Exploring the Venom Proteome of the Western Diamondback Rattlesnake, Crotalus atrox, via Snake Venomics and Combinatorial Peptide Ligand Library Approaches

Abstract

We report the proteomic characterization of the venom of the medically important North American western diamondback rattlesnake, Crotalus atrox, using two complementary approaches: snake venomics (to gain an insight of the overall venom proteome), and two solid-phase combinatorial peptide ligand libraries (CPLL), followed by 2D electrophoresis and mass spectrometric characterization of in-gel digested protein bands (to capture and "amplify" low-abundance proteins). The venomics approach revealed approximately 24 distinct proteins belonging to 2 major protein families (snake venom metalloproteinases, SVMP, and serine proteinases), which represent 69.5% of the total venom proteins, 4 medium abundance families (medium-size disintegrin, PLA(2), cysteine-rich secretory protein, and l-amino acid oxidase) amounting to 25.8% of the venom proteins, and 3 minor protein families (vasoactive peptides, endogenous inhibitor of SVMP, and C-type lectin-like). This toxin profile potentially explains the cytotoxic, myotoxic, hemotoxic, and hemorrhagic effects evoked by C. atrox envenomation. Further, our results showing that C. atrox exhibits a similar level of venom variation as Sistrurus miliarius points to a "diversity gain" scenario in the lineage leading to the Sistrurus catenatus taxa. On the other hand, the two combinatorial hexapeptide libraries captured distinct sets of proteins. Although the CPLL-treated samples did not retain a representative venom proteome, protein spots barely, or not at all, detectable in the whole venom were enriched in the two CPLL-treated samples. The amplified low copy number C. atrox venom proteins comprised a C-type lectin-like protein, several PLA(2) molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin highly conserved across the animal kingdom. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N-terminal pyrrolidone carboxylic acid formation required in the maturation of bioactive peptides such as bradykinin-potentiating peptides and endogenous inhibitors of metalloproteases. Our findings underscore the usefulness of combinatorial peptide libraries as powerful tools for mining below the tip of the iceberg, complementing thereby the data gained using the snake venomics protocol toward a complete visualization of the venom proteome.

Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs

“…In addition, the characterization of a number of serine proteinase, snake venom metalloproteinases (SVMPs) and L-amino acid oxidase (LAO) fragments absent in the starting material indicated that proteolysis occurred during handling of the samples. Proteolysis during ProteoMiner™ treatment of C. atrox venom has been previously reported [22].…”

“…It is responsible for the majority of snakebite fatalities in northern Mexico and for the second greatest number in the USA after C. adamanteus [23]. This study showed the occurrence in the venom of protein spots barely, or not at all, detectable in the whole venom, including a C-type lectin-like protein, several PLA 2 molecules, PIII-SVMP isoforms, glutaminyl cyclase isoforms, and a 2-cys peroxiredoxin [22]. Peroxiredoxin and glutaminyl cyclase may participate, respectively, in redox processes leading to the structural/functional diversification of toxins, and in the N-terminal pyrrolidone carboxylic acid formation required in the maturation of bioactive peptides such as bradykinin-potentiating peptides and endogenous inhibitors of metalloproteases.…”

“…Recently we have employed solid-phase combinatorial peptide ligand libraries (CPLL) to explore the venom proteome of the western diamondback rattlesnake, Crotalus atrox [22]. Treatment of the venom with ProteoMiner™ beads under overloading conditions resulted in the capture and amplification of several low abundance proteins, complementing data gained using our snake venomics protocol [24][25][26] towards a more complete description of the venom proteome.…”

“…Very recently we have applied two solid-phase combinatorial hexapeptide ligand libraries to explore the occurrence of low-abundance proteins in the venom of Crotalus atrox [22]. C. atrox is the largest western rattlesnake and one of the more aggressive rattlesnake species found in North America.…”

An investigation of the evolution of the anguimorph lizard venom system

“…It has been clinically used to treat type 2 diabetes and to enhance plasma insulin secretion . Variation in venom profiles has been extensively documented between snake species of the same genus Calvete et al, 2007;Fry et al, 2005Fry et al, , 2002Gutierrez et al, 2008;Lomonte et al, 2008;Mackessy, 2010;Salazar et al, 2009;Sanz et al, , 2006Tashima et al, 2008;Wagstaff et al, 2009) and between individuals of the same species, with intraspecific differences found among different geographic localities (Fr et al, 2002;Boldrini-Franca et al, 2010;Castro et al, 2013;Daltry et al, 1996;Forstner et al, 1997;French et al, 2004;Sunagar et al, 2014), and between juveniles and adults (Daltry et al, 1996;Calvete et al, 2009;Lopez-Lozano et al, 2002;Mackessy, 1988).…”