzed to identify the recombinant xTTR produced by Pichia pastoris cells. These included binding to retinol-binding protein (RBP; A), as shown in the affinity chromatography of TTR on a human retinol-binding protein-Sepharose column [the xTTR was eluted by water (arrow)]; mobility in SDS-PAGE [B; the monomer of xTTR was expressed from pPIC3.5 (lane 2) and pPIC9 (lane 3) vectors; molecular mass markers are shown on lane 1]; purity and homogeneity of xTTR analyzed by mass spectrometry [C; the mass of xTTR monomer was found to be 15,013 Da]; molecular mass of xTTR [D; determined by fast-protein liquid chromatography in a Superose-12 column; calibration of column was with bovine serum albumin (68 kDa), ovalbumin (45 kDa), myoglobin (16.7 kDa), and cytochrome c (12 kDa); the calculated molecular mass of xTTR was 66 kDa (indicated by an arrow)]; and cross-reactivity with antiserum generated against TTRs from other species shown in Western analysis (E). Molecular mass markers (lane 1). Yeast culture (lane 2), pooled unbound fraction (lane 3), and pooled fractions eluted (lane 4) from the RBP-Sepharose column with water. Analysis was done by SDS-PAGE (15% polyacrylamide gel) followed by transferring proteins onto nitrocellulose membrane. Molecular mass markers were detected by Amido black. The primary antibody was a rabbit antiserum against a mixture of human, wallaby, and chicken TTRs (1:5,000). The second antibody was horseradish-peroxidase-conjugated sheep anti-rabbit immunoglobulin (1:10,000). Detection by enhanced chemiluminescence. The arrow indicates the position of the recombinant xTTR subunit.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Analysis of binding of recombinant xTTR to thyroid hormones. Scatchard plot for L-thyroxine (T4) binding (B), Scatchard plot for 3,5,3'-triiodothyronine (T3) binding (A), and competitive inhibition for T3 binding (C). One-hundred nanomoles of xTTR were incubated with [125I]T4 or [125I]T3 in the presence of varied concentrations of unlabeled hormone (see MATERIALS AND METHODS) at 4°C overnight. Free hormone was separated from the TTR-bound hormone by filtering the incubation mixture through a layer of methyl cellulose-coated charcoal. All corrections including those for nonspecific binding were applied before performing the Scatchard analysis. The affinity (Kd) of the recombinant xTTR for T4 (Fig. 6B) was 508 ± 34 nM and for T3 (Fig. 6A) 248 ± 19 nM. The ratio of Kd T3 to Kd T4 was 0.49. For competitive inhibition of [125I]T3 binding to the purified xTTR by thyronine analogs (Fig. 6C), purified xTTR was incubated with 0.1 nM [125I]T3 for 1 h at 4°C in the presence of various concentrations of D-T3 (), T3 (), 3, 3', 5-triiodothyroacetic acid (), or T4 ().

Evolution of the NH₂-terminus by changes of mRNA splicing. Changes in the primary structure of TTR occurred during evolution predominately within the first 10 amino acids from the NH₂-terminus (for reviews, see Refs. 54 and 55). The NH₂-terminal segment of the TTR subunit is longer and more hydrophobic in avian and reptilian than in eutherian TTRs. X. laevis TTR has an even longer and more hydrophobic NH₂-terminal region than avian and reptilian TTRs.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   The nucleotide and derived amino acid sequence of TTR precursor mRNA from X. laevis at the exon 1/intron 1 and intron 1/exon 2 borders compared with those from other vertebrates. The nucleotide sequences of the exon 1/intron 1 (A) and intron 1/exon 2 (B) border regions of TTR genomic DNA from X. laevis were determined as described in MATERIALS AND METHODS. The 5' and 3' splice sites of intron 1 of xTTR were aligned with those from human (38, 60), buffalo rat (3, 17, 18), mouse (62, 63), tammar wallaby (3), eastern grey kangaroo (3), stripe-faced dunnart (3), short-tailed grey opossum (3), and white leghorn chicken (3) TTR genes. The splice sites are indicated by thick double-headed arrows. The consensus recognition sequences for splicing (39) are indicated in bold above the position of the splice sites in human TTR precursor mRNA. Nucleotides identical with those in the consensus sequence for the 3' splice site branch point are underlined. Nucleotides in exons are in bold upper case; those in introns are in lower case. *, The same base is found in this position as in human TTR precursor mRNA. Negative numbers indicate amino acids of the human TTR presegment; positive numbers indicate amino acids of the mature protein. -, -, -, -, And - are introduced into the numbering of amino acid residues to indicate the 5 additional residues at the 5' end of exon 2 observed in xTTR, which are absent from human TTR. The NH2-terminal amino acids, determined by Edman degradation of the mature proteins or of the recombinant TTR, are indicated by a box open at the right end.

Evolution of the TTR gene. A most parsimonious phylogenetic tree was constructed from the TTR amino acid sequences available in protein data banks and the derived amino acid sequence of TTR from X. laevis. Sea bream TTR was used as the outgroup. The obtained most parsimonious tree is shown in Fig. 8. X. laevis and R. catesbeiana TTRs are in the same evolutionary branch. The close similarity of the bootstrap numbers for the pairs of rat and mouse and R. catesbeiana and X. laevis reflect the great similarity of TTR amino acid sequences in each of the pairs. TTRs in X. tropicalis and X. laevis or in other X. laevis species would be still more similar.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Phylogenetic tree resulting from maximum parsimony analysis of mature TTR amino acid sequences from 19 vertebrate species. The fish (Sparus aurata) TTR sequence was used as the out group. The consensus tree is shown with bootstrap numbers based on 500 replicates.

View larger version (40K): [in this window] [in a new window]   Fig. 9.   A: comparison of TTR sequences with amino acid sequences [deduced from open reading frames (ORF)] most similar to TTRs. Mature protein sequences from 16 vertebrate species, including representatives from eutherians, marsupials, birds, reptiles, and amphibians, were aligned (sources of the sequences other than xTTR can be found in Refs. 54, 55, and 67). Amino acid sequences (deduced from ORFs) most similar to TTRs were aligned below that of xTTR. Amino acids identical to those in xTTR are indicated by asterisks. Gaps were introduced to aid alignment. The numbering of amino acids is based on that of human TTR. Amino acids present in sequences from marsupials, birds, reptiles, and amphibians that are not present in human TTR are designated - to -. Features of secondary structure of human TTR are indicated above its sequence (-helix and -sheets), singly underlined residues are in the core of the subunit, and doubly underlined residues are in the thyroid hormone-binding channel (9). Positions that have an identical amino acid in 16 TTRs and the 5 ORFs (*) or in which all amino acids are similar (+) or in which only 2 amino acids are tolerated (^) are indicated. B: comparison of hydrophobicity plots of xTTR with ORFs from C. elegans, E. coli, and S. pombe. Kyte-Doolittle hydrophobicity plots for the region of xTTR with similarity to the ORFs in A (a), S. pombe ORF in A (b), C. elegans Z ORF in A (c), and E. coli ORF in A (d). A scanning window of 7 amino acid residues was used.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Evolution of TTR function and structure. The evolutionarily oldest feature essential for life is compartmentation, i.e., the concentration of components and reactions in a compartment separated from the extracellular environment by membranes (43). Through the use of imported energy, the increase in entropy in the cellular compartment can then be kept smaller than that of the environment. With the evolution of multicellular organisms, communication across cell membranes became necessary. Lipid-soluble compounds were the obvious choice for the transfer of information across cell membranes. The intracellular aggregation of the signal compounds with a receptor protein can then control the interaction of the signal compound with intracellular systems. The observation that the nuclear receptors for all thyroid and steroid hormones form a single large superfamily of proteins suggests that the development of these regulator proteins occurred only once during evolution.

Timing and sites of TTR gene expression. Adult animals that express the TTR gene in the liver are all endothermic (45, 55). Endotherms appeared relatively recently during evolution. In the choroid plexus of the brain, the TTR gene was expressed well before the endotherms, namely in turtles (46) and lizards (1). Adult toads (2, 23) and bullfrogs (67) lack TTR gene expression in both liver and brain. Apparently, adult amphibians do not possess TTR (2, 23). However, the synthesis of a TTR with an unusual structure of the NH₂-termini was observed during development in the liver of bullfrog tadpoles (66). The brain and, in particular, the choroid plexus of tadpoles expressed the gene for a lipocalin but not the gene for TTR (67). Only traces of TTR mRNA were observed in nonhepatic tissues of lizards (1) and of sea breams (53).

xTTR synthesis in Pichia pastoris and xTTR properties. Concerted evolution of TTR structure and deiodinase gene expression? The high yield of xTTR production by Pichia pastoris allowed a detailed analysis of the properties of xTTR. The recombinant xTTR was shown to have the size and tetrameric structure expected of TTRs. It bound to human retinol-binding protein (a typical feature of TTRs). The amino acid sequence in its NH₂-terminal region was identical to that derived from the xTTR cDNA (Fig. 1).

Mechanism of evolution of TTR. The analysis of the splice site between intron 1 and exon 2 of precursor xTTR mRNA shows a further shift toward the 5' end of TTR mRNA compared with avian precursor TTR mRNA (3). The observations reported here for the xTTR gene make it likely that successive shifts in splice sites could be an evolutionary mechanism of general importance. Such shifts could produce stepwise changes in the properties of proteins, such as the affinity for a ligand. A stepwise "improvement" of protein function could be the point of operation of selection pressure. This would be an example for a molecular mechanism of positive neo-Darwinian evolution.

Perspectives

The type of functional pressure postulated for positive selection in a Darwinian mechanism of evolution should be investigated. Presumably, it would involve proteins coded for by the ORFs. Efficient synthesis of recombinant TTR in the yeast P. pastoris suggests possibilities for searching for TTR-like precursor proteins in prevertebrate organisms and for studying synthesis and properties of such proteins coded for by TTR genelike ORFs. Whether such proteins would form tetramers, bind thyroid hormones, and, if so, bind T₃ more strongly than T₄ (similar to xTTR but different than mammalian TTRs) is an interesting question.

Finally, the data reported here for the tissue pattern of TTR gene expression in Xenopus support the hypothesis that the need for a special thyroid hormone-binding protein system in the brain evolved at the stage of the reptiles and did not yet exist in amphibians. The identification of the features in the "postamphibian" vertebrate brain requiring such a thyroid hormone-binding protein system will be an exciting topic for future work. It is likely to provide an in-depth understanding of TTR function.