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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.
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