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Gen Comp Endocrinol
2020 Dec 01;299:113592. doi: 10.1016/j.ygcen.2020.113592.
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An aryl hydrocarbon receptor from the caecilian Gymnopis multiplicata suggests low dioxin affinity in the ancestor of all three amphibian orders.
Kazzaz SA
,
Giani Tagliabue S
,
Franks DG
,
Denison MS
,
Hahn ME
,
Bonati L
,
Powell WH
.
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The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Caudata) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. In X. laevis AHR1β, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilian Gymnopis multiplicata, representing the remaining extant amphibian order, Gymnophiona. G. multiplicata AHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable to X. laevis AhR1β (26.23 nM) and Ambystoma AHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both vertebrate groups that diverged earlier (teleost fish) and those that appeared more recently (other tetrapods). These findings suggest the hypothesis that AHRs with low TCDD affinity represent a characteristic that evolved in a common ancestor of all three extant amphibian groups.
Fig. 1. Phylogenetic analysis of G. multiplicata AHR. Amino acid sequences of bHLH and PAS domains of each AHR sequence were aligned in Clustal X2. A tree was inferred by the Neighbor-Joining method. It was rooted with the C. elegans AHR sequence, an invertebrate ortholog. Numbers at the branch points represent the bootstrap values based on 1000 samplings. Scale bar indicates degree of substitution (4 residues per 100). Accession numbers of the sequences and generic names of the species are found in Supplemental Information, Table S3.
Fig. 2. TCDD binding by G. multiplicata AHR. (A) Expression of each AHR in TNT reactions determined by Western blotting. (B) Velocity sedimentation analysis of TCDD binding. Synthetic AHR proteins or unprogrammed TNT lysates were incubated with 2 nM [3H]TCDD and fractionated on sucrose density gradients. Inset graph excludes mouse AHR and contains re-scaled y-axis. (C) Quantification of TCDD-specific binding revealed by sedimentation analysis. The radioactivity (disintegrations per minute) in fractions comprising each peak in panel B was summed. Specific binding is the difference between total binding (preparations containing an AHR) and nonspecific binding (preparation lacking AHR). The bar graph plots specific binding relative to that found for X. laevis AHR1β. Values represent means ± the standard error of four replicates. Values with identical labels do not differ significantly (One-way ANOVA with Tukey’s test for individual contrasts).
Fig. 3. TCDD-induced transactivation activity of G. multiplicata AHR. COS-7 cells were cotransfected with pGudLuc6.1 reporter construct, pRL-TK transfection control construct, and expression plasmids for X. laevis ARNT1 and each indicated AHR. Cells were treated with DMSO or TCDD for 18 h. Each plotted value represents the mean of at least three replicate assays ± standard error. (A) Transactivation activity of each AHR is given in relative luciferase units (RLU), the ratio of firefly to Renilla luciferase activity at each concentration of TCDD. Inset graph depicts G. multiplicata AHR with re-scaled y-axis. (B) Fractional induction. For each AHR, relative luciferase expression at each TCDD concentration was normalized to the maximal response, which was assigned a value of 1. Nonlinear regression was used to calculate EC50 values for each AHR. r2 values for the fitted curves are 0.81 for G. multiplicata AHR, 0.99 for A. mexicanum AHR, 0.86 for frog AHR1β, and 0.92 for the mouse AHR.
Fig. 4. FICZ-induced transactivation activity of G. multiplicata AHR. Fractional induction of reporter gene expression by each AHR following (A) 18-h or (B) 3-h exposure was determined as described for Fig. 3. Values represent mean ± standard error for at least four replicates. Panel A: r2 values for the fitted curves are 0.86 for G. multiplicata AHR, 0.91 for X. laevis AHR1β, 0.98 for A. mexicanum AHR, and 0.71 for mouse AHR. Panel B: r2 values for the fitted curves are 0.83 for G. multiplicata AHR, 0.83 for X. laevis AHR1β, 0.85 for A. mexicanum AHR, and 0.65 for mouse AHR.
Fig. 5. Sequence and structural model for the G. multiplicata AHR. (A) Sequence alignment produced by Clustal W. Only residues that differ from the caecilian sequence are shown; dots indicate conserved positions. Variable residues that protrude into the modeled binding cavity are boxed. Color scheme for residues: red, acidic; blue, basic; purple, polar; yellow, Cys; brown, aromatic; green, hydrophobic; orange, Ser, Thr; gray, Pro; white, Gly. Secondary structures attributed by DSSPcont (SS) are indicated below: light gray bars for helices and dark gray bars for β-strands. (B) Cartoon representation of the modeled G. multiplicata AHR LBD. Residues that both differ from the high-affinity mouse or chicken AHRs and protrude into the modeled binding cavity are labeled and shown as sticks. The light red shaded area delineates the molecular surface of the binding cavity identified by CASTp. (C) Comparison of cartoon renderings for modeled LBDs of AHRs: magenta for G. multiplicata AHR, green for A. mexicanum AHR, yellow for frog AHR1β, blue for chicken AHR, and gray for mouse AHR. Black labels indicate the conserved secondary structure elements identified by DSSPcont. Magnified inset image depicts position of potentially variable residues that protrude outside the binding pockets but may nonetheless affect binding cavity dimensions (see text, Section 4.1).
Figure S1. 14C-catalase marker fractionation. 14C-catalase standard (11.3S) was added to all sucrose density gradients in addition to the AHR proteins and 3H-TCDD. Gradients were fractionated and the radioactivity (dpm) was quantified over 15 minutes for each fraction by liquid scintillation counting. This standard served as a marker that could capture variability in protein mobility on the gradients and enable alignment of fractions from different gradients, since differences in elution position mirror the differences in 3H-TCDD peaks corresponding to specific binding of AHR. n=4 for each AHR.
Andersen,
Continuum secondary structure captures protein flexibility.
2002, Pubmed
Andersen,
Continuum secondary structure captures protein flexibility.
2002,
Pubmed
Beatty,
Toxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in larval and adult forms of Rana catesbeiana.
1976,
Pubmed
Case,
Electrophoretic patterns of certain proteins in caecilians (Amphibia: Gymnophiona).
1975,
Pubmed
Collier,
The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the mortality and growth of two amphibian species (Xenopus laevis and Pseudacris triseriata).
2008,
Pubmed
,
Xenbase
Denison,
Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor.
2011,
Pubmed
Dundas,
CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.
2006,
Pubmed
Ema,
Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors.
1994,
Pubmed
Farmahin,
Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds.
2013,
Pubmed
Fiser,
Modeling of loops in protein structures.
2000,
Pubmed
Fort,
Evaluation of the developmental toxicity of five compounds with the frog embryo teratogenesis assay: Xenopus (FETAX) and a metabolic activation system.
1989,
Pubmed
,
Xenbase
Fraccalvieri,
Comparative analysis of homology models of the AH receptor ligand binding domain: verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor.
2013,
Pubmed
Frueh,
Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression.
2001,
Pubmed
Giani Tagliabue,
Modeling the binding of diverse ligands within the Ah receptor ligand binding domain.
2019,
Pubmed
Gu,
The PAS superfamily: sensors of environmental and developmental signals.
2000,
Pubmed
Hahn,
Unexpected diversity of aryl hydrocarbon receptors in non-mammalian vertebrates: insights from comparative genomics.
2006,
Pubmed
Hahn,
Evolutionary conservation of the vertebrate Ah (dioxin) receptor: amplification and sequencing of the PAS domain of a teleost Ah receptor cDNA.
1995,
Pubmed
Hahn,
Diversity as Opportunity: Insights from 600 Million Years of AHR Evolution.
2017,
Pubmed
Hahn,
Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family.
1997,
Pubmed
Hahn,
Biomarkers and bioassays for detecting dioxin-like compounds in the marine environment.
2002,
Pubmed
Han,
Comparison of recombinant cell bioassays for the detection of Ah receptor agonists.
2004,
Pubmed
Hay,
Phylogenetic relationships of amphibian families inferred from DNA sequences of mitochondrial 12S and 16S ribosomal RNA genes.
1995,
Pubmed
Karchner,
The molecular basis for differential dioxin sensitivity in birds: role of the aryl hydrocarbon receptor.
2006,
Pubmed
Karchner,
AHR1B, a new functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes.
2005,
Pubmed
Lavine,
Aryl hydrocarbon receptors in the frog Xenopus laevis: two AhR1 paralogs exhibit low affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
2005,
Pubmed
,
Xenbase
Lee,
Molecular and functional characterization of a novel aryl hydrocarbon receptor isoform, AHR1β, in the chicken (Gallus gallus).
2013,
Pubmed
Martí-Renom,
Comparative protein structure modeling of genes and genomes.
2000,
Pubmed
McIntosh,
Mammalian Per-Arnt-Sim proteins in environmental adaptation.
2010,
Pubmed
Motto,
New aryl hydrocarbon receptor homology model targeted to improve docking reliability.
2011,
Pubmed
Nebert,
Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis.
2000,
Pubmed
Odio,
Specific ligand binding domain residues confer low dioxin responsiveness to AHR1β of Xenopus laevis.
2013,
Pubmed
,
Xenbase
Oka,
Molecular cloning and characterization of the aryl hydrocarbon receptors and aryl hydrocarbon receptor nuclear translocators in the American alligator.
2016,
Pubmed
Pandini,
Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis.
2009,
Pubmed
Pardo,
Stem caecilian from the Triassic of Colorado sheds light on the origins of Lissamphibia.
2017,
Pubmed
Pezdirc,
PCB accumulation and tissue distribution in cave salamander (Proteus anguinus anguinus, Amphibia, Urodela) in the polluted karstic hinterland of the Krupa River, Slovenia.
2011,
Pubmed
Poland,
Analysis of the four alleles of the murine aryl hydrocarbon receptor.
1994,
Pubmed
Propst,
Evaluation of the developmental toxicity of benzo[a]pyrene and 2-acetylaminofluorene using Xenopus: modes of biotransformation. Stover Group.
1997,
Pubmed
,
Xenbase
Puga,
The transcriptional signature of dioxin in human hepatoma HepG2 cells.
2000,
Pubmed
Pyron,
A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians.
2011,
Pubmed
Ramadoss,
Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells.
2004,
Pubmed
Rannug,
The tryptophan derivative 6-formylindolo[3,2-b]carbazole, FICZ, a dynamic mediator of endogenous aryl hydrocarbon receptor signaling, balances cell growth and differentiation.
2018,
Pubmed
Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed
San Mauro,
Life-history evolution and mitogenomic phylogeny of caecilian amphibians.
2014,
Pubmed
Seok,
Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex.
2017,
Pubmed
Session,
Genome evolution in the allotetraploid frog Xenopus laevis.
2016,
Pubmed
,
Xenbase
Shen,
Statistical potential for assessment and prediction of protein structures.
2006,
Pubmed
Shoots,
An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.
2015,
Pubmed
,
Xenbase
Sippl,
Recognition of errors in three-dimensional structures of proteins.
1993,
Pubmed
Soshilov,
Ligand promiscuity of aryl hydrocarbon receptor agonists and antagonists revealed by site-directed mutagenesis.
2014,
Pubmed
Vajda,
Effects of steroids and dioxin (2,3,7,8-TCDD) on the developing wolffian ducts of the tiger salamander (Ambystoma tigrinum).
2005,
Pubmed
Van den Berg,
The 2005 World Health Organization reevaluation of human and Mammalian toxic equivalency factors for dioxins and dioxin-like compounds.
2006,
Pubmed
Wincent,
The suggested physiologic aryl hydrocarbon receptor activator and cytochrome P4501 substrate 6-formylindolo[3,2-b]carbazole is present in humans.
2009,
Pubmed
Wu,
Hox cluster characterization of Banna caecilian (Ichthyophis bannanicus) provides hints for slow evolution of its genome.
2015,
Pubmed
Zardoya,
On the origin of and phylogenetic relationships among living amphibians.
2001,
Pubmed
,
Xenbase
Zhang,
Mitogenomic perspectives on the origin and phylogeny of living amphibians.
2005,
Pubmed
Zhang,
A mitogenomic perspective on the phylogeny and biogeography of living caecilians (Amphibia: Gymnophiona).
2009,
Pubmed
