Evolutionary Glycomics: A Comprehensive Study of Vertebrate Host Serum/Plasma Glycome Using Orthogonal Glycomics Techniques

Abstract

Eukaryotic Vertebrate host glycosylation machinery has been subject to immense evolutionary pressure. Besides, being crucial for host sustenance, glyco-epitopes are also key-regulators of inter-species pathogen transmission. Significant evolutionary events such as loss of N-glycolyl neuraminic acid (Neu5Gc) or alpha Galactose (α-Gal) epitopes in species such as humans have been relevant for species development and have significantly impacted the ongoing arms race between pathogen and their hosts. Comprehensive knowledge on Vertebrate species-specific glycosylation is still scattered, thereby leaving a substantial gap in our understanding of their glycome evolution. Serum/plasma N- and O-glycomes of 35 different Vertebrate species (mammals, marsupials, birds, reptiles and fish) were analysed by two orthogonal glycomics methods, Porous Graphitised Carbon nano- Liquid Chromatography coupled to Electrospray Ionisation Tandem Mass Spectrometry (PGC nano-LC ESI-MS/MS) and Matrix Associated Laser Desorption Ionisation Time-of-flight Mass Spectrometry (MALDI TOF MS). Sialic acid diversity was additionally captured using High Performance Liquid Chromatography (HPLC) analyses of 1,2 Diamino Methylenedioxybenzene dichloride (DMB)- labelled sialic acids. Vertebrate serum/plasma glycome predominantly contained complex di-, mono sialylated glycans. Core fucosylation of these N-glycans was strongly species dependent, as were the non-reducing end modifications. Most species showed a preference for incorporation of either N-acetyl neuraminic acid (NeuAc) or NeuGc, with exceptions such as rats that use both. Anseriformes (Goose, Duck) N-glycans carried GalNAc-GlcNAc (LacDiNAc) epitopes that, however, were absent in Galliformes (Chicken, Turkey). Reptilians (e.g. Green sea turtles) had high levels of α-Gal epitopes, and Saltwater Crocodile uniquely showed high amounts of oligomannose N-glycans in serum. This largest, vertebrate species-specific serum/plasma glycan and MS/MS spectral data is currently in the process of being made available open access through our collaboration with glycan focussed database such as Glyconnect and UniCarbDB.