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 post-translational 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.

Keywords: blood pressure; cardiovascular; cardiovascular disease; glycobiology; glycoprotein; glycosylation; heart; heart failure; natriuresis; natriuretic peptide; peptide hormone; post-translational modification (PTM).

Figure 1.

Summary of identified O-glycosites on natriuretic peptides. A, schematic representation of NPs with previously reported O-glycosites (yellow squares with diagonal line) and sites identified in this study (yellow squares with bold, black frame). The NP pro-part is shown in blue, and the mature bioactive hormone is shown in red. Receptor binding involving the cyclic part of the bioactive hormones and their cognate receptors is shown on the right. Scissors indicate corin or furin cleavage sites. A conserved serine in the mature NPs is highlighted in boldface red type and with an asterisk and marked with a white box on BNP and CNP. The six amino acids upstream of the proprotein convertase activation sites are shown with previously identified O-glycosites highlighted in red. B, analysis of NP sequence conservation. The top panel shows conservation among all NPs in five species. The three bottom panels show the conservation tracks for each NP as indicated. The conserved serine in the disulfide ring of ANP is highlighted in red and with an asterisk. Dark gray, complete conservation; medium and light gray, less conservation; white, no conservation. All alignments were performed in ClustalW using the NP proprotein sequences of Homo sapiens, Mus musculus, Xenopus laevis, O. mykiss, and A. japonica.

Figure 2.

Identification of circulating O-glycosylated NT-proANP in human plasma. A, Western blotting with pAb proANP_1–16 of plasma from three plasma samples with high endogenous proANP concentrations subjected to deglycosylation with a broad specificity of neuraminidase and O-glycanase. Band intensities are shown above or below the respective bands. B, Western blotting with pAb proANP_1–16 of plasma treated only with neuraminidase and immunoprecipitated (IP) with pAb proANP_1–16. Arrows, 9–10 kDa bands; arrows with one asterisk, 14 kDa bands; arrow with two asterisks, 12–13 kDa band.

Figure 3.

Proteolytic stability of ANP glycoforms. A and B, in vitro cleavage analysis of equal amounts of ANP and O-glycosylated ANP (Tn-ANP_S117, Tn-ANP_S123, or Tn-ANP_S117,S123) with either neprilysin (A) or insulin-degrading enzyme (B) in a time course up to 24 h. ANP peptide sequence with GalNAc glycosylation and initial neprilysin and insulin-degrading enzyme proteolytic cleavage sites (vertical lines) and their expected masses after cleavage are shown at the top of each panel. Samples were taken at the indicated time points, and the product development was monitored by MALDI-TOF MS. Intact ANP is indicated by arrows, and products formed by proteolytic activity have either lower or slightly higher masses. Initial cleavage by neprilysin opens the disulfide ring, which increases the mass of intact ANP with 18 Da, whereas initial cleavage by insulin-degrading enzyme leads to a loss of 466 Da. After the initial cleavage events, ANP is further degraded to products of lower masses. Assays were repeated 2–3 times with similar results, respectively. Mock treatment did not affect the ANP signal over time (not shown).

Figure 4.

Analysis of natriuretic peptide receptor type A activation with ANP glycoforms. A, illustration of the stepwise enzymatic synthesis of ANP glycopeptides. Tn-ANP was synthesized using Fmoc solid-phase synthesis and used for further glycan elongation to produce T-ANP using Drosophila core 1 synthase (dC1GalT1), to ST-ANP using human α-2,3-sialyltransferase 1 (ST3Gal1), and to diST using human GalNAc α-2,6-sialyltransferase 1 (ST6GalNAc1). B, table summary of peptides and glycopeptides used in receptor activation assays. All peptides were synthesized with a disulfide bridge. C, NPR-A activation assay using ANP and glycosylated ANP variants on Ser¹¹⁷ and CNP as control. D, NPR-A activation assay using ANP and glycosylated ANP variants on Ser¹²³ and CNP control peptide. Receptor activation was measured by an increase in cGMP content in HEK293 cells stably expressing NPR-A stimulated by increasing concentrations of peptides as indicated. The receptor activation data in C and D are directly comparable, since the experiment was performed simultaneously on the same plates. The same ANP and CNP data are shown in both C and D for purposes of clarity and ease of comparison. Error bars, S.E.

Figure 5.

Acute in vivo actions of O-glycosylated ANP. ANP or O-glycosylated ANP with sialylated core 1 glycan on either Ser¹¹⁷ or Ser¹²³ was infused into rats for 60 min followed by a post-infusion clearance period of 30 min, and mean arterial pressure (MAP) (A), urinary output (UV) (B), and urinary sodium excretion (UNaV) (C) were evaluated in the study. *, p < 0.01 versus ST-ANP_S117; #, p < 0.05 versus ST-ANP_S123; $, p < 0.05 versus ST-ANP_S117; ***, p < 0.0001 versus all groups; **, p < 0.001 versus all groups (two-way ANOVA, Tukey's post hoc tests). Error bars, S.D.