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

Michel Degueldre,Julien Echterbille,Nicolas Gilles,Loïc Quinton,Gilles Mourier,Nicolas Smargiasso,Edwin De Pauw,Christian Damblon,Charlotte Gouin

doi:10.3390/ijms18112453

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.

Highlights

Snake venoms are classically composed of large toxins that affect the central nervous, the cardiac, and the neuromuscular systems
Several peptide sequences were determined when sufficient informative fragments (a, b, and/or y-) were detected. This structural work reports the unusual presence of post-translational modification (PTM) in snake venom peptidomes, such as hydroxyproline, O-glycosylation, and S-cysteinylation
All these PTMs have already been described in cone snail venom, this is the first time that a study highlights such modifications in snake venom

Summary

Introduction

Snake venoms are classically composed of large toxins that affect the central nervous, the cardiac, and the neuromuscular systems. It was demonstrated several decades ago that these properties can be exploited by deriving these toxic peptides into selective molecular scalpels or innovative drugs Following this idea, the venom of Dendroaspis angusticeps (Eastern Green Mamba) has been analyzed by bi-dimensional liquid chromatography and mass spectrometry, revealing more than 300 different toxins (5–10 kDa, [1]). Several were deeply studied and led to the discovery of innovative pharmacological tools, such as ligands of various subtypes of muscarinic receptors [2,3], selective ligands of α1a-adrenoreceptors (ρ-Da1a, [1]), or α2-adrenoreceptors (ρ-Da1b, [4]) Another toxin, called mambaquaretine-1, was isolated and characterized for its potency to selectively antagonize vasopressin type 2 receptors [5]. These peptides are atypical due to their limited size and due to their high number of prolines and their glycosylation

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