Natalia N. Tamm

Processing of Pro–Brain Natriuretic Peptide Is Suppressed by O-Glycosylation in the Region Close to the Cleavage Site

BACKGROUND Processing of the brain natriuretic peptide (BNP) precursor, proBNP, is a convertase-dependent reaction that produces 2 molecules--the active BNP hormone and the N-terminal part of proBNP (NT-proBNP). Although proBNP was first described more than 15 years ago, very little is known about the cellular mechanism of its processing. The study of proBNP processing mechanisms is important, because processing impairments could be associated with the development of heart failure (HF). METHODS The biochemical properties of recombinant proBNP and NT-proBNP and the same molecules derived from the blood of HF patients were analyzed by gel-filtration chromatography, site-directed mutagenesis, and different immunochemical methods with a panel of monoclonal antibodies (MAbs). RESULTS Part of the proBNP molecule (amino acid residues 61-76) located near the cleavage site was inaccessible to specific MAbs because of the presence of O-glycans, whereas the same region in NT-proBNP was completely accessible. We demonstrated that a convertase (furin) could effectively cleave deglycosylated (but not intact) proBNP. Of several mutant proBNP forms produced in a HEK 293 cell line, only the T71A variant was effectively processed in the cell. CONCLUSIONS Only proBNP that was not glycosylated in the region of the cleavage site could effectively be processed into BNP and NT-proBNP. Site-directed mutagenesis enabled us to ascertain the unique suppressing role of T71-bound O-glycan in proBNP processing.

Processing of Pro-B-Type Natriuretic Peptide: Furin and Corin as Candidate Convertases

BACKGROUND B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. METHODS We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. RESULTS Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1-32, whereas corin-mediated cleavage led to the production of BNP 4-32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. CONCLUSIONS Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.

Human Pro–B-Type Natriuretic Peptide Is Processed in the Circulation in a Rat Model

BACKGROUND The appearance of B-type natriuretic peptide (BNP) in the blood is ultimately caused by proteolytic processing of its precursor, proBNP. The mechanisms leading to the high plasma concentration of unprocessed proBNP are still poorly understood. The goals of the present study were to examine whether processing of proBNP takes place in the circulation and to evaluate the clearance rate of proBNP and proBNP-derived peptides. METHODS We studied the processing of human proBNP in the circulation and the clearance rate of proBNP and proBNP-derived peptides (BNP and N-terminal fragment of proBNP, NT-proBNP) in rats by injecting the corresponding peptides and analyzing immunoreactivity at specific time points. Glycosylated and nonglycosylated proBNP and NT-proBNP were used in the experiments. We applied immunoassays, gel filtration, and mass spectrometry (MS) techniques to analyze the circulation-mediated processing of proBNP. RESULTS ProBNP was effectively processed in the circulation into BNP (1-32) and various truncated BNP forms as confirmed by gel filtration and MS analysis. Glycosylation of proBNP close to the cleavage-site region suppressed its processing in the circulation. The terminal half-life for human glycosylated proBNP was 9.0 (0.5) min compared with 6.4 (0.5) min for BNP. For NT-proBNP, the terminal half-lives were 15.7 (1.4) min and 15.5 (1.3) min for glycosylated and nonglycosylated forms, respectively. CONCLUSIONS In rats, processing of human proBNP to active BNP occurs in the circulation. The clearance rate of proBNP is quite similar to that of BNP. These observations suggest that peripheral proBNP processing may be an important regulatory step rather than mere degradation.

Diagnostic utility of a single-epitope sandwich B-type natriuretic peptide assay in stable coronary artery disease: Data from the Akershus Cardiac Examination (ACE) 1 Study

OBJECTIVES To assess the merit of a novel single-epitope sandwich (SES) assay specific to the stable part of BNP in patients with reversible myocardial ischemia as post-translational modifications of BNP may influence assay performance. DESIGN AND METHODS We measured BNP concentration by a conventional assay and the SES-BNP assay in 198 patients referred for myocardial perfusion imaging (MPI). BNP concentration was determined before and immediately after exercise stress testing, and 1.5 and 4.5h later. Patients were categorized according to MPI results. RESULTS BNP concentration was higher with both assays at all time points in patients with reversible myocardial ischemia (n=19) compared to the other patients (n=179). Measuring BNP after stress testing or calculating the changes in BNP concentration did not improve diagnostic accuracy compared to baseline measurements: SES-BNP: AUC 0.71 (95% CI 0.58-0.84) vs. conventional BNP: 0.71 (0.59-0.83), p=0.96. By linear regression analysis, reversible myocardial ischemia was significantly associated with baseline SES-BNP concentration (p=0.043), but not with measurements by the conventional assay (p=0.089). In multivariate logistic regression models, only baseline measurement with the SES-BNP assay was significantly associated with reversible myocardial ischemia: odds ratio [logarithmical transformed BNP] 2.00 (95% CI 1.16-3.47), p=0.013. The SES-BNP assay, but not the conventional BNP assay, reclassified a significant proportion of the patients towards their correct category on top of the best clinical model of our data set: NRI=0.47, p=0.04. CONCLUSIONS The SES-BNP assay was significantly associated with reversible myocardial ischemia as assessed by several statistical indices, while a conventional BNP assay was not.

Searching for a BNP standard: Glycosylated proBNP as a common calibrator enables improved comparability of commercial BNP immunoassays.

BACKGROUND Circulating B-type natriuretic peptide (BNP) is widely accepted as a diagnostic and risk assessment biomarker of cardiac function. Studies suggest that there are significant differences in measured concentrations among different commercial BNP immunoassays. The purpose of our study was to compare BNP-related proteins to determine a form that could be used as a common calibrator to improve the comparability of commercial BNP immunoassay results. METHODS BNP was measured in 40 EDTA-plasma samples from acute and chronic heart failure patients using five commercial BNP assays: Alere Triage, Siemens Centaur XP, Abbott I-STAT, Beckman Access2 and ET Healthcare Pylon. In parallel with internal calibrators from each manufacturer, six preparations containing BNP 1-32 motif a) synthetic BNP, b) recombinant BNP (E. coli), c) recombinant nonglycosylated proBNP (E. coli), d) recombinant His-tagged (N-terminal) nonglycosylated proBNP (E. coli), e) recombinant glycosylated proBNP (HEK cells), and f) recombinant glycosylated proBNP (CHO cells) were also used as external calibrators for each assay. RESULTS Using the internal standards provided by manufacturers and for five of six external calibrators, up to 3.6-fold differences (mean 1.9-fold) were observed between BNP immunoassays (mean between-assay CV 24.5-47.2%). A marked reduction of the between-assay variability was achieved, when glycosylated proBNP expressed in HEK cells was used as the common calibrator for all assays (mean between-assay CV 14.8%). CONCLUSIONS Our data suggest that recombinant glycosylated proBNP could serve as a common calibrator for BNP immunoassays to reduce between-assay variability and achieve better comparability of BNP concentrations of commercial BNP immunoassays.