title: Comparative venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado: Identification of a novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®.
abstract: Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America.
  
  
  CONCLUSIONS
  Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms.
authors.affiliation: School of Biological Sciences, University of Northern Colorado, 501 20th Street, CB 92, Greeley, CO, 80639, USA; Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Saviola
authors.affiliation: Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.
authors.forename: Davinia
authors.initials: D
authors.lastname: Pla
authors.affiliation: Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.
authors.forename: Libia
authors.initials: L
authors.lastname: Sanz
authors.affiliation: Department of Biology, University of Texas at Arlington, Arlington, TX 76010, USA.
authors.forename: Todd A
authors.initials: TA
authors.lastname: Castoe
authors.affiliation: Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. Electronic address: jcalvete@ibv.csic.es.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Calvete
authors.affiliation: School of Biological Sciences, University of Northern Colorado, 501 20th Street, CB 92, Greeley, CO, 80639, USA. Electronic address: stephen.mackessy@unco.edu.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Mackessy
mesh_terms: D000818:Animals; D000997:Antivenins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003120:Colorado; D017839:Crotalus / Q000502:physiology*; D005260:Female; D005843:Geography; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D050830:L-Amino Acid Oxidase / Q000737:chemistry; D008297:Male; D010455:Peptides / Q000737:chemistry; D054467:Phospholipases A2 / Q000737:chemistry; D040901:Proteomics; D012910:Snake Venoms / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
chemical_list: D000997:Antivenins; C431183:Crotalidae Polyvalent immune Fab; D007140:Immunoglobulin Fab Fragments; D010455:Peptides; D012910:Snake Venoms; D050830:L-Amino Acid Oxidase; D054467:Phospholipases A2; D015032:Zinc
keywords: Antivenomics; Myotoxin a; Snake venom proteomics; Venom ontogenetic change