In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom

Abstract

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.

Deciphering the Molecular Diversity of an Ant Venom Peptidome through a Venomics Approach

“…Since the O -glycosylation of peptide toxins has seldom been found in animal venoms, we decided to further investigate the type of O -linked HexNAc in U 17 -Tb1a, U 17 -Tb1b and U 17 -Tb1c by assessing the MS/MS fragmentation of their oxonium ions. Indeed, the MS/MS spectrum yields specific fragments which enables the identification of the isomeric forms of sugars .…”

“…The nonglycosylated counterparts of these three peptides were also present in the venom at a slightly lower proportion as Tb-2467, Tb-2570 and Tb-2650 peptides, respectively. Since the O-glycosylation of peptide toxins has seldom been found in animal venoms, 27 we decided to further investigate the type of O-linked HexNAc in U 17 -Tb1a, U 17 -Tb1b and U 17 -Tb1c by assessing the MS/MS fragmentation of their oxonium ions. Indeed, the MS/MS spectrum yields specific fragments which enables the identification of the isomeric forms of sugars.…”

“…Nevertheless, Oglycosylated AMPs, named formaecins, have been isolated in the hemolymph of the ant Myrmecia gulosa and were significantly more active than their nonglycosylated counterparts. 50 Interestingly, several O-glycosylated peptides have been identified in the venom of cone snails, 51 scorpions, 52 snakes, 27 and wasps. 53 None of these other glycosylated peptides exhibit any significant sequence homology with the glycosylated myrmicitoxins described in the present study.…”

The Need for Next-Generation Antivenom for Snakebite Envenomation in India

“…The advent of electrospray ionization [67] and matrix-assisted laser desorption ionization mass spectrometry [68,69] ionization techniques have helped to delineate the venom proteome in a different way. Based on these ionization strategies, several mass spectrometers have been extensively used to characterize the venom proteome of several snake species, including the 'big four' snake species in India [12,48,65,[70][71][72][73][74][75][76]. The venom proteome of several snake species, including the 'big four' snake species in India, has been explored using various mass spectrometry-based proteomics workflows [33,35,40,42,48,77].…”