Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage

Abstract

Edited by F. Anne StephensonThe ␤ 1 -adrenergic receptor (␤ 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for ␤-adrenergic antagonists, such as ␤-blockers, relatively little is yet known about its regulation. We have shown previously that ␤ 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates ␤ 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of ␤ 1 AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the ␤AR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of ␤ 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. ␤-Adrenergic receptors (␤ARs)4 are G protein-coupled receptors (GPCRs) that activate intracellular signaling pathways mainly via the stimulatory G s protein after binding of agonists such as adrenaline and noradrenaline (1, 2). The ␤ARs exist as three subtypes, ␤ 1 , ␤ 2 , and ␤ 3 , and the former two are important in the regulation of the excitation-contraction coupling of the myocardium. The ␤ 1 AR is the predominant ␤AR subtype expressed in the heart and the main mediator of the endogenous catecholamine-stimulated positive chronotropy and inotropy (1, 2). Thus, it is the most important target receptor for ␤-adrenergic antagonists, also called ␤-blockers, which are widely used in the treatment of cardiac diseases, such as chronic heart failure, coronary artery disease, arrhythmias, and hypertension. However, these therapeutic agents have limited effectiveness in some patients and also exert adverse effects. Consequently, there is a growing need to better understand the underlying mechanisms in cardiac function and disease to develop alternative and more individualized treatment options that can improve clinical outcomes.During chronic heart failure, a persistent compensatory increase of catecholamines causes ␤ 1 AR desensitization and down-regulation. The density of ␤ 1 ARs at the plasma membrane is reduced by 50%, whereas that of ␤...

Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

“…Covalent modifications of these mucintype glycans have been reported, such as the O-acetylation of neuraminic acid (1,23,34), and the sulfation of different Gal and GlcNAc units (23,35,36). Mucin-type O-glycosylation has been implicated in a wide variety of biological processes, such as interaction with pathogens (37,38), cell adhesion (39 -41), and proteolytic processing (40,(42)(43)(44)(45). Altered glycosylation patterns have been linked to different diseases.…”

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans

“…O-glycosylation was shown to interfere with the action of proprotein convertases on several pro-hormones and receptors (Goth et al, 2015, Kato et al, 2006, May et al, 2003, Schjoldager et al, 2010, Goth et al, 2017). This appears not to be the case for OCN as its O-glycosylation does not interfere with its processing by furin in vitro and in vivo.…”

“…C1GALT1C1 knockout HEK293sc (HEK293 simple cell or COSMC) cells and GALNTs deficient HEK293 cells were generated using Zinc finger nuclease (ZFN) gene editing as described previously (Goth et al, 2015, Steentoft et al, 2013, Steentoft et al, 2011, Goth et al, 2017. Cells were transfected using Lipofectamine 2000 reagent and secretion was performed over 24 hours in EMEM supplemented with PS and 22 µM VK1.…”

The half-life of the bone-derived hormone osteocalcin is regulated throughO-glycosylation in mice, but not in humans

“…O-glycosylation was shown to interfere with the action of proprotein convertases on several pro-hormones and receptors (Goth et al, 2015, Kato et al, 2006, May et al, 2003, Schjoldager et al, 2010, Goth et al, 2017). This appears not to be the case for OCN as its O-glycosylation does not interfere with its processing by furin in vitro and in vivo.…”

“…In some experiments, CHO-ldlD culture, transfection and secretion media was supplemented with 0.1 mM galactose and/or 1 mM Nacetylgalactosamine (GalNAc) to rescue the O-glycosylation defect as previously reported (Kingsley et al, 1986) Human embryonic kidney cells HEK293 were originally purchased from ATCC. COSMC knockout HEK293sc (HEK293 simple cell) cells and GALNTs deficient HEK293 cells were generated using Zinc finger nuclease (ZFN) gene editing as described previously (Goth et al, 2015, Steentoft et al, 2013, Steentoft et al, 2011, Goth et al, 2017. Cells were transfected using Lipofectamine 2000 reagent and secretion was performed over 24 hours in EMEM supplemented with PS and 22 µM VK1.…”