The Evolution of the Thyroid Hormone Distributor Protein Transthyretin in the Order Insectivora, Class Mammalia

Porntip Prapunpoj1,*, Samantha J. RichardsonGo,*, Luca Fumagalli{dagger} and Gerhard Schreiber*

*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia; and
{dagger}Institut d'Ecologie, Laboratoire de Zoologie et d'Ecologie Animale, Bâtiment de Biologie, Lausanne, Switzerland

Abstract

Thyroid hormones are involved in the regulation of growth and metabolism in all vertebrates. Transthyretin is one of the extracellular proteins with high affinity for thyroid hormones which determine the partitioning of these hormones between extracellular compartments and intracellular lipids. During vertebrate evolution, both the tissue pattern of expression and the structure of the gene for transthyretin underwent characteristic changes. The purpose of this study was to characterize the position of Insectivora in the evolution of transthyretin in eutherians, a subclass of Mammalia. Transthyretin was identified by thyroxine binding and Western analysis in the blood of adult shrews, hedgehogs, and moles. Transthyretin is synthesized in the liver and secreted into the bloodstream, similar to the situation for other adult eutherians, birds, and diprotodont marsupials, but different from that for adult fish, amphibians, reptiles, monotremes, and Australian polyprotodont marsupials. For the characterization of the structure of the gene and the processing of mRNA for transthyretin, cDNA libraries were prepared from RNA from hedgehog and shrew livers, and full-length cDNA clones were isolated and sequenced. Sections of genomic DNA in the regions coding for the splice sites between exons 1 and 2 were synthesized by polymerase chain reaction and sequenced. The location of splicing was deduced from comparison of genomic with cDNA nucleotide sequences. Changes in the nucleotide sequence of the transthyretin gene during evolution are most pronounced in the region coding for the N-terminal region of the protein. Both the derived overall amino sequences and the N-terminal regions of the transthyretins in Insectivora were found to be very similar to those in other eutherians but differed from those found in marsupials, birds, reptiles, amphibians, and fish. Also, the pattern of transthyretin precursor mRNA splicing in Insectivora was more similar to that in other eutherians than to that in marsupials, reptiles, and birds. Thus, in contrast to the marsupials, with a different pattern of transthyretin gene expression in the evolutionarily "older" polyprotodonts compared with the evolutionarily "younger" diprotodonts, no separate lineages of transthyretin evolution could be identified in eutherians. We conclude that transthyretin gene expression in the liver of adult eutherians probably appeared before the branching of the lineages leading to modern eutherian species.

Introduction

All mammals probably descended from small insectivorous species living in the Cretaceous period about 100 MYA. Marsupials and placental mammals both probably originated from a small Cretaceous shrewlike form (for review, see Young 1981Citation , p. 431). The disappearance of the dinosaurs at the end of the Cretaceous period about 65 MYA led to the "evolutionary explosion" of mammalian species (see Colbert and Morales 1991Citation , p. 262). Small insectivorous mammals such as shrews, hedgehogs, and moles maintained in their morphological structure some traits probably already present in their reptilelike mammalian ancestors. The size of the brain remained relatively small. The tympanic bone in the insectivores still forms a simple ring around the middle area, unlike the large capsulelike structure found in higher mammals. In modern representatives of the order Insectivora, teeth and feet appear to have also retained many of the structures typical of their insectivore ancestors (for details see Colbert and Morales 1991Citation , p. 263).

In the last 10 years, new insight has been obtained into the evolution of the distribution system for thyroid hormones in vertebrates (for review, see Schreiber and Richardson 1997Citation ). Thyroid hormones are involved in the regulation of growth, differentiation, and metabolism. They are more soluble in lipid membranes than in water. It is necessary to prevent the permeation into and subsequent accumulation of thyroid hormones in membranes. This permeation would lead to the depletion of thyroid hormones from the circulating medium in the vascular compartment. For example, it has been shown with in vitro perfused rat liver that the addition of thyroid hormone–binding proteins is necessary to counteract the disappearance of thyroid hormones from the perfusion medium and to ensure the uniform distribution of thyroid hormones throughout the liver lobule (Mendel et al. 1987Citation ). Three such thyroid hormone–binding proteins are found in the blood of vertebrates: thyroxine-binding globulin, transthyretin (TTR), and albumin. Thyroxine-binding globulin is found only in the blood of larger eutherians (Larsson, Pettersson, and Carlström 1985Citation ). Albumin occurs in the blood of all vertebrates. TTR shows very characteristic changes during the evolution of vertebrates. These changes fall into two groups, namely, those in the tissue pattern of TTR gene expression and those in TTR gene structure. In Australian polyprotodont marsupials, reptilians, monotremes, amphibians, and fish, TTR was not found in the blood of adult individuals (Achen et al. 1992Citation ; Richardson et al. 1994, 1997Citation ). Most likely, expression of the TTR gene in the liver of adult animals appeared relatively late and independently in the vertebrate lineages leading to eutherians, birds, and diprotodont marsupials (for review, see Schreiber and Richardson 1997Citation ). During development, but not during adulthood, when thyroid hormone levels are high, the TTR gene is also expressed in the liver of tadpoles (Yamauchi et al. 1998Citation ) and fish (Santos and Power 1999Citation ). To our knowledge, a systematic study of the expression of the TTR gene during the development of reptiles or diprotodont marsupials has not yet been reported. Here, we investigated whether the TTR gene was expressed in the liver of adult individuals representing several families of the order Insectivora. Modern Insectivora species are most similar in morphology to the common insectivorelike ancestors of all eutherians.

The structure of TTR is well conserved in vertebrate species. An exception is the N-terminal region of the TTR subunit. For this segment of TTR, which is located at the entrance of the tunnel containing the thyroid hormone–binding sites, a systematic change can be found, from a longer, relatively hydrophobic N-terminal region of the TTR subunit of reptiles and birds to a shorter, more hydrophilic N-terminal region in the TTR subunits of the eutherians. The structure of the subunit in marsupials is midway between reptiles/birds and eutherians in character (for review, see Schreiber and Richardson 1997Citation ). Here, we report the identification of TTR in serum from hedgehogs, shrews, and moles and the cloning and structural analysis of the TTR cDNA for hedgehogs and shrews, deduce the amino acid sequence, and determine the position of TTR in Insectivora in the pattern of vertebrate TTRs.

A series of stepwise single base mutations at the splice site between intron 1 and exon 2 has been suggested as a possible mechanism for the evolution of the N-terminal region of the TTR subunit (Aldred, Prapunpoj, and Schreiber 1997Citation ). Therefore, the base sequence of genomic DNA in the region coding for the splice sites at the flanks of the first intron of precursor TTR mRNA was determined for hedgehogs and shrews. The obtained data give a clear answer about the position of modern insectivore species in the evolution of the splicing of precursor TTR mRNA.

Materials and Methods

Chemicals, Serum, Genomic DNA, and RNA Sources
L[125I]-thyroxine (1.2 Ci/mg) was obtained from NEN Dupont, SepPak C-18 cartridges from Millipore Waters, thin-layer chromatography plates from Merck, the Enhanced Chemiluminescence kit from Amersham, anti-rabbit Ig raised in sheep from Silenus, Melbourne, Australia, and X-ray film from Eastman-Kodak. All reagents were of analytical grade.

Human (Homo sapiens) serum was obtained from one of the investigators (G.S.); Sorex ornatus californicus serum was obtained from J. Patton, Museum of Vertebrate Zoology, University of California at Berkeley; Talpa europaea serum was obtained from P. Licht, University of California at Berkeley; Sorex araneus serum and livers were obtained from P. Vogel, University of Lausanne, Switzerland; and Erinaceus europaeus serum, genomic DNA, and total liver RNA were obtained from R. Lawn, Stanford, Calif.

Preparation of L[125I]-Thyroxine
Commercially available L[125I]-thyroxine was found to contain up to 5% 125I on the reference date. Therefore, 125I-thyroxine was separated from 125I and other degradation products by reversed phase chromatography using a SepPak C-18 cartridge column (Mendel et al. 1989Citation ), and the purification was checked by thin-layer chromatography (Pardridge and Mietus 1980Citation ).

Determination of Thyroxine-Binding Proteins in Serum
For each species analyzed, 10 µl of serum was incubated with 1 fmol 125I-thyroxine (2.4 nCi) at room temperature for 1 h. Two aliquots (5 and 2 µl) were analyzed in the same nondenaturing 10% polyacrylamide gel and 0.05 M Tris-HCl (pH 8.9) at 4°C (for details, see Richardson et al. 1994Citation ). Following electrophoresis, lanes containing 5 µl of serum were subjected to autoradiography to detect the positions of 125I-thyroxine bound to serum proteins, and lanes containing 2 µl serum were stained with Coomassie Brilliant Blue to visualize the positions of migration of the serum proteins.

Western Analysis for the Presence of Transthyretin in Serum
For each species analyzed, 2 µl of serum was separated in a 0.1% SDS polyacrylamide gel, with a stacking gel of 4.5% acrylamide (pH 6.8) and a resolving gel of 15% acrylamide (pH 8.6) (Laemmli and Favre 1973Citation ), then transferred onto a nitrocellulose membrane. Nonspecific binding sites on the membrane were blocked with skimmed milk diluted with phosphate buffer. The primary antibody was 1:5,000 antiserum raised in a rabbit against a mixture of transthyretins purified from serum from a human (H. sapiens), Tammar wallabies (Macropus eugenii) and chickens (Gallus gallus). The secondary antibody was 1:10,000 anti-rabbit Ig. Enhanced chemiluminescence against X-ray film was used for detection.

Purification of Hedgehog TTR
TTR was purified from hedgehog (E. europaeus) serum as described previously for the purification of TTR from mountain brushtail possum serum (Richardson et al. 1994Citation ). Briefly, 500 µl of hedgehog serum which had been incubated with a tracer amount of 125I-thyroxine was subjected to electrophoresis in a nondenaturing 10% polyacrylamide gel (pH 8.6) at 4°C. After exposure to X-ray film at -70°C, the gel was thawed and the region corresponding to the signal of 125I-thyroxine bound to TTR was excised. The gel was homogenized in 0.5 M sodium phosphate and 0.15 M NaCl (pH 7.4) at 4°C, diluted to 40 ml with the same buffer, and rotated for extraction for 2 days per extraction for a total of two extractions. The supernatants were pooled and concentrated using an Amicon Diaflo apparatus with a YM-10 membrane and then using an Amicon Centricon-10 ultrafiltration unit. The method for determination of the amino acid sequence of the purified protein was described in Duan et al. (1991)Citation .

Cloning and Sequencing of TTR cDNA from Hedgehogs
Total RNA was isolated from hedgehog (E. europaeus) liver by extraction of the homogenate with guanidinium thiocyanate-phenol-chloroform as described by Chomczynski and Sacchi (1987)Citation . Polyadenylated RNA was purified from total RNA by affinity chromatography using oligo dT30 nucleotides covalently bound to polystyrene latex beads (oligotex mRNA kit from Qiagen). The first cDNA strand was synthesized with avian myeloblastosis virus reverse transcriptase. After treatment with RNase H, the second cDNA strand was synthesized with Escherichia coli DNA polymerase I. A hedgehog liver cDNA library was then constructed in {lambda}gt11 according to the method of Young and Davis (1983a, 1983bCitation ). The library was screened with 32P-labeled rat TTR cDNA (Duan, Cole, and Schreiber 1989Citation ). Four positive clones with an insert size of 0.6 kb were selected for sequence determination. Polymerase chain reaction using the recombinant bacteriophage DNA as a template, {lambda}gt11 forward (5'-GGTGGCGACGACTCCTGGAGCCCG-3') as 5' primer, {lambda}gt11 reverse (5'-TTGACACCAGACCAACTGGTAATG-3') as 3' primer, and AmpliTherm DNA polymerase (Epicentre Technologies) was carried out to amplify an insert fragment. The entire sequence of cDNA was determined for both strands by the dideoxynucleotide chain termination method (Sanger, Nicklen, and Coulson 1977Citation ) using a kit with T7 Sequenase DNA polymerase (Amersham).

Cloning and Sequencing of TTR cDNA from Shrews
RNA was extracted from shrew liver homogenate, polyadenylated RNA was prepared and transcribed into cDNA, and double-stranded cDNA was synthesized as described above for the hedgehog system. A cDNA library was constructed in phage vector {lambda}MOSElox according to the method described by Palazzolo et al. (1990)Citation using the cDNA rapid-cloning module {lambda}MOSElox (Amersham, RPN1716). Phage recombinants were subcloned in vivo by infection of E. coli ER1647. Then, the library was screened for the TTR cDNA with 32P-labeled rat TTR cDNA. The shrew TTR cDNA was determined for both strands by the dideoxynucleotide chain termination method as mentioned above.

Splicing of the Precursor TTR mRNA in Insectivore Mammals
Genomic DNA of the shrew was prepared from an adult liver by phenol extraction of tissue homogenate, as described by Sambrook, Fritsch, and Maniatis (1989)Citation . Polymerase chain reactions were carried out to amplify TTR genomic DNA fragments containing the exon 1/intron 1 and intron 1/exon 2 regions. Oligonucleotide primers consisting of transthyretin cDNA segments near the 3' end of exon 1 (5'-ACCGTCTATTCCTCCTTTGCCTTG-3' for hedgehogs or 5'-CGCCTCCTTCTCCTCTGCCTGG-3' for shrews) and complementary to sequences near the 5' end of exon 2 (5'-ATACTTTCACAGCCACATTGACTG-3' for hedgehogs or 5'-GGACTGCCTTGAACAGCATTTAGG-3' for shrews) were used in the polymerase chain reactions. Polymerase chain reactions were carried out in 0.1 ml reaction mixture containing 1,000 ng of genomic DNA, 10 pmol of each primer, and 0.5 U of Taq DNA polymerase. Amplification started with denaturation for 5 min at 94°C, followed by 30 cycles of annealing at 55°C for 30 s, extension at 72°C for 1 min, and denaturation at 94°C for 30 s. A final amplification step was performed for extension at 72°C for 5 min. The polymerase chain reaction mixture was analyzed in a 1% "low-melting" agarose gel and purified by phenol/chloroform extraction (Sambrook, Fritsch, and Maniatis 1989Citation ).

Synthesis of Shrew TTR in Pichia pastoris
BamHI and EcoRI sites were introduced by polymerase chain reaction into shrew TTR cDNA such that the cleavage by BamHI occurred immediately before nucleotide 2, and that by EcoRI occurred immediately after the stop codon TGA (positions 443–445) of the shrew TTR cDNA. The PCR product was ligated with the Pichia expression vector pPIC3.5. The recombinant vector was linearized with SalI and introduced into P. pastoris strain GS115 by electroporation. The synthesis of the recombinant shrew TTR was carried out in glycesol-containing medium. Cells were grown and induced with methanol (5% final concentration) at 30°C for 3 days.

Determination of N-Terminal Sequence of the Recombinant Shrew TTR
To determine the N-terminal sequence of shrew TTR, the shrew TTR gene was expressed in P. pastoris. TTR secreted into the medium was purified by affinity column chromatography on human retinol-binding protein coupled to Sepharose 4B (Larsson, Pettersson, and Carlström 1985Citation ). The N-terminal amino acid sequence of the recombinant TTR was determined on a commercial basis by the Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia.

Parsimony Analysis
The program PAUP*, version 4.0b2 (Swofford 1999Citation ), was employed to perform maximum-parsimony analysis from 17 complete (i.e., including presegment) TTR amino acid sequences (corresponding to a total number of 381–459 nucleotides), with the sequence of sea bream (Sparus aurata) transthyretin as an outgroup. The two forms of serine, whose codons are not adjacent in the genetic code, were considered two different states, and gaps were treated as additional character states. Tree bisection-reconnection branch swapping with 1,000 random input orders was used. Support for specific nodes was evaluated by bootstrap analysis with 1,000 replicates.

Results

Analysis of Thyroxine-Binding Proteins in Serum from Insectivora
Albumin has been identified as a thyroxine distributor protein in serum of adult individuals from 150 species of vertebrates (Schreiber et al. 1993Citation ; Richardson et al. 1994Citation ). TTR was found in serum from adult eutherians and birds and in serum from adult Australian diprotodont marsupials, but not in serum from adult polyprotodont Australian marsupials (Richardson et al. 1994Citation ). The question arose as to when, along the lineages leading to eutherians, diprotodont marsupials, and birds, transthyretin gene expression in the liver of adult individuals first appeared. We suggested that TTR gene expression first appeared in the livers of the diprotodont marsupials, shortly after the branching of the lineages leading to the Diprotodonta and the Polyprotodonta (Richardson et al. 1994Citation ). We found TTR in serum of ratites, the group of extant birds which diverged earliest from the avian lineage (Chang et al. 1999Citation ). Here, we investigated TTR synthesis in the liver of adult individuals from several species in the eutherian order Insectivora. The modern extant Insectivora are believed to be most similar in morphology to the insectivorous mammal-like common ancestors of the eutherians. We analyzed serum from four species of the order Insectivora (hedgehogs, two shrew species, and moles) for their patterns of thyroxine distributor proteins and, specifically, for the presence of TTR.

To investigate the thyroxine distributor proteins in the blood of adult Insectivora, serum from shrews (two species: S. araneus and S. ornatus californicus), moles (T. europaea), and hedgehogs (E. europaeus) was analyzed for the presence of thyroxine-binding proteins by electrophoresis followed by autoradiography. For comparison, human serum was included in the analysis. The results revealed albumin to be a thyroxine distributor protein in each species studied (fig. 1A) . Serum from the mole had TTR in addition to albumin, and serum from the hedgehog had albumin, TTR, and two proteins migrating as globulins, which bound 125I-thyroxine. The serum sample from both species of shrew showed only a single band for proteins binding 125I-thyroxine. The positions of migration of these bands were similar to those of the respective albumins. It is possible that these bands also contain TTR. Human serum showed the presence of albumin, TTR, and thyroxine-binding globulin.



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Fig. 1.—Analysis of thyroxine distributor proteins in serum of species in the order Insectivora. A, Identification of thyroxine-binding proteins in serum. Aliquots of serum from a human (Homo sapiens), shrews (Sorex araneus and Sorex ornatus californicus), a mole (Talpa europaea), and a hedgehog (Erinaceus europaeus) were incubated with a tracer amount (0.6 fmol) of 125I-thyroxine at room temperature for 1 h prior to separation in a nondenaturing polyacrylamide gel (pH 8.6). Duplicate samples were analyzed: 2 µl for staining of proteins with Coomassie Brilliant Blue (Coo) and 5 µl for detection of binding of 125I-thyroxine to proteins by autoradiography (AR). The positions of albumins (open triangles), TTRs (filled-in triangles), and thyroxine-binding globulin (asterisk) are indicated. B, Western analysis of serum for the presence of TTR. Aliquots of 2 µl serum from a human, shrews (S. araneus and S. ornatus californicus), a mole (T. europaea), and a hedgehog (E. europaeus) were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. Antiserum had been generated against a mixture of purified TTRs from human, wallaby, and chicken sera. The positions of TTRs are indicated by a bracket. Molecular weight markers were from Pharmacia: phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and {alpha}-lactalbumin (14,400). Bands in the high-molecular-weight region are due to nonspecific binding of the antibody

 
In some eutherian species (e.g., pig and cow) TTR comigrates with albumin in nondenaturing gels. To confirm the presence of TTR in serum indicated by binding of 125I-thyroxine to proteins, serum samples from two species of shrew (S. araneus and S. ornatus californicus), a mole (T. europaea), and a hedgehog (E. europaeus) were then analyzed for the presence of transthyretin by Western blotting. A signal corresponding to TTR was evident in lanes containing serum from a human, shrews, a mole, and a hedgehog (fig. 1B ).

Purification and Identification of N-Terminal Amino Acid Residues of Hedgehog TTR
It is not possible to unambiguously identify the border between the presegment and the mature protein from nucleotide sequence data. The beginning of the polypeptide chain has to be determined by analysis of the protein. Therefore, TTR was purified from serum of hedgehogs as described in Materials and Methods. An attempt was made at sequencing from the N-terminus by Edman degradation. The second amino acid was found to be proline. No unambiguous sequence could be obtained following the proline residue.

Cloning and Sequencing of TTR cDNA from Hedgehogs
A hedgehog liver cDNA library was constructed and screened for TTR cDNA clones as described in Materials and Methods. Four positive clones 0.6 kb long were selected for sequencing. The entire nucleotide sequence of hedgehog TTR cDNA was determined for both strands with overlapping fragments. The TTR cDNA clone is 595 nt in length, followed by 34 polyadenylate residues with one G interrupting at position 606. The start codon ATG is present 16 bases downstream of the 5' end of the cDNA while the first stop codon, TGA, is located at bases 451–453. The whole coding sequence is 435 nt long (GenBank accession number AF251940). Three additional in-frame stop codons were found in the 3' untranslated region of the cDNA. One of these, TAA, is located in the consensus polyadenylation sequence AATAAA at 12 bp upstream of the polyA segment. The deduced amino acid sequence of hedgehog TTR cDNA was aligned with that of human TTR. By comparison with the amino acid sequence of human TTR, two amino acid residues were "missing" from the first 10 amino acid residues at the N-terminus of hedgehog mature TTR. The molecular mass of the hedgehog TTR subunit, calculated from the amino acid sequence deduced from the cDNA sequence, is 15,844.

Cloning and Nucleotide Sequence Analysis of Shrew TTR cDNA
RNA was extracted from shrew (S. araneus) liver and a cDNA library, and TTR cDNA clones were prepared as described in Materials and Methods. DNA was prepared from four isolated plasmid clones. T7 gene10 primer, T7 terminator primer, and two internal primers were used to determine the sequences for both strands of the insert. TTR cDNA obtained from shrew liver consisted of 553 nt (GenBank accession number AJ223149). The reading frame started with ATG at nucleotides 2–4, was followed by an open reading frame of 437 nt, and ended with stop codon TGA at nucleotides 443–445. Shrew TTR cDNA has the same number of nucleotides coding for the mature protein as human TTR. TTR cDNA from shrews contained only one additional in-frame stop codon in an untranslated region preceding the polyA segment, and this was located in the consensus polyadenylation sequence ATTAAA, which is 16 bp upstream of the 5' end of the polyadenylate segment. The molecular mass of the shrew TTR deduced from the cDNA is 15,943.

Identification of the N-Terminal Amino Acid Sequence of Shrew TTR
For the unambiguous identification of the synthesized protein and the location of the presegment/mature protein border, recombinant shrew TTR was purified from the yeast culture supernatant by affinity chromatography, and the N-terminal amino acid sequence was determined as described in Materials and Methods. The first five amino acids obtained were glycine, proline, threonine, glycine, and threonine.

Splicing of the TTR Precursor mRNA in Hedgehogs and Shrews
The nucleotide sequence of the genomic TTR DNA was determined in the region coding for the 5' end of hedgehog and shrew TTR precursor mRNAs. Oligonucleotide primers corresponding to sequences near the exon 1/exon 2 border of the TTR cDNAs were designed. They were used in a polymerase chain reaction using genomic DNA as the template, leading to the synthesis of DNA segments containing the exon 1/intron 1 and the intron 1/exon 2 regions of insectivore TTR precursor mRNAs. The comparison of the genomic DNA nucleotide sequence with the TTR cDNA sequences gave the locations of the splice sites.

In order to obtain maximum amino acid sequence alignment of hedgehog TTR with human TTR, two gaps were inserted after amino acid position 3. The splice site at the 3' end of intron 1 of hedgehog and shrew TTR genes occurred in the position -{alpha} at the 5' end of exon 2 (fig. 2A ). Apparently, a single base change from U to G changed the histidine codon, CAU, in position -{alpha} of marsupials into the 3' splice site recognition sequence CAG during evolution of eutherian TTR from a more marsupial TTR-like protein. In hedgehog TTR precursor mRNA, the splice site is located between amino acid positions 3 and 6. Splicing at the 5' end of intron 1 of TTR precursor mRNAs from both hedgehogs and shrews was found to occur at the site corresponding to amino acid position 3 of the mature protein (fig. 2B ), identically to previously studied vertebrate species (Aldred, Prapunpoj, and Schreiber 1997Citation ).



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Fig. 2.—Nucleotides flanking exon 1/exon 2 splice sites of TTR mRNA in Insectivora and other vertebrates. The nucleotide sequences of the intron 1/exon 2 (A) and exon 1/intron 1 (B) border regions of TTR genomic DNA from hedgehogs and shrews were determined as described in Materials and Methods. The 5' and 3' splice sites of intron 1 of hedgehog and shrew TTR precursor mRNAs were aligned with those from human (Mita et al. 1984Citation ; Tsuzuki et al. 1985Citation ), buffalo rat (Fung et al. 1988Citation ; Duan, Cole, and Schreiber 1989Citation ; Aldred, Prapunpoj, and Schreiber 1997Citation ), mouse (Wakasugi et al. 1985Citation ; Wakasugi, Maeda, and Shimada 1986Citation ), Tammar wallaby (Aldred, Prapunpoj, and Schreiber 1997Citation ), eastern gray kangaroo (Aldred, Prapunpoj, and Schreiber 1997Citation ), stripe-faced dunnart (Aldred, Prapunpoj, and Schreiber 1997Citation ), short-tailed gray opossum (Aldred, Prapunpoj, and Schreiber 1997Citation ), and white leghorn chicken TTR precursor mRNAs (Aldred, Prapunpoj, and Schreiber 1997Citation ). The splice sites are indicated by two-ended arrows. The consensus recognition sequences for splicing (Moore, Query, and Sharp 1993Citation ) are indicated in bold above the positions of the splice sites in human TTR precursor mRNA. Nucleotides identical to those in the consensus sequences for the 3' splice site branch point are underlined. Nucleotides in exons are in bold uppercase letters; those in introns are in lowercase letters. The deduced amino acid sequences are given below the nucleotide sequences. Asterisks indicate that the same base is found in a 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. -{alpha}, -ß and -{gamma} were introduced into the numbering of amino acid residues to indicate the additional residues at the 5' end of exon 2 observed in chicken TTR which are absent from human TTR (Aldred, Prapunpoj, and Schreiber 1997Citation ). The N-terminal amino acids, determined by Edman degradation of the mature proteins, are indicated by a box open at the right end

 
Comparison of the Structures of TTR Genes in Vertebrates
The derived amino acid sequences of TTRs from hedgehogs (E. europaeus) and shrews (S. araneus) were aligned with the sequences of TTRs from 24 vertebrate species (fig. 3 ). Hedgehog TTR had the residues at positions 4 and 5 (numbering for human TTR sequence) "missing." The comparison of hedgehog and shrew mature TTR amino acid sequences with those from other species revealed the following relative identities: humans, 87% and 86%; pigs, 87% and 85%; sheep, 87% and 85%; rabbits, 86% and 84%; rats, 85% and 85%; mice, 83% and 82%; wallabies, 72% and 69%; kangaroos, 71% and 69%; sugar gliders, 71% and 69%; dunnarts, 70% and 68%; South American gray opossums (Monodelphis domestica), 72% and 70%; chickens, 75% and 72%; lizards, 68% and 67%; bullfrogs, 60% and 58%; and seabreams, 52% and 50%, respectively.



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Fig. 3.—Comparison of the structures of TTRs in vertebrates. Amino acid sequences and those derived from cDNA sequences are aligned for 24 species (17 complete and 7 N-terminal sequences). The sequence for human TTR is written using the single-letter amino acid abbreviation. X indicates that an amino acid could not be unambiguously identified by Edman degradation. Those residues in other species with identical amino acids are indicated by asterisks. Gaps were introduced to aid alignment. Features of secondary structure of human TTR are indicated above the sequences. The numbering of residues is based on that for human TTR: negative numbers refer to residues in the presegment, positive numbers represent residues of the mature protein; -{alpha}, -ß, and -{gamma} were introduced to indicate positions of residues in noneutherian species. Singly underlined residues are in the core of the subunit. Doubly underlined residues are in the central thyroid hormone–binding site. # = site of exon boundary. The sources of the TTR sequences are as follows: human (Mita et al. 1984Citation ), rhesus monkey (van Jaarsveld et al. 1973Citation ), pig (Duan et al. 1995bCitation ), sheep (Tu et al. 1989Citation ), rabbit (Sundelin et al. 1985Citation ), rat (Duan, Cole, and Schreiber 1989Citation ), mouse (Wakasugi et al. 1985Citation ), wallaby (Macropus eugenii; Brack et al. 1994Citation ; Richardson et al. 1994Citation ), kangaroo (Macropus giganteus; Schreiber et al. 1993Citation ; Aldred, Prapunpoj, and Schreiber 1997Citation ), brushtail possum (Trichosurus caninus; Richardson et al. 1994Citation ), sugar glider (Petaurus breviceps), southern hairy-nosed wombat (Lasiorhinus latifrons; Richardson et al. 1994Citation ), stripe-faced dunnart (Sminthopsis macroura), South American short-tailed gray opossum (Monodelphis domestica; Duan et al. 1995aCitation ), opossum (Didelphis virginiana; Richardson, Wettenhall, and Schreiber 1996Citation ), chicken (Duan et al. 1991Citation ), pigeon, emu, and ostrich (Chang et al. 1999Citation ), shingleback lizard (Tiliqua rugosa; Achen et al. 1993Citation ), bullfrog (Rana catesbeiana; Yamauchi et al. 1998Citation ), and seabream (Sparus aurata; Santos and Power 1999Citation )

 
Discussion

The earliest placental mammals seem to have appeared during the Cretaceous period, only a little later than the dinosaurs. They were, and remained, small insectivorous species until the demise of the dinosaurs and the beginning of the radiation of mammals at the end of the Cretaceous and the early Tertiary. The fossil record suggests that shape, size, and bone structure of the early Insectivora species were not too different from those of the small Insectivora species living today. A decisive selection advantage favoring evolution of mammals might have been endothermy, i.e., the capability of maintaining a relatively constant body temperature, independent of that of the environment. Endothermy is also the common feature of most vertebrate species hitherto known to express the TTR gene in the liver of adult individuals. In monotremes and some polyprotodont marsupials, which do not show TTR gene expression in the liver of adults although they are endothermic, body temperatures are lower, and there is a greater tendency for a decrease in body temperature in response to a colder environment (Nardone, Wilber, and Musacchia 1952;Citation Grigg, Augee, and Beard 1992Citation ) than in eutherians with a greater capacity for heat production (standard metabolisms are 142.3 and 203.7 kJ/kg0.75 in monotremes and marsupials, respectively, compared with 288.8, 347.4, and 598.5 kJ/kg0.75 in eutherians and nonpasserine and passerine birds, respectively; Dawson and Hulbert 1970Citation ). It is likely that the synthesis of TTR in adult liver was one of the early events in the evolution of eutherians. The results reported here show that all studied modern Insectivora possessed substantial amounts of TTR in their blood, indicating the synthesis of TTR in their liver. In contrast to the situation for marsupial species, an eutherian species lacking TTR gene expression in liver was not found.

After the cloning of TTR cDNA from the hedgehog and the shrew, the nucleotide sequence of the TTR gene could be determined and the primary structure of the TTR subunit could be predicted. Southern analysis of rat genomic DNA and cloned TTR cDNA had shown earlier that the TTR gene was probably present in rats as a single gene copy (Fung et al. 1988Citation ). There is only one TTR gene locus on chromosome 18 in humans (Wallace et al. 1985Citation ; Loughna, Bennett, and Moore 1995Citation ; Yankowitz et al. 1998Citation ), on chromosome 4, region C6-D1, in mice (Qiu, Shimada, and Cheng 1992Citation ), and on chromosome 24 in bovines (Larsen, Womack, and Kirkpatrick 1996Citation ). Identical nucleotide sequences were obtained for TTR cDNA clones from cDNA libraries prepared from either rat brain or rat liver (Duan, Cole, and Schreiber 1989Citation ). Therefore, using TTR sequences for the mature proteins obtained from either liver- or brain-derived cDNA libraries from 17 species, a maximum-parsimony search was performed with seabream TTR as the outgroup. Three equally most-parsimonious trees of 469 steps resulted from the parsimony analysis. The bootstrap 50% majority-rule consensus tree (1,000 replicates with random addition of sequences) is shown in figure 4. The tree and its confidence values show that all eutherians are monophyletic, including the Insectivora. It is interesting that the position of lizard (Tiliqua rugosa) TTR is significantly closer to avian and mammalian TTRs than to bullfrog TTR.



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Fig. 4.—Maximum-parsimony search for 17 TTR amino acid sequences in vertebrates. The 17 complete amino acid sequences of the TTRs (including the presegment sequences) presented in figure 3. were used for calculation of the most-parsimonious trees. Seabream (Sparus aurata) served as an outgroup. A strict consensus tree of the three most-parsimonious (469-step) trees is shown. Numbers indicate bootstrap percentages based on 1,000 replicates. Branch lengths are not drawn to scale. "JL" indicates the appearance of the expression of the TTR gene in the juvenile liver during development, "CP" indicates the first appearance of TTR gene expression in the choroid plexus, and "AL" indicates the presence of TTR gene expression in the liver of adult individuals

 
It is also interesting that the TTR gene is expressed in the liver of the outgroup (seabream) and the evolutionarily oldest species of the tree (bullfrog) only transiently during development. In adult animals, TTR gene expression appeared in the choroid plexus at a much earlier stage (branching into birds/reptiles and marsupials/eutherians) than TTR gene expression in adult liver (eutherians and marsupials). The fact that birds, diprotodont marsupials, and eutherians are linked by tree segments leading to species not showing TTR gene expression in adult liver suggests that TTR gene expression in adult liver evolved independently in at least three instances.

Construction of a yeast expression vector containing TTR cDNA in P. pastoris allowed the synthesis of enough shrew TTR for determination of the N-terminal amino acid sequence. Previously, we showed that the N-terminal region of TTR has the greatest rate of change during the evolution of TTR in mammals. The comparison of the N-terminal region of insectivore TTRs with that of reptilian/bird and eutherian TTRs showed that the TTR gene and the protein in insectivores is shorter and more hydrophilic, i.e., more "eutherian-like" than "marsupial-like."

The splice site for shrew precursor TTR mRNA was found to be similar to that in other eutherians. The process of shortening the N-terminal region of the TTR subunit by moving the splice site between exon 1 and 2 in the 3' direction has gone even further for hedgehog TTR than for human, rat, and mouse TTRs.

In summary, both gene expression pattern and structure of TTR in Insectivora are very similar to those in other eutherians. The hedgehog TTR gene, for example, is not more closely related to a putative mammalian ancestor TTR gene than is the human TTR gene. Marsupial TTR genes, particularly those from polyprotodont marsupials, are probably more similar to the common ancestor TTR gene than are the Insectivora TTR genes.

Conclusions

There has been an increase in extracellular thyroid hormone distributor proteins, for both the number of proteins and their concentration in blood, during the evolution of the vertebrates. Fish, amphibians, reptiles, and some polyprotodont marsupials have albumin as their only thyroid hormone distributor protein in serum, whereas birds, eutherians, diprotodont marsupials, and some American polyprotodont marsupials have TTR in addition to albumin (Richardson et al. 1994Citation ; Richardson, Wettenhall, and Schreiber 1996Citation ). A third thyroid hormone distributor protein, thyroxine-binding globulin, is found in "larger eutherians" (Larsson, Pettersson, and Carlström 1985Citation ). Some marsupial species have a globulin which binds thyroxine, but the identity of this globulin is not known (Richardson et al. 1994Citation ). In each case, the additional distributor protein has had higher affinity for thyroid hormones than have the existing distributor proteins (i.e., TTR has higher affinity for thyroid hormones than albumin, and thyroxine-binding globulin has higher affinity for thyroid hormones than TTR does.). We suggested that the onset of hepatic TTR synthesis correlated with the development of homeothermy and the increase in lipid volume to body mass ratio (for review, see Schreiber and Richardson 1997Citation ).

TTR is the only thyroid hormone distributor protein synthesized in the brain. It is synthesized by the choroid plexus of reptiles, birds, and mammals (Harms et al. 1991; Achen et al. 1992, 1993Citation ). We suggested that the onset of TTR synthesis in the choroid plexus was correlated with the first appearance of the cortex, resulting in an increase in lipid volume (see Schreiber and Richardson 1997Citation ). A multicomponent network system has evolved in vertebrates, ensuring appropriate distribution of the lipophilic thyroid hormones in environments with increased lipid volume in which the loss of thyroid hormones from extracellular space by permeation into membranes is prevented by binding of the hormones to special proteins. We suggest the name "thyroid hormone distributor proteins" for these.

Larger eutherians have three thyroid hormone distributor proteins in their blood. The affinities of these three proteins differ by several orders of magnitude, resulting in "buffering" of the free thyroxine level over a wide range between the concentration of free thyroxine in blood and the maximum solubility of thyroxine in aqueous solution at physiological pH (see Schreiber and Richardson 1997Citation ).

Acknowledgements

We are very grateful to R. Lawn, Stanford University, for hedgehog serum, genomic DNA, and liver RNA; to J. Patton, University of California at Berkeley, for shrew serum; to P. Licht, University of California at Berkeley, for mole serum; and to P. Vogel, University of Lausanne, for shrew tissues.

Footnotes

Claudia Kappen, Reviewing Editor

1 Present address: Department of Biochemistry, Prince of Songkla University, Hat-Tai, Songkla, Thailand. Back

2 Abbreviation: TTR, transthyretin. Back

3 Keywords: transthyretin evolution thyroid hormone distribution hedgehog shrew mole Back

4 Address for correspondence and reprints: Samantha J. Richardson, Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: s.richardson{at}biochemistry.unimelb.edu.au Back

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Accepted for publication April 11, 2000.