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

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. O-glycosylation is an abundant post-translational modification but its relevance for bioactive peptides is unclear. Here, the authors detect O-glycans on almost one third of the classified peptide hormones and show that O-glycosylation can modulate peptide half-lives and receptor activation properties.This work was supported by the Novo Nordisk Foundation, the Lundbeck Foundation, Danish National Research Foundation Grant DNRF107

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

15 pags, 8 figs, 1 tab. -- This article contains supplementary material (Table S1, Figs. S1–S4, and Data Sets S1–S4.1)The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11–mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11–mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.This work was supported by the Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, the Kirsten og Freddy Johansen Fonden, the Lundbeck Foundation, the A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, the Mizutani Foundation, the Novo Nordisk Foundation, the Danish Research Council Sapere Aude Research Talent Grant (to K. T. S.), and the Danish National Research Foundation (DNRF107). The authors declare that they have no conflicts of interest with the contents of this articl

G protein-coupled receptors in the sweet spot:glycosylation and other post-translational modifications

Abstract Post-translational modifications (PTMs) are a fundamental phenomenon across all classes of life and several hundred different types have been identified. PTMs contribute widely to the biological functions of proteins and greatly increase their diversity. One important class of proteins regulated by PTMs, is the cell surface expressed G protein-coupled receptors (GPCRs). While most PTMs have been shown to exert distinct biological functions, we are only beginning to approach the complexity that the potential interplay between different PTMs may have on biological functions and their regulation. Importantly, PTMs and their potential interplay represent an appealing mechanism for cell and tissue specific regulation of GPCR function and may partially contribute to functional selectivity of some GPCRs. In this review we highlight examples of PTMs located in GPCR extracellular domains, with special focus on glycosylation and the potential interplay with other close-by PTMs such as tyrosine sulfation, proteolytic cleavage, and phosphorylation

A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

Regulated shedding of the ectodomain of cell membrane proteins by proteases is a common process that releases the extracellular domain from the cell and activates cell signaling. Ectodomain shedding occurs in the immediate extracellular juxtamembrane region, which is also where O-glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell models and in vivo in mouse Galnt2 knockouts. The study provides compelling evidence for a wider role of site-specific O-glycosylation in ectodomain shedding

The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality

Heterotaxy (Htx) is a disorder of left-right (LR) body patterning, or laterality, that is associated with major congenital heart disease(1). The etiology and mechanism underlying most human Htx is poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer (LRO), where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals(2–6). The mechanism that specifies these two cilia types remains unknown. We now show that the GalNAc-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with Htx(7), and now demonstrate, in Xenopus, that galnt11 activates Notch signaling. GALNT11 O-glycosylates NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus LRO cilia and show that galnt11-mediated notch1 signaling modulates the spatial distribution and ratio of motile and immotile cilia at the LRO. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. In contrast, Notch overexpression decreases this ratio mimicking the ciliopathy, primary ciliary dyskinesia. Together, our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the LRO to determine laterality and identifies a novel mechanism for human Htx