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, 6 (6), e20997

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums That Eat Pitvipers


Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums That Eat Pitvipers

Sharon A Jansa et al. PLoS One.


The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Phylogenetic trees of opossums.
A. The phylogeny of didelphids resulting from a mixed-model Bayesian analysis of a combined-data matrix comprising DNA sequences from five nuclear protein-coding genes and morphological data . Nodes that received Bayesian posterior probability values ≥0.95 in this analysis are indicated with black circles. B. The topology from A excluding Lestodelphys and Caluromysiops, (for which no vWF sequences are available). Branch lengths are shown as the estimated number of amino acid substitutions in vWF, assuming the JTT model of amino acid substitution as implemented in PAML . Taxa that are known to eat pitvipers are indicated in bold; those that are known to exhibit resistance to pitviper venom are indicated with an asterisk. Metachirus (indicated with a dagger) has been challenged with pitviper venom but does not exhibit resistance. Branches that were included in the foreground for branch-site tests are shown with solid heavy lines. Venom resistance of Chironectes is unknown; therefore, this taxon was included in one set of branch-site tests and excluded from the other (indicated with a dashed heavy line). For the purpose of this analysis, Didelphis marsupialis includes its dubiously distinct sister taxon D. aurita.
Figure 2
Figure 2. Structure of vWF showing botrocetin-binding sites.
Schematic showing the structure of the mature vWF protein and its constituent domains (A, B, C, D, and CK; modified from [39]). Amino-acid residues are numbered 1–2050 corresponding to the human vWF sequence, with the A1 domain spanning residues 478–728. The region sequenced from opossums for this study includes part of the A1 and A2 domains and spans residues 524–843 (indicated with a grey box). The botrocetin-binding region (indicated with a black box) is located in the A1 domain and spans residues 623–671. Aligned amino-acid sequences of this region are shown for five placental taxa (Homo, Mus, Canis, Talpa, and Dugong) as well as members of the opossum tribe Didelphini (including species of Didelphis, Philander, Lutreolina, and Chironectes) and its sister taxon Metachirini (Metachirus nudicaudatus). Amino acids that are identical to vWF sequence from Homo are shaded in grey. The 12 amino-acid residues (positions 628, 629, 632, 635, 636, 639, 643, 660, 661, 664, 667, and 668) identified as critical for botrocetin binding in Mus are indicated with red dots below the sequences.
Figure 3
Figure 3. Structure and functional analyses of the vWF-A1 domain.
A. The structure of the mouse vWF A1 domain complexed with botrocetin (Protein Data Bank file 1U0O ; image realized using Geneious v.5.0.3 [78]). The two chains of botrocetin are shown as a dark grey trace model. The vWF A1 domain is shown as a light grey spacefill model, with residues that are involved in botrocetin binding shown in color (yellow, red, or blue). Amino acid residues identified as being under positive selection in the lineage of venom-resistant opossums (Didelphini) are shown in red (P≥0.95) or yellow (0.5<P<0.95). Residues that are colored blue are involved in botrocetin binding but are not inferred to be under positive selection in opossums. B. Box plots of the absolute value of change in amino acid charge (top) and hydrophobicity (bottom) between venom-resistant and non-resistant taxa for sites of the vWF-A1 domain that bind botrocetin and those that do not. C. A site-by-site sliding window analysis along the vWF-A1 domain showing the average change in charge (solid line) and hydrophobicity (dashed line) between resistant and non-resistant taxa. Botrocetin-binding sites are indicated with pale grey bars, sites that bind platelet glycoprotein Ibα are dark grey, and sites that are under positive selection in venom-resistant opossums are indicated with red asterisks.
Figure 4
Figure 4. Change in amino-acid properties as a function of distance from botrocetin-binding sites.
Plots of the absolute value of the average change in amino-acid charge (a) and hydrophobicity (d) between resistant and non-resistant taxa as a function of distance from a known botrocetin-binding site. Solid dots correspond to values at known botrocetin-binding sites; open circles indicate other sites in the A1 domain. For both physicochemical properties (charge, hydrophobicity), the magnitude of change is negatively correlated with distance from a known botrocetin-binding site. To test the significance of this correlation, we analyzed 1000 replicate datasets in which magnitude of change in each physicochemical property was randomized across the sequence. Histograms show the distribution of slope values for the best-fit regression lines through scatterplots of change in charge (b, c) or change in hydrophobicity (e, f) as a function of these randomly permuted distances. Permutations were performed with (b, e) and without (c, f) botrocetin-binding sites included. Dashed lines indicate the limits of the 95% confidence interval; solid lines correspond to the slope of the best-fit regression line based on the unpermuted data.

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