Discovery of O-glycans on atrial natriuretic peptide (ANP) that affect both its proteolytic degradation and potency at its cognate receptor

Abstract

Atrial natriuretic peptide (ANP) is a peptide hormone that in response to atrial stretch is secreted from atrial myocytes into the circulation, where it stimulates vasodilatation and natriuresis. ANP is an important biomarker of heart failure where low plasma concentrations exclude cardiac dysfunction. ANP is a member of the natriuretic peptide (NP) family, which also includes the B-type natriuretic peptide (BNP) and the C-type natriuretic peptide. The proforms of these hormones undergo processing to mature peptides, and for proBNP, this process has previously been demonstrated to be regulated by O-glycosylation. It has been suggested that proANP also may undergo posttranslational modifications. Here, we conducted a targeted O-glycoproteomics approach to characterize O-glycans on NPs and demonstrate that all NP members can carry O-glycans. We identified four O-glycosites in proANP in the porcine heart, and surprisingly, two of these were located on the mature bioactive ANP itself. We found that one of these glycans is located within a conserved sequence motif of the receptor-binding region, suggesting that O-glycans may serve a function beyond intracellular processing and maturation. We also identified an O-glycoform of proANP naturally occurring in human circulation. We demonstrated that site-specific O-glycosylation shields bioactive ANP from proteolytic degradation and modifies potency at its cognate receptor in vitro. Furthermore, we showed that ANP O-glycosylation attenuates acute renal and cardiovascular ANP actions in vivo. The discovery of novel glycosylated ANP proteoforms reported here significantly improves our understanding of cardiac endocrinology and provides important insight into the etiology of heart failure.

A Single Membrane Protein Required for Atrial Secretory Granule Formation

“…In neurons, a significant fraction of the forward trafficking from ER to dendritic recycling endosomes and the plasma membrane occurs even when the Golgi apparatus has been disrupted by BFA and many surface receptors have immature N-linked glycans, consistent with Golgi bypass (61). A Golgi by-pass pathway would allow production of proANP, which does not require N-glycosylation, is not extensively O-glycosylated and is cleaved only at the time of secretion (57).…”

“…We used HEK293 cells, which lack secretory granules and any of the features unique to cardiomyocytes, to test the hypothesis that PAM alters the trafficking of proANP at an early stage in the secretory pathway. ProANP synthesis requires formation of an intrachain disulfide bond, but does not require N-glycosylation, endoproteolytic cleavage or extensive O-glycosylation, making it difficult to track its movement through the secretory pathway (57). proANP-Emerald (58,59) was transiently expressed in HEK293 cells and in HEK293 cells stably expressing PAM1 (PAM/HEK) at levels similar to those observed in adult mouse atrium ( Fig.…”

Glycan Mimetics from Natural Products: New Therapeutic Opportunities for Neurodegenerative Disease

“…However, the degradation function of these enzymes was determined by O-glycosylation status of their substrates. Studies showed that site-specific O-glycosylation shields bioactive atrial natriuretic peptide (ANP) from proteolytic degradation by insulin-degrading enzyme and neprilysin [81]. In addition, an increase in tyrosine-linked glycan on Aβ fragments has been identified in the CSF samples of AD patients and was specifically found on short Aβ 1-15 and Aβ 1-20 [82].…”

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

“…Peptide hormones are synthesized as precursor proteins that may undergo regulated proteolytic processing during their transport through the secretory pathway to become mature hormones (involving enzymes such as carboxypeptidases 4 , proprotein convertases (PCs) 5 , and C-terminal α-amidation 6 ). In addition, a number of other posttranslational modifications (PTMs) have been identified, including tyrosine sulfation 7 , Nterminal acetylation 8 , phosphorylation 9 , and glycosylation 10 . Unlike constitutively secreted proteins, most peptide hormones are stored within the cell.…”

“…Recent sensitive mass spectrometry (MS)-based studies have identified alpha-amidation, acetylation, and phosphorylation on select peptide hormones 9,14 . However, given that the vast majority of proteins trafficking the secretory pathway undergo O-glycosylation, it is surprising that only very few O-glycosylated peptide hormones have been identified to date 10,[15][16][17][18][19] .…”