Toxicon 99 (2015) 68e72
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Why the honey badger don't care: Convergent evolution of venomtargeted nicotinic acetylcholine receptors in mammals that survive venomous snake bites Danielle H. Drabeck a, b, *, Antony M. Dean a, 1, Sharon A. Jansa a, b a b
Department of Ecology, Evolution, and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States J. F. Bell Museum of Natural History, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2014 Received in revised form 3 February 2015 Accepted 17 March 2015 Available online 18 March 2015
Honey badgers (Mellivora capensis) prey upon and survive bites from venomous snakes (Family: Elapidae), but the molecular basis of their venom resistance is unknown. The muscular nicotinic cholinergic receptor (nAChR), targeted by snake a-neurotoxins, has evolved in some venom-resistant mammals to no longer bind these toxins. Through phylogenetic analysis of mammalian nAChR sequences, we show that honey badgers, hedgehogs, and pigs have independently acquired functionally equivalent amino acid replacements in the toxin-binding site of this receptor. These convergent amino acid changes impede toxin binding by introducing a positively charged amino acid in place of an uncharged aromatic residue. In venom-resistant mongooses, different replacements at these same sites are glycosylated, which is thought to disrupt binding through steric effects. Thus, it appears that resistance to snake venom aneurotoxin has evolved at least four times among mammals through two distinct biochemical mechanisms operating at the same sites on the same receptor. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Convergent evolution Venom resistance Honey badger Nicotinic acetylcholine receptor Mellivora capensis
1. Introduction Among the handful of mammals known to be resistant to venomous snake bites, the honey badger (Mellivora capensis) has a near legendary ability to attack venomous snakes (Elapdiae and Viperidae) and survive their toxic bites (Begg et al., 2003; Hughes et al., 2013; Waxman, 2014). However, the mechanisms by which resistant species, including honey badgers, are rendered invulnerable to snake bites are largely unknown. Most research into mammalian venom resistance has focused on blood-serum factors that neutralize venom metalloproteinases and phospholipases (e.g., Catanese and Kress, 1992; Lovo-Farah et al., 1996; Melo and SuarezKurtz, 1988; Menchaca and Perez, 1981; Neves-Ferreira et al., 2010; Perez et al., 1979; Tarng et al., 1986). Comparatively few studies have focused on the role that modified venom targets play in conferring toxin resistance. These venom targets are
* Corresponding author. Department of Ecology, Evolution, and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States. E-mail address: [email protected] (D.H. Drabeck). 1 Laboratory of Microbial Evolution, Sun Yat-sen University, 201 He Danqing Hall No. 135 Xingangxi Road, Guangzhou 510275, PR China. http://dx.doi.org/10.1016/j.toxicon.2015.03.007 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
physiologically important protein receptors that no longer bind venom toxins, yet retain the ability to bind their endogenous ligands (Barchan et al., 1992, 1995; Jansa and Voss, 2011). The muscular nicotinic acetylcholine receptor (nAChR), a wellcharacterized transmembrane receptor that mediates synaptic transmission from nerves to muscles, is targeted by a-neurotoxins present in the venom of elapid and hydrophid snakes (Barchan et al., 1995; Neumann et al., 1986). Two mammals known to survive elapid bites, the Egyptian mongoose (Herpestes ichneumon) and the hedgehog (Erinaceus concolor), have mutations in the a1subunit of their nAChR proteins that eliminate binding of the krait (Bungarus multicinctus) venom toxin a-bungarotoxin (Fig. 1B) (Asher et al., 1997, 1998; Barchan et al., 1992; Barchan et al. 1995; Haggerty and Froehner, 1981; Kao et al., 1984; Takacs et al., 2001, 2004). Cobra-nAChR receptors (Naja spp.) also fail to bind a-neurotoxins, are immune to their own venom, and have convergently acquired similar amino acid replacements (Takacs et al., 2001, 2004). Honey badgers have been observed to survive bites from puff adders (Bitis spp.) (Colleen Begg, pers. comm.), have been anecdotally reported to survive arterial injections of black mamba venom (Rousseau, 1982), and have a diet comprising up to 25%
D.H. Drabeck et al. / Toxicon 99 (2015) 68e72
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Fig. 1. (A) The evolutionary tree of mammals depicting relationships among species that have been sequenced for the a1 subunit of the nicotinic acetylcholine receptor gene (CHRNA1). The two trees are topologically identical; ancestral-state reconstructions of amino-acid sites 187 (left-hand tree) and 189 (right-hand tree) are indicated with colored branches (legend upper left). The derived amino acid is given at the branch terminus for species that differ from the ancestral condition (tryptophan and phenylalanine for sites 187 and 189, respectively). Species that are known to survive envenomation by elapid snakes (honey badger, mongoose, hedgehog) or have been shown experimentally to have lost (mongoose, hedgehog) binding between a-bungarotoxin and the nAChR receptor are shown in red; the reduced binding ability of human is indicated with an asterisk. (B) Alignment of the a-bungarotoxin-binding region of nAChR (Barchan et al., 1995) for the 11 mammal species that have lost one or both ancestral aromatic residues at sites 187 and 189. The reconstructed ancestral placental sequence is shown for reference as is the sequence from cobra. Dots indicate sequence identity with this ancestral sequence. (C) Molecular model (Dellisanti et al., 2007a, b; UniProt Accession 2QC1) of the a1 subunit of the nicotinic acetylcholine receptor (spacefill model) shown binding with a-bungarotoxin (green ribbon model). Sites 187 and 189 are shown as the ancestral amino acid for placental mammals.
venomous snakes, including puff adders (Bitis arietans) and cape cobras (Naja nivea) (Begg et al., 2003). However, the biochemical basis of their resistance to neurotoxic venom has not been examined. To investigate the molecular basis of resistance to neurotoxic venoms in honey badgers, we examined the region of the nAChR receptor that contains the a-neurotoxin binding site and which has been previously implicated in venom resistance (Haggerty and Froehner, 1981; Kao et al., 1984). We asked whether the honey badger has independently acquired the same amino acid changes that alter toxin binding in other neurotoxin-resistant species, specifically, the presence of non-aromatic residues at position 187 and 189 of the nAChR a1 subunit. We sequenced this region of the nAChR receptor from honey badgers and other closely related but venom-susceptible mustelids. To extend the comparative context for the study, we retrieved 45 mammalian DNA sequences of the a1 subunit of the muscle cholinergic receptor gene (CHRNA1) from publically available sequence databases. We then employed a comparative phylogenetic approach to infer the molecular evolution of this receptor across a wide range of mammalian species, including those with known resistance and susceptibility to elapid venom. 2. Materials and methods Whole blood samples from four individuals of M. capensis were provided by the San Diego Zoo Institute for Conservation Research (2 samples) and the Fort Wayne Zoo (2 samples) (Fort Wayne, IN). We also sequenced five additional carnivores that are not known to be resistant to any snake venoms. Tissue samples from Lontra
canadensis (J.F. Bell Museum tissue collection number MP137), Procyon lotor (MP444), Mustela erminae (MP409, MP410), and Mustela vison (MP0083) were obtained from the Bell Museum of Natural History (University of Minnesota); tissue samples of Taxidea taxus, were kindly gifted by Dr. Emily Latch (University of Wisconsin, Milwakee). Genomic DNA was extracted from these samples using a QIAGEN DNeasy kit according to the standard protocols for blood and animal tissue (Qiagen, Inc., Valencia, CA, USA). We designed primers to amplify an 850 bp piece of the alpha subunit of the muscular nicotinic acetylcholine receptor gene (CHRNA1) that included the ligand binding site corresponding to residues 122e205 of the protein sequence. Polymerase chain reactions (PCRs) were carried out in 25 uL reactions using 1.0 ml of 10 mM ACH_F1 (50 -TGCAGATGGTGACTTTGCCATTGTCAAG-30 ) primer solution, 1.0 ml of 10 mM ACH_R1 (50 AGTCTGTGGGCAGGTAGAACACC-30 ) primer solution, 0.125 uL GoTaq polymerase (Promega Inc.), and recommended concentrations of GoTaq Green Buffer, MgCl2, and dNTPs. Reactions were performed for thirty cycles of melting at 94 C for 30 s, followed by annealing at 58 C for 15 s, and extension at 72 C for 90 s. Reactions were preceded by a 2 min denaturation at 94 C and included a final extension at 72 C for 7 min. Amplified PCR products were sequenced by Beckman Coulter Genomics on an ABI 3730XL DNA Analyzer using BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). Resulting sequences were assembled, edited, and aligned using Geneious version 5 (Drummond et al., 2010). Sequences generated for this report have been submitted to GenBank. We searched GenBank and Ensembl for all available mammalian CHRNA1 sequences (Supplemental Table 1). We aligned our mustelid DNA sequences along with these sequences using MUSCLE
70
D.H. Drabeck et al. / Toxicon 99 (2015) 68e72
(Edgar, 2004) with default parameters specified by Geneious (v 5.5) and translated the resulting aligned DNA sequences to amino acids (Supplementary Figure 1). We used recently published mammalian phylogenies (Meredith et al., 2011; Koepfli et al., 2008) to construct a tree describing well-established evolutionary relationships among the taxa that had CHRNA1 sequences. We used this phylogeny to infer ancestral amino acids using parsimony analysis as implemented in MESQUITE (Maddison and Maddison, 2011) and as the basis for tests of positive selection on the CHRNA1 gene. We tested for selection on the CHRNA1 gene in the three lineages that are known to be resistant to snakebite (H. ichneumon, Erinaceus sp., and M. capensis) using the branch-site tests for selection in the codeml program of PAML 4.8 (Yang, 2007). We identified these three lineages on the mammalian phylogeny as “foreground” branches that could have sites under selection and designated the remainder of the tree as “background” branches. The selection test compares the difference in ln-likelihood values of a model that allows a proportion of positively selected sites on foreground branches (u2 > 1) with one that does not allow positive selection on those branches by fixing u2 ¼ 1 (Zhang et al., 2005). We also used a Bayes-Empirical-Bayes (BEB) method to identify sites in the protein that had a high posterior probability of being under positive selection (Yang et al., 2005). 3. Results and discussion Phylogenetic analysis reveals that most placental mammals have aromatic residues at sites W187 and F189, and that the ancestral state for both of these sites optimizes as an aromatic amino acid (Fig. 1A). Selection tests strongly support a model that allows for a proportion of positively selected sites for CHRNA1 in Herpestes, Mellivora, and Erinaceus, the three lineages that are known to be resistant to snake venoms and to eat venomous snakes (2D[ ¼ 12.9, df ¼ 1, p
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Why the honey badger don't care: Convergent evolution of venomtargeted nicotinic acetylcholine receptors in mammals that survive venomous snake bites Danielle H. Drabeck a, b, *, Antony M. Dean a, 1, Sharon A. Jansa a, b a b
Department of Ecology, Evolution, and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States J. F. Bell Museum of Natural History, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 September 2014 Received in revised form 3 February 2015 Accepted 17 March 2015 Available online 18 March 2015
Honey badgers (Mellivora capensis) prey upon and survive bites from venomous snakes (Family: Elapidae), but the molecular basis of their venom resistance is unknown. The muscular nicotinic cholinergic receptor (nAChR), targeted by snake a-neurotoxins, has evolved in some venom-resistant mammals to no longer bind these toxins. Through phylogenetic analysis of mammalian nAChR sequences, we show that honey badgers, hedgehogs, and pigs have independently acquired functionally equivalent amino acid replacements in the toxin-binding site of this receptor. These convergent amino acid changes impede toxin binding by introducing a positively charged amino acid in place of an uncharged aromatic residue. In venom-resistant mongooses, different replacements at these same sites are glycosylated, which is thought to disrupt binding through steric effects. Thus, it appears that resistance to snake venom aneurotoxin has evolved at least four times among mammals through two distinct biochemical mechanisms operating at the same sites on the same receptor. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Convergent evolution Venom resistance Honey badger Nicotinic acetylcholine receptor Mellivora capensis
1. Introduction Among the handful of mammals known to be resistant to venomous snake bites, the honey badger (Mellivora capensis) has a near legendary ability to attack venomous snakes (Elapdiae and Viperidae) and survive their toxic bites (Begg et al., 2003; Hughes et al., 2013; Waxman, 2014). However, the mechanisms by which resistant species, including honey badgers, are rendered invulnerable to snake bites are largely unknown. Most research into mammalian venom resistance has focused on blood-serum factors that neutralize venom metalloproteinases and phospholipases (e.g., Catanese and Kress, 1992; Lovo-Farah et al., 1996; Melo and SuarezKurtz, 1988; Menchaca and Perez, 1981; Neves-Ferreira et al., 2010; Perez et al., 1979; Tarng et al., 1986). Comparatively few studies have focused on the role that modified venom targets play in conferring toxin resistance. These venom targets are
* Corresponding author. Department of Ecology, Evolution, and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, United States. E-mail address: [email protected] (D.H. Drabeck). 1 Laboratory of Microbial Evolution, Sun Yat-sen University, 201 He Danqing Hall No. 135 Xingangxi Road, Guangzhou 510275, PR China. http://dx.doi.org/10.1016/j.toxicon.2015.03.007 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
physiologically important protein receptors that no longer bind venom toxins, yet retain the ability to bind their endogenous ligands (Barchan et al., 1992, 1995; Jansa and Voss, 2011). The muscular nicotinic acetylcholine receptor (nAChR), a wellcharacterized transmembrane receptor that mediates synaptic transmission from nerves to muscles, is targeted by a-neurotoxins present in the venom of elapid and hydrophid snakes (Barchan et al., 1995; Neumann et al., 1986). Two mammals known to survive elapid bites, the Egyptian mongoose (Herpestes ichneumon) and the hedgehog (Erinaceus concolor), have mutations in the a1subunit of their nAChR proteins that eliminate binding of the krait (Bungarus multicinctus) venom toxin a-bungarotoxin (Fig. 1B) (Asher et al., 1997, 1998; Barchan et al., 1992; Barchan et al. 1995; Haggerty and Froehner, 1981; Kao et al., 1984; Takacs et al., 2001, 2004). Cobra-nAChR receptors (Naja spp.) also fail to bind a-neurotoxins, are immune to their own venom, and have convergently acquired similar amino acid replacements (Takacs et al., 2001, 2004). Honey badgers have been observed to survive bites from puff adders (Bitis spp.) (Colleen Begg, pers. comm.), have been anecdotally reported to survive arterial injections of black mamba venom (Rousseau, 1982), and have a diet comprising up to 25%
D.H. Drabeck et al. / Toxicon 99 (2015) 68e72
69
Fig. 1. (A) The evolutionary tree of mammals depicting relationships among species that have been sequenced for the a1 subunit of the nicotinic acetylcholine receptor gene (CHRNA1). The two trees are topologically identical; ancestral-state reconstructions of amino-acid sites 187 (left-hand tree) and 189 (right-hand tree) are indicated with colored branches (legend upper left). The derived amino acid is given at the branch terminus for species that differ from the ancestral condition (tryptophan and phenylalanine for sites 187 and 189, respectively). Species that are known to survive envenomation by elapid snakes (honey badger, mongoose, hedgehog) or have been shown experimentally to have lost (mongoose, hedgehog) binding between a-bungarotoxin and the nAChR receptor are shown in red; the reduced binding ability of human is indicated with an asterisk. (B) Alignment of the a-bungarotoxin-binding region of nAChR (Barchan et al., 1995) for the 11 mammal species that have lost one or both ancestral aromatic residues at sites 187 and 189. The reconstructed ancestral placental sequence is shown for reference as is the sequence from cobra. Dots indicate sequence identity with this ancestral sequence. (C) Molecular model (Dellisanti et al., 2007a, b; UniProt Accession 2QC1) of the a1 subunit of the nicotinic acetylcholine receptor (spacefill model) shown binding with a-bungarotoxin (green ribbon model). Sites 187 and 189 are shown as the ancestral amino acid for placental mammals.
venomous snakes, including puff adders (Bitis arietans) and cape cobras (Naja nivea) (Begg et al., 2003). However, the biochemical basis of their resistance to neurotoxic venom has not been examined. To investigate the molecular basis of resistance to neurotoxic venoms in honey badgers, we examined the region of the nAChR receptor that contains the a-neurotoxin binding site and which has been previously implicated in venom resistance (Haggerty and Froehner, 1981; Kao et al., 1984). We asked whether the honey badger has independently acquired the same amino acid changes that alter toxin binding in other neurotoxin-resistant species, specifically, the presence of non-aromatic residues at position 187 and 189 of the nAChR a1 subunit. We sequenced this region of the nAChR receptor from honey badgers and other closely related but venom-susceptible mustelids. To extend the comparative context for the study, we retrieved 45 mammalian DNA sequences of the a1 subunit of the muscle cholinergic receptor gene (CHRNA1) from publically available sequence databases. We then employed a comparative phylogenetic approach to infer the molecular evolution of this receptor across a wide range of mammalian species, including those with known resistance and susceptibility to elapid venom. 2. Materials and methods Whole blood samples from four individuals of M. capensis were provided by the San Diego Zoo Institute for Conservation Research (2 samples) and the Fort Wayne Zoo (2 samples) (Fort Wayne, IN). We also sequenced five additional carnivores that are not known to be resistant to any snake venoms. Tissue samples from Lontra
canadensis (J.F. Bell Museum tissue collection number MP137), Procyon lotor (MP444), Mustela erminae (MP409, MP410), and Mustela vison (MP0083) were obtained from the Bell Museum of Natural History (University of Minnesota); tissue samples of Taxidea taxus, were kindly gifted by Dr. Emily Latch (University of Wisconsin, Milwakee). Genomic DNA was extracted from these samples using a QIAGEN DNeasy kit according to the standard protocols for blood and animal tissue (Qiagen, Inc., Valencia, CA, USA). We designed primers to amplify an 850 bp piece of the alpha subunit of the muscular nicotinic acetylcholine receptor gene (CHRNA1) that included the ligand binding site corresponding to residues 122e205 of the protein sequence. Polymerase chain reactions (PCRs) were carried out in 25 uL reactions using 1.0 ml of 10 mM ACH_F1 (50 -TGCAGATGGTGACTTTGCCATTGTCAAG-30 ) primer solution, 1.0 ml of 10 mM ACH_R1 (50 AGTCTGTGGGCAGGTAGAACACC-30 ) primer solution, 0.125 uL GoTaq polymerase (Promega Inc.), and recommended concentrations of GoTaq Green Buffer, MgCl2, and dNTPs. Reactions were performed for thirty cycles of melting at 94 C for 30 s, followed by annealing at 58 C for 15 s, and extension at 72 C for 90 s. Reactions were preceded by a 2 min denaturation at 94 C and included a final extension at 72 C for 7 min. Amplified PCR products were sequenced by Beckman Coulter Genomics on an ABI 3730XL DNA Analyzer using BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). Resulting sequences were assembled, edited, and aligned using Geneious version 5 (Drummond et al., 2010). Sequences generated for this report have been submitted to GenBank. We searched GenBank and Ensembl for all available mammalian CHRNA1 sequences (Supplemental Table 1). We aligned our mustelid DNA sequences along with these sequences using MUSCLE
70
D.H. Drabeck et al. / Toxicon 99 (2015) 68e72
(Edgar, 2004) with default parameters specified by Geneious (v 5.5) and translated the resulting aligned DNA sequences to amino acids (Supplementary Figure 1). We used recently published mammalian phylogenies (Meredith et al., 2011; Koepfli et al., 2008) to construct a tree describing well-established evolutionary relationships among the taxa that had CHRNA1 sequences. We used this phylogeny to infer ancestral amino acids using parsimony analysis as implemented in MESQUITE (Maddison and Maddison, 2011) and as the basis for tests of positive selection on the CHRNA1 gene. We tested for selection on the CHRNA1 gene in the three lineages that are known to be resistant to snakebite (H. ichneumon, Erinaceus sp., and M. capensis) using the branch-site tests for selection in the codeml program of PAML 4.8 (Yang, 2007). We identified these three lineages on the mammalian phylogeny as “foreground” branches that could have sites under selection and designated the remainder of the tree as “background” branches. The selection test compares the difference in ln-likelihood values of a model that allows a proportion of positively selected sites on foreground branches (u2 > 1) with one that does not allow positive selection on those branches by fixing u2 ¼ 1 (Zhang et al., 2005). We also used a Bayes-Empirical-Bayes (BEB) method to identify sites in the protein that had a high posterior probability of being under positive selection (Yang et al., 2005). 3. Results and discussion Phylogenetic analysis reveals that most placental mammals have aromatic residues at sites W187 and F189, and that the ancestral state for both of these sites optimizes as an aromatic amino acid (Fig. 1A). Selection tests strongly support a model that allows for a proportion of positively selected sites for CHRNA1 in Herpestes, Mellivora, and Erinaceus, the three lineages that are known to be resistant to snake venoms and to eat venomous snakes (2D[ ¼ 12.9, df ¼ 1, p
