General and Comparative Endocrinology 169 (2010) 167–173

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General and Comparative Endocrinology

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Molecular cloning and characterization of a type 3 iodothyronine deiodinasein the pine snake Pituophis deppei

Patricia Villalobos, Aurea Orozco ⇑, Carlos Valverde-RInstituto de Neurobiología, Departamento de Neurobiología Celular y Molecular, Universidad Nacional Autónoma de México, Campus UNAM, Juriquilla, Querétaro,Qro 76230, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 September 2009Revised 3 August 2010Accepted 6 August 2010Available online 13 August 2010

Keywords:Type III deiodinaseThyroid hormoneReptiles

0016-6480/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ygcen.2010.08.001

⇑ Corresponding author. Fax: +52 442 238 1038.E-mail address: aureao@servidor.unam.mx (A. Oro

The three distinct but related isotypes of the iodothyronine deiodinase family: D1, D2, and D3, have beenamply studied in vertebrate homeotherms and to a lesser extent in ectotherms, particularly in reptiles.Here, we report the molecular and kinetic characteristics of both the native and the recombinant hepaticD3 from the pine snake Pituophis deppei (PdD3). The complete PdD3 cDNA (1680 bp) encodes a protein of287 amino acids (aa), which is the longest type 3 deiodinase so far cloned. PdD3 shares 78% identity withchicken and 71% with its other orthologs. Interestingly, the hinge domain in D3s, including PdD3, is richin proline. This structural feature is shared with D1s, the other inner-ring deiodinases, and deserves fur-ther study. The kinetic characteristics of both native and recombinant PdD3 were similar to thosereported for D3 in other vertebrates. True Km values for T3 IRD were 9 and 11 nM for native and recom-binant PdD3, respectively. Both exhibited a requirement for a high concentration of cofactor (40 mMDTT), insensitivity to inhibition by PTU (>2 mM), and bisubstrate, sequential-type reaction kinetics. Insummary, the present data demonstrate that the liver of the adult pine snake P. deppei expresses D3. Fur-thermore, this is the first report of the cloning and expression of a reptilian D3 cDNA. The finding of hepa-tic D3 expression in the adult pine snake P. deppei is consistent with results in adult piscine species inwhich the dietary T3 content seems to regulate liver deiodinase expression. Thus, our present results sup-port the proposal that hepatic D3 in adult vertebrates plays a sentinel role in avoiding an inappropriateoverload of exogenous T3 secondary to feeding in those species that devour the whole prey.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Iodothyronine deiodination is the essential first step in theintracellular fine-tuning of thyroid hormone (TH) bioactivity.Accordingly, with the notable exception of reptiles, the physiolog-ical, biochemical, and molecular properties of at least three distinctisotypes of iodothyronine deioidinases (D1, D2, and D3) have beencharacterized in all vertebrate classes over the past 15 years (forreview see Bianco et al., 2002; Orozco and Valverde-R, 2005;Gereben et al., 2008). D2 converts the prohormone T4 to the morebiologically active T3 via outer-ring deiodination (ORD). On the otherhand, through inner-ring deiodination (IRD), D3 catalyzes the con-version of T4 and T3 to their inactive metabolites, reverse T3 (rT3)and 3,30-diiodothyronine (3,30-T2), respectively. D1 catalyzes bothORD and IRD; thus, depending on the thyroid status, it generateseither active or inactive TH. In the case of reptilian Ds, the availableinformation is scarce and pertains almost exclusively to the acti-

ll rights reserved.

zco).

vating pathway. Initial in vivo experiments in thyroidectomizedlizards suggested T4 to T3 conversion (Kar and Chandola-Saklani,1985), and deiodination in reptiles became firmly establishedwhen total ORD activity was partially characterized in different tis-sues of a snake species (Wong et al., 1993). Subsequently, the com-plete kinetic characterization of D1 and D2 activities has beenreported in various tissues in turtle (Hugenberger and Licht,1999), lizard (Fenton and Valverde-R, 2000), and crocodile(Shepherdley et al., 2002a). In contrast, the reptilian IRD pathwayhas been minimally studied, and the available information isincomplete and inconclusive. Indeed, Hugenberger and Licht(1999) reported TH monodeiodinase activity in turtle whichproduced rT3 from T4. Although the authors suggested that thisenzymatic activity was more similar to D3 than to D2, they namedthis enzyme ‘‘monodeiodinase with high affinity for T4”. Studies insaltwater crocodiles (Crocodylus porosus) reported no detectable T3

IRD activity in any tissue of juveniles (Shepherdley et al., 2002a),but described T3 IRD in the embryonic liver, an activity which de-creased toward hatching (Shepherdley et al., 2002b). In this samecontext, although the cDNAs for the three deiodinase isotypes havebeen cloned from mammals, birds, amphibians, and fish (for

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review Bianco et al., 2002; Orozco and Valverde-R, 2005; Gerebenet al., 2008), the reptilian mRNAs for this family of selenoenzymesremain unidentified. In the present study we report the cloningand expression of a D3 cDNA from the liver of the pine snake Pit-uophis deppei as well as the kinetic analysis of both the nativeand the cloned protein.

2. Materials and methods

2.1. Animals

Six adult male Mexican pine snakes (P. deppei), a species ende-mic to the central highland of Mexico, were used in the presentstudy. Pine snakes were collected from our university groundsin Juriquilla, Querétaro, Mexico, and ranged from 70 to 120 cmin total length. To avoid variability due to gender differences, onlymales were kept and acclimated for 20 days at 25 ± 1 �C in thelaboratory under a 12:12 h photoperiod. Pine snakes were fedtwice a week with live neonatal mice. Animals were treatedaccording to the procedures reviewed and approved by the Ani-mal Welfare Committee of our institute. Prior to decapitation pinesnakes were anesthetized in a CO2 chamber, and the livers weredissected and immediately divided into pools to determine deio-dinase activity and T3 content and to extract RNA. Pools werequickly frozen in liquid nitrogen and stored at �70 �C until use.Brain, heart, muscle, and gut were also collected to analyze D3tissue distribution.

2.2. Cloning, sequencing, and expression

Total RNA was isolated from liver using TRIzol reagent (Invitro-gen), and 10 lg was reverse transcribed using Superscript RNAaseH reverse transcriptase (Invitrogen) with an oligo (dT) containingan adaptor sequence (50-GACTTCAGGCTAGCATCGATCCATGGGTCGACT � 19-30) used for subsequent amplification. Based on the con-sensus sequences of deiodinases from different vertebrate classes, apair of degenerate primers was designed (50-TYGGNWSNTGYACNTGACC-30 and 50-AARAAYTTNCGGGTNGGYAG-30), and atouch-down RT-PCR was performed (hot start: 40 s at 94 �C; 40 sat 69–41 �C, descending 1 �C every 2 cycles; 1 min at 72 �C � 35 cy-cles). The amplified fragment was subcloned into pGEM-T (Prome-ga), transformed into DH5a competent cells, and sequenced. Basedon this sequence, specific primers were designed to perform 30 ra-pid amplification of the cDNA end (30-RACE) in a series of nestedPCRs. 30-RACEs were performed with a specific sense primer (50-ATGGCGCGCCTGCGCGCCTTCGAGCGCCTGGCCACGCGCT-30) and ananti-sense primer complementary to the adaptor sequence of theoligo (dT) primer. Initially, we tried to extend the 50-end by 50-RACEstrategy tailing the cDNA with dCTP using terminal transferase, andthen an oligo-dG-polylinker primer in combination with specificprimers in a series of nested amplifications to generate the 50-end. This was unsuccessful since the high content of G and C inthe 50 region hampered the pine snake D3 cloning by standard 50-RACE methods. We then used a specific sense primer (50-TTTTTACTAGTATGCTCCACTCGCTGGGCGCTCACACC-30) based onthe 50-end of D3 from the chicken genome (Accession No.NM_001122648) containing a SpeI site. The sequence from thecloned fragments was used to design new anti-sense oligonucleo-tides to verify the start site sequence in independent RT reactions.The full deduced amino acid sequence of the snake D3 clone wasverified by aligning it with other D3s, and mRNA secondary struc-ture was predicted using the MFOLD program (http://bio-info.math.rpi.edu/~zukerm). Once the entire P. deppei cDNA(PdD3) sequence was determined, a pair of oligonucleotides specificfor the 50 and 30 flanking regions of the cDNA was designed contain-

ing a SpeI and Eco91I site. The full, amplified cDNA was digestedwith SpeI and Eco91I (MBI Fermentas) and ligated (T4 ligase:Promega) into the vector pXENEX1 designed for expression ofmRNA in Xenopus laevis oocytes (Jeziorski et al., 1998). The cDNAwas verified by sequencing, linearized with HindIII (New EnglandBiolabs), purified, and used to transcribe capped mRNA with T7

polymerase (Ambion). Female X. laevis were anesthetized byhypothermia, and 2 or 3 ovarian lobes were removed through anabdominal incision and placed into Barths’ solution. Stage V–VIoocytes (n = 30) were microinjected with 50 nL (300 ng/oocyte)of mRNA and incubated for 4 days at 18 �C in Barths’ solutionwith daily medium changes. Oocytes were then harvested, homog-enates prepared, and recombinant deiodinase activity was assayed.It is important to note that uninjected oocytes do not expressdetectable deiodinase activity (data not shown). D3 activity ofcrude homogenates of the oocytes was measured as previously de-scribed, and kinetic characterization was performed using T3 assubstrate.

2.3. Hepatic microsomes

Preparation of microsomal fractions was based on a previouslydescribed method (Mol et al., 1997) that was modified for theseexperiments. Briefly, liver was homogenized in 10 volumes of buf-fer (0.25 M sucrose (Sigma), 10 mM Hepes (Sigma), 1 mM DTT(Invitrogen), pH 7) and centrifuged for 10 min at 3000g. The super-natant was then centrifuged for 30 min at 10,000g, and the result-ing supernatant was again centrifuged for 90 min at 40,000g. Thefinal pellet was resuspended in five volumes of buffer, snap-frozenin aliquots, and stored at �80 �C. Protein concentrations weredetermined using the Bradford protein assay reagent (BioRad) withBSA as the standard.

2.4. Deiodinase activity assays

All experiments were performed in duplicate, and each exper-iment was repeated a minimum of three times. The products fromthe deiodination reaction were separated by descending paperchromatography as previously described (Fenton et al., 1997;Mayorga et al., 2008). Reactions were stopped by the additionof ethanol and centrifuged (5 min at 12,000g); the radiolabeleddeiodination products were resolved, and [125I]-3,30-T2 wascounted in a gamma scintillation counter (Cobra II, Auto-Gamma).T3 IRD activity was assayed in hepatic microsomal preparations(pool of six animals) or crude homogenates of X. laevis oocytespreviously injected with the transcribed PdD3 mRNA for the na-tive and recombinant enzymes, respectively. In brief: the totalvolume of the reaction mixture was 100 lL and included a solu-tion of [125I]-T3 [specific activity: 1200 lCi/lg (Perkin Elmer LifeSciences)], plus non-radioactive thyronine (Sigma), protein (0,0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) and cofactor (dithiothreitol(DTT, 25, 50, 75, 100, and 200 mM)). Other parameters varied tooptimize PdD3 activity were temperature (5, 10, 20, 30, 37, and40 �C); incubation time (0.5, 1, 1.5, 2, 2.5, and 3 h), and pH (6,7, 8, and 9). After optimal conditions were established, the sensi-tivity to PTU inhibition was assayed. To determine PdD3 substrateaffinity, the inhibition of 2 nM [125I]-T3 IRD deiodination by unla-beled T3, T4, or rT3 was assessed over a wide range of concentra-tions (10, 100, and 1000 nM). These iodothyronines are potentialsubstrates for deiodinases, thus can inhibit T3 ORD competition.Michaelis–Menten constants for T3 were calculated using anunlabeled substrate concentration range of 1.5, 3, 6, 12, and24 nM. To establish the kinetic mechanism, double reciprocalplots of deiodination rates as a function of substrate (T3: 1.5, 3,6, 12, and 24 nM) at fixed cofactor concentrations (DTT: 25, 50,and 100 mM) were constructed. The kinetic constants for both

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enzymes were calculated using a nonlinear regression Michaelis–Menten analysis (GraphPad Prism version 5.00 Software). Sensi-tivity to inhibition by PTU (0.5–10 mM; (Sigma)) was measuredin the presence of 2 nM 125I–T3 and 50 mM DTT. The inhibitionconstant (Ki) was calculated using the equation of dose-depen-dent inhibition.

2.5. Radioimmunoassay of iodothyronines (RIA)

To determine intrahepatic T3 levels, individual tissues werehomogenized, and iodothyronines were extracted as previouslydescribed (Garcia et al., 2004). In brief: tissues (average wetweight 100 ± 7 mg) were homogenized in 10 volumes of metha-nol:ammonium hydroxide (99:1) solution. The homogenates werecentrifuged for 10 min at 700g. The supernatants were collected,evaporated in a speed vacuum, and suspended 1:5 (initial w/v)in assay buffer Tris–HCl (0.05 M; pH 8.6). Intratissular contentof T3 was measured by RIA as previously described (Orozco etal., 1992). The inter- and intra-assay coefficients of variation were9.5% and 6.6%, respectively. The incubation mixture containedassay buffer, a working dilution (1:20,000) of anti-T3 serum(Sigma), T3 standard (standard curve, T3: 12.5–800 ng/dL), theradioactive solution (10 pg/100 lL of [125I]-T3 plus 10 mg/10 mLof 8-anilino-1-naphthalene sulfonic acid (Sigma)), and 50 lL ofthe experimental sample. Free and antibody-bound radioactiveT3 were separated using 0.5% activated charcoal/ dextran solution(Sigma).

Fig. 1. (A) Nucleotide and deduced amino acid sequences of Pituophis deppei D3 cDNselenocysteine at position 149. Sec* indicates stop codon (UGA). The SECIS sequence andstructure of the SECIS (form 2) from Pituophis deppei D3 mRNA.

3. Results and discussion

3.1. Cloning and structural characteristics of pine snake D3

As depicted in Fig. 1A, the complete PdD3 cDNA encompasses1680 bp and includes an open reading frame of 861 bp that con-tains an in-frame TGA codon (position 445–447 bp) for the inser-tion of the amino acid selenocysteine (SeC). The 30-UTR of 819 bpcomprises a SeC insertion sequence (SECIS) of 66 bp beginning atnucleotide 1375, and is located 271 bp upstream of the poly(A).The nucleotide sequence of this SECIS is 68% identical to that ofchicken D3 and contains the consensus sequence required forthe correct incorporation of SeC during translation (Berry et al.,1991). The hypothetical secondary structure of this element cor-responds to that of a form 2 (Fig. 1B). A consensus polyadenyla-tion signal (AATAAA) begins at nucleotide 1628, 17 bp upstreamof the poly(A) tail (Genbank Accession No. GQ862344). Whenaligned with the different vertebrate D3 sequences available inGenbank, P. deppei PdD3 cDNA shares 78% identity with chickenand 71% with the rest of its orthologs (Fig. 2), while the percentidentity between vertebrate paralogs D1 and D2 was 59 and 55,respectively (data not shown). Furthermore, as in the case of itsvertebrate orthologs (Fig. 2), in PdD3 the amino acid residues(140–155) that flank the active site are highly conserved, as arethe histidine residues 181 and 198, which are essential for sub-strate binding (Toyoda et al., 1994). Besides these common struc-tural features which are relevant for its function, when compared

A. The deduced amino acid sequence predicts a protein of 287 amino acids withthe polyadenylation signal in the 30-UTR are underlined. (B) The proposed secondary

Fig. 2. Alignment of Pituophis deppei D3 amino acid sequence and its orthologs. The position of selenocysteine (149) is indicated as U. The alignment includes the followingsequences from Genbank: Homo sapiens (NM_001362), Macaca mulatta (NM_001122649), Mus musculus (AF426023), Rattus norvegicus (U24282), Bos Taurus(NM_001010993), Ovis aries (AY656759), Sus scrofa (AY533208), Gallus gallus (NM_001122648), Xenopus laevis (L28111), Xenopus tropicalis (NM_001113667), Ranacatesbeiana (L41731), Neoceratodus forsteri (AY339982), Tetraodon rubripes (AB360769), Oreochromis niloticus (Y11111), Sparus aurata (00888896), Solea senegalensis(AM902722), Paralichthys olivaceus (AB362423), Chiloscyllium punctatum (EU275162).

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to its other vertebrate counterparts the entire arrangement ofPdD3 exhibits some discrete variations that may bear functionalimportance. Thus, PdD3 is the longest (287 aa) type 3 deiodinase

so far reported (mammals (�278 aa); chicken (274 aa); amphib-ians (268–271 aa); fish (258–271 aa)). On the other hand, struc-tural and functional studies indicate that all three members of

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Fig. 3. Effect of (A) protein concentration, (B) time, (C) temperature, (D) pH, (E) cofactor concentration, and (F) PTU concentration on microsomal hepatic PdD3 activity.Results represent the means of three separate assays using microsomes pooled from six adult animals. Except as indicated otherwise, assay conditions were: [125I]-T3, 2 nM;rT3, 3 lM; DTT, 50 mM; protein, 50 lg/tube, PTU, 2 mM; pH, 7.0; incubation for 1 h at 30 �C.

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the deiodinase family are integral membrane proteins whosestructures share conserved functional domains critical for theirhomodimerization and catalytic activity (Toyoda et al., 1995; Ba-qui et al., 2000; Callebaut et al., 2003; Vivek Sagar et al., 2008).The proposed model is based on hydrophobic cluster analysis ofhuman deiodinases and locates a hinge domain 30 of the trans-membrane domain and 50 of the globular domain. Available ami-no acid sequence data from vertebrate D2s show no prolineresidues in the hinge region, while in D1s and most importantlyin D3s the number of prolines is high. This observation suggeststhat the presence of proline residues could be a specific featureof IRD-catalyzing enzymes (Fig. 2). Moreover, the number of pro-line residues in the hinge domain of D3s increases through phy-logeny (fish, amphibians (2–3); snake, chicken (5); mammals(6)). This aspect could be of evolutionary importance and war-rants further attention.

3.2. Kinetic analysis of the native and the recombinant protein

As shown in Fig. 3, optimal assay conditions for native and re-combinant PdD3 activity determination were attained with a 1 hincubation at 30 �C, at pH 7.0, 50 mM DTT and 0.5 mg/mL protein,a concentration that falls within the linear part of the proteincurve. Under these assay conditions, the calculated Ki of PTU inhi-bition upon T3 IRD was 4.8 mM. Both recombinant and native he-patic PdD3 activity exhibited kinetic characteristics similar tothose reported for D3 in other vertebrates. Thus, the apparent Km

values for T3 IRD were 9.8 and 9.4 nM for native and recombinantPdD3, respectively, while the Vmax value for hepatic microsomalactivity was 689 fmol [125I]-3,30-T2/mg min (Fig. 4A and B). Thefamily of curves obtained from double reciprocal plots at differentT3 and DTT concentrations intersect at a single point, indicatingthat both PdD3 enzymes exhibit the kinetics of a bisubstrate,

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Fig. 4. Kinetic analysis of native and recombinant PdD3. (A) and (B) T3 kinetics (concentration range of 1.5, 3, 6, 12, and 24 nM). (C) and (D) Kinetics at different T3 (T3: 1.5, 3,6, 12, and 24 nM) and DTT (25, 50, and 100 mM) concentrations. Insert shows double reciprocal plots. (E) and (F) Inhibition of 2 nM [125I]-T3 deiodination by increasingconcentrations of T4, T3, and rT3 (10, 100, and 1000 nM). In (A)–(D), activity is expressed as fmol [125I]-3,30-T2/mg min. In (C) and (F), activity is expressed as a percentage ofthe control. Except as indicated otherwise, assay conditions were: [125I]-T3, 2 nM; rT3, 3 lM; DTT, 50 mM; protein, 50 lg/tube, PTU, 2 mM; pH, 7.0; incubation for 1 h at 30 �C.

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sequential-type reaction (Fig. 4C and D). Both the recombinant andnative PdD3 enzymes had the same substrate affinity; T3 > T4 > rT3,(apparent Km values of 9.6, 675, and 3254 nM, respectively) (Fig. 4Eand F).

Hepatic D3 expression in most vertebrates is limited to embry-onic life. However, studies from our laboratory and others havedisclosed the presence of hepatic D3 expression in adult piscinespecies (Oreochromis niloticus, (Sanders et al., 1997), Neoceratodusforsteri (Sutija et al., 2004), Oncorhynchus mykiss (Bres et al.,2006), and Chiloscyllium punctatum (Mayorga et al., 2008)). Thefinding of hepatic D3 expression in the adult pine snake P. deppeifits well with our hypothesis that D3 participates in preventingan inappropriate systemic overload of exogenous T3 after feeding(Mayorga et al., 2008). Indeed, D3 could exert a protective rolewhen substantial amounts of exogenous thyroid hormones are in-gested by those predatory species that devour whole prey. P. deppei

feeds on live rodents in the wild, and, as indicated above, the spec-imens that were kept in our facilities were also fed with live neo-natal mice. Although glucoronidation or sulfation are importantinactivating pathways in the peripheral metabolism of thyroid hor-mones, very little is known about their functional interplay in THeconomy (Visser, 1996; Wu et al., 2005). In fact, available informa-tion suggests that conjugation pathways are of little physiologicalimportance in protecting thyroid hormone homeostasis wheneuthyroid adult organisms are challenged with a surplus of exoge-nous TH both in mammals (Kaptein et al., 1997) and in fish (Finn-son and Eales, 1999). Thus, the present findings are in agreementwith the suggestion that dietary T3 content may regulate liverdeiodinase activity in snake, as it does in fish species (Eales andFinnson, 1991; Sweeting and Eales, 1992; MacLatchy and Eales,1993; Bres et al., 2006; Mol et al., 1999; Van der Geyten et al.,2005; Plate et al., 2002; Plohman et al., 2002). In support of this

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levels.

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notion is the fact that the concentration of intratissular T3 washighest in the gut, while D3 activity was at least sevenfold higherin the liver (Fig. 5).

Acknowledgments

We gratefully acknowledge Anaid Antaramian, Leonor Casanovaand Elizabeth Vázquez Gómez for their technical assistance. Wealso thank Dr. Dorothy Pless for critically reviewing the manu-script. This work was partially supported by Grants UNAM-PAPIIT203409 and CONACYT 080420.

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