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.

Keywords: Dendroaspis angusticeps; glycopeptidome; glycotoxins; venomics.

Figure 1

Venn diagram of compounds detected and the mass distribution of glycosylated peptides.

Figure 2

NUPLC-MS/MS spectra of peptide bearing O-glycans containing (A) sialic acid and (B) fucose. Carbohydrates are represented by the nomenclature proposed by Harvey et al. [13]. Oxonium ions are flagged by arrows.

Figure 3

MS/MS spectra of two glycoforms of the same peptide sequence: MALDI-PSD spectrum for glycoform A; nESI-MS/MS spectrum for glycoform B. Arrows indicate fragment ions identified in the mass spectra: a-type in green, y-type in blue and b-type in red. Carbohydrates are represented by the nomenclature proposed by Harvey et al. [13]. Oxonium ions are flagged by arrows.

Figure 4

Mass spectra of the S-Cys-GlycoDa-1 fraction in MALDI-MS acquisition. (A) crude fraction; (B) reduced fraction; and (C) reduced and alkylated fraction.

Figure 5

De novo sequencing of the m/z 2596 peptide in the S-Cys-GlycoDa-1 fraction; * internal fragments. Arrows indicate fragment ions identified in the mass spectra: a-type in green, y-type in blue and b-type in red. Carbohydrates are represented by the nomenclature proposed by Harvey et al. [13].

Figure 6

2D-NMR spectra of GlycoDa-12 extract pH 4.5 (HSQC-TOCSY) in blue and GlycoDa-12 extract pH 4.5 (HSQC) in red.

Figure 7

Edited ¹³C–¹H HSQC: overlay of the natural extract GlycoDa-12 (CH, CH₃: blue/CH₂: cyan) and the synthesized peptide with cysteine oxidized (CH, CH₃: orange/CH₂: red). The methyl signal for threonine, NAcetyl hexose, leucine, and isoleucine are indicated in blue boxes. The 10 hexose CH are observed in pink boxes. CHα for the two cysteines, the histidine, the hydroxyl-proline, and the threonine are labeled.

Figure 8

BLAST result for GlycoDa-12 toxin. In red, conserved cysteines, in blue amino acids which are different than database sequences.