Differential evolution and neofunctionalization of snake venom metalloprotease domains

Abstract

Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

Fig. 1.

Snake venom metalloproteinase (SVMP) phylogeny with mid-point rooting.

Fig. 2.

Sequence alignment of Psammophis mossambicus propeptide domain selectively expressed variants (1. A7X4E9, 2. A7X4D4, 3. A7X4B5, 4. A7X4C9, 5. A7X4C4, 6. A7X4C0, 7. A7X443, A7X437, 9. A7X488, 10. A7X473, 11. A7X480, 12. A7X468, 13. A7X452, 14. A7X484, 15. A7X476, 16. A7X457, 17. A7X447, 18. A7X497, 19. A7X4A6, 20. A7X461, 21. A7X493, 22. A7X4B0, 23. A7X465), Echis propeptide domain selectively expressed variants (GR950192 Echis pyramidum leakeyi and GR948204 Echis coloratus) and Echis multidomain SVMP (Q2UXQ6 Echis ocellatus and E9KNB4 Echis carinatus sochureki). Signal peptide is shown in lowercase, metalloprotease domain in straight underline, disintegrin domain in wavy underline.

Fig. 3.

Comparison of evolutionary selection pressures acting on the propeptide regions of (A) Echis coloratus multidomain, (B) Echis spp. monodomain and (C) Psammophis mossambicus monodomain SVMPs. The corresponding site-wise synonymous and non-synonymous mutations are shown in green and red bars, respectively while the omega is shown in gold. Model 8 computed omega values and the number of positively-selected sites (PP ≥ 0.95, Bayes-Empirical Bayes approach) are also indicated. Domains are: C, cysteine-rich; D, disintegrin; p, protease; PP, pro-protein (PP); and S, signal peptide.

Fig. 4.

Three-dimensional homology model of Echis coloratus SVMP, depicting the locations of positively selected amino acid sites (shown in red) detected by site-model 8 analyses for different domains. The omega values (Codeml: Mgene with option G) and the number of positively selected sites (Model 8, PP ≥ 0.95, Bayes-Empirical Bayes approach) are also indicated for the respective domains. A plot of amino acid positions (x axis) against accessible surface area (ASA) ratio (y axis) indicating the positions of amino-acids (exposed or buried) in the crystal structure of Echis coloratus SVMP is presented. Residues with an ASA ratio of more than 50% (above blue line) are considered to be exposed to the surrounding solvent whereas those with a ratio lesser than 20% (bellow the red line) are considered to be buried. The co-ordinates of positively selected sites in the protease, disintegrin-like and cysteine-rich domains are shown as big green, blue, and orange circles, respectively. Three-dimensional structures of each SVMP domain depicting the locations of positively selected sites (in red) along with the model 8 omega are also presented.

Fig. 5.

Inhibition of human a7 nAChR by proline-rich SVMP pre-pro only domain variants from Psammophis mossambicus. Pm1 and Pm2 concentration-dependently inhibited a7 nAChR endogenously expressed in SH-SY5Y human neuroblastoma cells in a high-throughput FLIPR Ca²⁺ imaging assay. Inset, the IC50s for inhibition of a7 nAChR by Pm1 and Pm2 were determined as 11.99 μm (pIC50 4.921 ± 0.058) and 11.83 μm (pIC50 4.927 ± 0.094), respectively. Data are presented as mean ± S.E. with n = 3 replicates from 3 independent assays.

Fig. 6.

Sequence alignment of 1. JX467171 Azemiops feae, JX467172, 2. Azemiops feae, 3.B0VXV8 Sistrurus catenatus edwardsii, 4. Q90Y12 Crotalus durissus terrificus, 5. Q2PE51 Crotalus durissus terrificus, 6. Q27J49 Lachesis muta, 7. P01021 Gloydius blomhoffii, 8. P68515 Bothrops insularis, 9. Q9PW56 Bothrops jararaca, 10. P0C7P5 Protobothrops flavoviridis.