An aryl hydrocarbon receptor from the caecilian Gymnopis multiplicata suggests low dioxin affinity in the ancestor of all three amphibian orders

Highlights

• A caecilian aryl hydrocarbon receptor exhibits low dioxin binding and sensitivity.

• The protein’s ligand-binding domain resembles frog and salamander AHRs in structure and function.

• These findings suggest that low dioxin affinity of AHR evolved in a common ancestor of all three extant amphibian groups.

Abstract

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.

Introduction

The aryl hydrocarbon receptor (AHR), a member of the Per-ARNT-Sim (bHLH/PAS) family of proteins (McIntosh et al., 2010), is a ligand-activated transcription factor that mediates the toxicity of a wide range of environmental contaminants, including chlorinated dioxin-like compounds and polynuclear aromatic hydrocarbons (Denison et al., 2011). The unliganded receptor is part of a cytoplasmic complex with hsp90, p23, and AIP (Murray and Perdew, 2011). Agonist binding triggers translocation to the nucleus, shedding of these chaperones, and dimerization with the ARNT protein. The AHR:ARNT heterodimer binds to cognate enhancer sequences (Seok et al., 2017) and alters transcription of numerous target genes (Frueh et al., 2001, Puga et al., 2000). Well-known gene targets include the “AHR gene battery,” encoding phase I and phase II detoxification enzymes such as the cytochrome P450 family 1 members (CYP1s) and UDP glucuronosyltransferase, glutathione S transferase, and quinone reductase (Nebert et al., 2000). Induced over two orders of magnitude, CYP1s are frequently used as a biomarker of exposure to AHR agonists (Hahn, 2002). AHR also exerts biological effects through “non-classical” mechanisms involving interactions with additional signaling pathways and nuclear proteins (Denison et al., 2011).

AHR can be activated by structurally diverse agonists of both xenobiotic and endogenous origin. The prototypical agonist in mechanistic toxicological studies of AHR is the industrial contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Among the most toxic of the planar halogenated aromatic hydrocarbons, TCDD binds vertebrate AHRs with relatively high affinity (Van den Berg et al., 2006). Another potent AHR agonist, 6-formylindolo[3,2-b]carbazole (FICZ), has endogenous sources, forming in vivo from tryptophan as a photoproduct, an oxidation product, or as a metabolite produced by gut bacteria (Rannug and Rannug, 2018). FICZ binds AHR with even higher affinity than TCDD, but it is rapidly metabolized by the detoxification enzymes induced by exposure (Wincent et al., 2009).

Dioxin toxicity varies substantially between different vertebrate species or populations. Many studies link differences in AHR ligand binding affinity to these variations. Invertebrate AHRs typically do not bind TCDD, and the animals are largely insensitive to its toxic effects (Hahn et al., 2017). TCDD-sensitive mouse strains express the high-affinity AHR^b-1 allele, while toxicity is lower in strains expressing AHR^d, which encodes a protein that binds TCDD with 4- to 10-fold lower affinity, comparable to the human receptor (Ema et al., 1994, Poland et al., 1994, Ramadoss and Perdew, 2004). Differential affinity for AHR also underlies wide-ranging variations in TCDD toxicity between many bird species (Farmahin et al., 2013, Karchner et al., 2006). Amphibian receptors display less robust xenobiotic binding (Lavine et al., 2005, Shoots et al., 2015), with X. laevis AHRs exhibiting K_d values for TCDD 20- to 50-fold higher than those of many fish, birds, and mammals (Karchner et al., 2005, Karchner et al., 2006). Amphibians are concomitantly much less sensitive to toxicity of dioxin-like chemicals (Beatty et al., 1976, Collier et al., 2008, Jung and Walker, 1997, Pezdirc et al., 2011, Vajda and Norris, 2005) and PAHs (Fort et al., 1989, Propst et al., 1997).

To elucidate the structural basis for differences in TCDD affinity, ligand-binding domains (LBDs) have been characterized in AHRs from animals across the spectrum of TCDD sensitivity. A comparative study of AHR1 from the chicken (Gallus gallus) and common tern (Sterna hirundo) identified two residues—I324 and S380—that are critical for high-affinity binding by the receptor from chicken, the species more sensitive to TCDD toxicity (Karchner et al., 2006). In mice, a single polymorphism (A375V) reduces TCDD affinity 4–5 fold in the AHR^d allele vs. AHR^b-1; the LBD from human AHR contains valine at the homologous position (Ema et al., 1994, Poland et al., 1994, Ramadoss and Perdew, 2004). Additional signature residues conferring high-affinity binding in mouse AHR (Pandini et al., 2009) and AHRs from other species (Fraccalvieri et al., 2013) were identified by homology modeling and confirmed using site-directed mutagenesis. Similar approaches were employed to explain the low TCDD affinity of amphibian AHRs. Three amino acids within the LBD of X. laevis AHR1β confer low-affinity binding—N325, A354, and A370 (Odio et al., 2013). Homologous residues are present in AHR from X. tropicalis (Order Anura; frogs and toads) and Ambystoma mexicanum (Order Caudata; salamanders; (Shoots et al., 2015). These shared sequence elements and functional properties suggest that low TCDD affinity emerged in the common ancestor of these two extant amphibian groups. Is it possible that low TCDD affinity appeared even earlier in amphibian evolution?

Caecilians, the legless amphibians, comprise Order Gymnophiona, the earliest of the extant orders to diverge from the common lineage (Hay et al., 1995, Pyron and Wiens, 2011, Zardoya and Meyer, 2001, Zhang and Wake, 2009, Zhang et al., 2005). Therefore, caecilians offer an opportunity to probe the timing of emergence of low affinity AHRs during amphibian evolution. If the caecilians possess a low-affinity AHR, then this phenotype likely arose in an ancestor of all extant groups. Alternatively, if caecilian AHRs bind dioxin-like compounds with high affinity—similar to AHRs from both previously- and more recently-derived vertebrate groups—then low-affinity binding must have evolved in the common ancestor of frogs and salamanders after the caecilian divergence. This study sought to address this question. In this first characterization of a caecilian AHR, we report the sequence, pharmacological properties, and a structural model of the ligand-binding domain of a receptor derived from Gymnopis multiplicata (the Varagua caecilian). Comparative studies of low-affinity amphibian AHRs will help discern the pleiotropic roles of AHR in development, physiology, and toxicology from both general and taxon-specific perspectives.