Effect of Sialylated <i>O</i>-Glycans in Pro–Brain Natriuretic Peptide Stability

Jiang, Jingjing; Pristera, Nicole; Wang, Wei; Zhang, Xiumei; Wu, Qingyu

doi:10.1373/clinchem.2009.140558

Cited by 36 publications

(30 citation statements)

References 40 publications

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“…Transfection, Immunoprecipitation, and Western BlottingPlasmids were transfected into HEK 293 and HL-1 cells using FuGENE (Roche Diagnostics) or Lipofectamine 2000 (Invitrogen) reagents, as described previously (25,26). Conditioned medium was collected after 48 -72 h, and recombinant proteins were immunoprecipitated with an anti-V5-antibody.…”

Section: Methodsmentioning

confidence: 99%

Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin

Jiang

Wang

et al. 2011

Journal of Biological Chemistry

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Corin is a cardiac membrane protease that activates natriuretic peptides. It is unknown how corin function is regulated. Recently, soluble corin was detected in human plasma, suggesting that corin may be shed from cardiomyocytes. Here we examined soluble corin production and activity and determined the proteolytic enzymes responsible for corin cleavage. We expressed human corin in HEK 293 cells and detected three soluble fragments of ϳ180, ϳ160, and ϳ100 kDa, respectively, in the cultured medium by Western blot analysis. All three fragments were derived from activated corin molecules. Similar results were obtained in HL-1 cardiomyocytes. Using protease inhibitors, ionomycin and phorbol myristate acetate stimulation, small interfering RNA knockdown, and site-directed mutagenesis, we found that ADAM10 was primarily responsible for shedding corin in its juxtamembrane region to release the ϳ180-kDa fragment, corresponding to the near-entire extracellular region. In contrast, the ϳ160-and ϳ100-kDa fragments were from corin autocleavage at Arg-164 in frizzled 1 domain and Arg-427 in LDL receptor 5 domain, respectively. In functional studies, the ϳ180-kDa fragment activated atrial natriuretic peptide, whereas the ϳ160-and ϳ100-kDa fragments did not. Our data indicate that ADAM-mediated shedding and corin autocleavage are important mechanisms regulating corin function and preventing excessive, potentially hazardous, proteolytic activities in the heart. Natriuretic peptides act as a cardiac endocrine mechanism to regulate blood volume and pressure (1, 2). Corin is a serine protease that activates atrial natriuretic peptides (ANP) 2 and B-type natriuretic peptides in the heart (3, 4). The physiological importance of corin function has been shown in mouse models, in which disruption of the corin gene causes hypertension and cardiac hypertrophy (5, 6). In humans, population studies have identified polymorphisms (T555I/Q568P) in the corin gene in African Americans who had high blood pressure and cardiac hypertrophy (7-9). The polymorphisms were shown to alter corin protein structure and impair its biological activity in functional studies (10). In patients with heart failure (HF), the corin variants were associated with poor natriuretic peptide processing and worse clinical outcomes (11).Corin belongs to the type II transmembrane serine protease family, a subclass of trypsin-like enzymes defined by an N-terminal transmembrane domain and a C-terminal protease domain (12, 13). The transmembrane domain anchors the proteases on the cell surface, localizing the biological activities at specific sites. Under physiological and/or pathological conditions, type II transmembrane serine proteases can be shed from the cell surface. Soluble forms of enteropeptidase, hepsin and matriptases, for example, have been reported (14 -17). Recently, soluble corin was detected in human blood, indicating that corin is shed from the heart (18 -20). Interestingly, plasma corin levels were lower in patients with severe HF compared with those of normal...

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Section: Methodsmentioning

confidence: 99%

Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin

Jiang

Wang

et al. 2011

Journal of Biological Chemistry

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“…Thus, O-glycosylation in the PC site is essential for preventing inactivation, whereas some degree of inactivation at the PC site is equally important. More recently, it was also found that furin processing of the brain natriuretic peptide precursor, pro-brain natriuretic peptide, was blocked by O-glycosylation (7,8). These examples suggest that site-specific O-glycosylation directed by GalNAc-transferase isoforms play a direct regulatory role in PC processing.…”

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confidence: 95%

O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3

Schjoldager

Vester-Christensen

Bennett

et al. 2010

Journal of Biological Chemistry

123

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The angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of the endothelial and lipoprotein lipases and a promising drug target. ANGPTL3 undergoes proprotein convertase processing (RAPR 224 2TT) for activation, and the processing site contains two potential GalNAc O-glycosylation sites immediately C-terminal (TT 226 ). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification of Thr 226 adjacent to the proprotein convertase processing site in ANGPTL3. We demonstrated that GalNAc-T2 glycosylation of Thr 226 in a peptide with the RAPR 224 2TT processing site blocks in vitro furin cleavage. The study demonstrates that ANGPTL3 activation is modulated by O-glycosylation and that this step is probably controlled by GalNAc-T2.Site-specific O-glycosylation is emerging as a novel mechanism that regulates the action of proprotein convertase (PC) 2 processing of proteins (1). PC processing is crucial in regulating many fundamental biological pathways (2). O-Glycans in or immediately adjacent to the recognition sites of PCs can interfere with proteolysis required for activation or inactivation of proteins normally undergoing processing. We recently presented the first example of O-glycosylation-mediated regulation of the PC processing of the phosphaturic factor FGF23 (1), which results in functional inactivation of FGF23 and direct inhibition of the FGFR receptor by the released C-terminal fragment (3). Deficiency in intact FGF23 leads to the rare syndromes familial tumoral calcinosis and hyperostosis-hyperphosphatemia associated with hyperphosphatemia (4). Deficiency in FGF23 can be caused by either mutations in the UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferase T3 (GalNAc-T3) that O-glycosylates the PC recognition site (RHTR 179 ) of FGF23 or by mutations in FGF23 resulting in loss of secretion (5). Importantly, mutations in the PC recognition site of FGF23 that completely block cleavage lead to the mirror disease autosomal dominant hypophosphatemic Ricketts (6). Thus, O-glycosylation in the PC site is essential for preventing inactivation, whereas some degree of inactivation at the PC site is equally important. More recently, it was also found that furin processing of the brain natriuretic peptide precursor, pro-brain natriuretic peptide, was blocked by O-glycosylation (7,8). These examples suggest that site-specific O-glycosylation directed by GalNAc-transferase isoforms play a direct regulatory role in PC processing.ANGPTL3 (angiopoietin-like protein 3) expressed by the liver is an imp...

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“…Adding complexity to the model, proBNP has also been identified as a glycoprotein, with 7 serine and threonine residues in the N-terminal part of the peptide (proBNP 39 -50 ) having an oligosaccharide attachment (23 ). Glycosylation influences the processing of proBNP , as it inhibits the binding of proteases to cleavage sites (5,8,9 ), but glycosylation also influences NT-proBNP measurements for assays with the capture antibody in the N-terminal and middle region of the molecule (10,11 ). This includes the commercially available NT-proBNP assay from Roche Diagnostics (Elecsys proBNP II), as this assay cannot bind glycosylated NTproBNP 1-76 molecules (10,11 ).…”

Section: Discussionmentioning

confidence: 99%

“…The stability of intracellular proBNP has been attributed to glycosylation. Indeed, the sugar moieties attached to the N-terminal part of proBNP cover cleavage sites, limiting the binding of proteases to the molecule and thus the processing of proBNP (5,8,9 ). In addition, sugar moieties in the N-terminal fragment of proBNP influence antibody binding of the commercially available assay for detection of circulating NT-proBNP (10,11 ).…”

confidence: 99%

Influence of Glycosylation on Diagnostic and Prognostic Accuracy of N-Terminal Pro–B-Type Natriuretic Peptide in Acute Dyspnea: Data from the Akershus Cardiac Examination 2 Study

et al. 2015

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BACKGROUND The N-terminal part of pro–B-type natriuretic peptide (NT-proBNP) is glycosylated, but whether glycosylation influences the diagnostic and prognostic accuracy of NT-proBNP measurements is not known. METHODS We measured NT-proBNP concentrations of 309 patients with acute dyspnea by use of standard EDTA tubes and EDTA tubes pretreated with deglycosylation enzymes. The primary cause of dyspnea was classified as heart failure (HF) or non-HF, and the diagnosis was adjudicated by 2 independent physicians. We collected information on all-cause mortality during follow-up. RESULTS In all, 142 patients (46%) were diagnosed with HF. NT-proBNP concentrations in nondeglycosylated samples distinguished HF patients from patients with non-HF related dyspnea [median 3588 (quartiles 1–3 1578–8404) vs 360 (126–1139) ng/L, P < 0.001], but concentrations were markedly higher in samples pretreated with deglycosylation enzymes (total NT-proBNP) [7497 (3374–14 915) vs 798 (332–2296) ng/L, P < 0.001]. The AUC to separate HF patients from patients with non-HF related dyspnea was 0.871 (95% CI 0.829–0.907) for total NT-proBNP compared with 0.852 (0.807–0.890) for NT-proBNP measurements in standard EDTA plasma. During a median follow-up of 816 days, 112 patients (36%) died. Both NT-proBNP and total NT-proBNP concentrations were associated with mortality in separate multivariate models, but only total NT-proBNP concentrations provided added value to the basic risk model of our dataset as assessed by the net reclassification index: 0.24 (95% CI 0.003–0.384). There was a graded increase in risk across total NT-proBNP quartiles, in contrast with the results for NT-proBNP measurements. CONCLUSIONS NT-proBNP concentrations were higher, and diagnostic and prognostic accuracy was improved, by pretreating tubes with deglycosylation enzymes.

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Effect of Sialylated O-Glycans in Pro–Brain Natriuretic Peptide Stability

Cited by 36 publications

References 40 publications

Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin

Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin

O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3

Influence of Glycosylation on Diagnostic and Prognostic Accuracy of N-Terminal Pro–B-Type Natriuretic Peptide in Acute Dyspnea: Data from the Akershus Cardiac Examination 2 Study

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