An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles

Thomas D Madsen,Zilu Ye,Shifa Jebari,Hiren J Joshi,Katrine T Schjoldager,Mette M Rosenkilde,Jens P Goetze,Cesar Martin,Sergey Y Vakhrushev,Daniel B Andersen,Rune E Kuhre,Tongzhong Ju,Christoffer K Goth,Lasse H Hansen,Jens J Holst,John Hintze

doi:10.1038/s41467-020-17473-1

Abstract

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

Highlights

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure
The latter were subjected to prediction of conservation in human neuropeptide orthologs by sequence alignment analysis and revealed that 374 out of 409 glycosites were predicted to be conserved in humans, based on conservative preservation of Ser/Thr residues within (±5) amino acid residues[25] (Supplementary Fig. 1b)
Surveying the distribution of the identified human and conserved O-glycosites across each peptide hormone precursor revealed that about half (223) of the 410 glycosites were located in the proprotein part, and the other half (187) were located on the mature peptide hormones (Fig. 1a, Table 1)

Summary

Introduction

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. While peptide hormone-based drugs benefit from being selective for their cognate receptors, the usage of native peptides as therapeutics is challenging due to the inherent instability giving rise to short half-lifes of the biologically active peptide form(s) on a scale of a few minutes[2]. The plasma concentration of most peptide hormones is in the lower picomolar range, which for technical reasons has challenged the peptidomic insight into the molecular composition of the mature forms of these hormones and their PTMs. Recent sensitive mass spectrometry (MS)-based studies have identified alpha-amidation, acetylation, and phosphorylation on select peptide hormones[9,14]. We identified O-glycosites on the atrial natriuretic peptide (ANP) hormone, and found that

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Conclusion