VENOMIC ANALYSIS OF THE LIZARD FROM GUATEMALA HELODERMA CHARLESBOGERTI (CAMPBELL & VANNINI, 1988), A GENOMIC-AID PROTEOME

Heloderma charlesbogerti (Hc) is a venomous lizard endemic to Guatemala, belonging to the Toxicofera clade. This genus comprises five large anguimorph lizards: H. alvarezi, H. charlesbogerti, H. exasperatum, H. horridum, and H. suspectum, which are found across the United States, Mexico, and Guatemala. Despite their toxicological and ecological importance, the venom composition and biological activity of these species remain largely unexplored. The recent publication of the complete genome of (Hc) provided a unique opportunity to investigate the proteome of its venom with unprecedented accuracy. In this study, we conducted the first integrative proteomic and functional analysis of Hc venom, combining reverse-phase high-performance liquid chromatography (RP-HPLC), SDS-PAGE, electrospray ionization quadrupole time-of-flight mass spectrometry (ESI/Q/TOF-MS), and biological activity tests. Venom was extracted from 20 individuals and pooled according to sex (male/female) and origin (captive/wild). While chromatographic profiles revealed slight differences between groups, all 20 individuals were used in the pool of the species’ venom proteome. A total of 40 distinct chromatographic peaks were detected. The first 18, corresponding to low molecular weight proteins, were digested in solution with trypsin and analyzed by MS. The remaining peaks were analyzed by SDS-PAGE, followed by in-gel digestion and MS. Using a custom protein database derived from the Hc genome, combined with the publicly available database from the NCBI “Toxins”. In addition to peak-based analysis, a shotgun proteomic approach was applied to the pooled venom samples, enabling the identification of less abundant proteins that were not retained in the HPLC fractions. We identified multiple toxin families including phospholipases A2, helokinestatins, CRiSPs, serine proteases, helofensins, and exendins, as well as other venom-associated proteins.  Biological assays revealed that Hc venom has significantly lower proteolytic and procoagulant activities than Bothrops asper (Ba) venom. However, it showed higher PLA₂ activity at lower concentrations. While Ba induced coagulation in 60 seconds at 0.1 µg/mL, Hc required >100 µg/mL. These results highlight the biochemical uniqueness of Hc venom, its potential for pharmacological applications, and the need for conservation efforts focused on this endangered species. This study expands the understanding of lizard venom diversity and offers a foundation for future toxinological and biomedical research.

This work was supported by the National Council for Scientific and Technological Development (CNPq) and by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ).