Alexey G. Katrukha

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

BACKGROUNDnProcessing 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).nnnMETHODSnThe 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).nnnRESULTSnPart 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.nnnCONCLUSIONSnOnly 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

BACKGROUNDnB-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.nnnMETHODSnWe 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.nnnRESULTSnReduction 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.nnnCONCLUSIONSnBoth 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.

Immunodetection of Glycosylated NT-proBNP Circulating in Human Blood

BACKGROUNDnBrain natriuretic peptide (BNP) or NT-proBNP (N-terminal fragment of BNP precursor) measurements are recommended as aids in diagnosis and prognosis of patients with heart failure. Recently it has been shown that proBNP is O-glycosylated in human blood. The goal of this study was to map sites on the NT-proBNP molecule that should be recognized by antibodies used in optimal NT-proBNP assays.nnnMETHODSnWe analyzed endogenous NT-proBNP by several immunochemical methods using a broad panel of monoclonal antibodies specific to different epitopes of the NT-proBNP molecule.nnnRESULTSnTreatment of endogenous NT-proBNP by a mixture of glycosidases resulted in significant improvement of the interaction between deglycosylated NT-proBNP and monoclonal antibodies (MAbs) specific to the mid-fragment of the molecule. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) were able to recognize glycosylated and deglycosylated protein with similar efficiency.nnnCONCLUSIONSnThe central part of endogenous NT-proBNP is glycosylated, making it almost invisible for the antibodies specific to the mid-fragment of the molecule. Thus sandwich assays using even one antibody (poly- or monoclonal) specific to the central part of the molecule could underestimate the real concentration of endogenous NT-proBNP. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) are the best candidates to be used in an assay for optimal NT-proBNP immunodetection.

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

BACKGROUNDnThe 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.nnnMETHODSnWe 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.nnnRESULTSnProBNP 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.nnnCONCLUSIONSnIn 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.

Novel Immunoassay for Quantification of Brain Natriuretic Peptide and Its Precursor in Human Blood

BACKGROUNDnBrain natriuretic peptide (BNP) is an unstable molecule that can rapidly lose immunologic activity in blood. Conventional sandwich BNP immunoassays use 2 antibodies specific to 2 different epitopes. Larger distances between epitopes are associated with a greater probability of proteolysis sites being located between the antibody-binding sites, and thus such assays have an increased susceptibility to underdetect BNP because of the increased likelihood of proteolytic degradation. The purpose of our study was to develop a sandwich immunoassay for the precise quantification of BNP and BNP precursor (proBNP) in human blood that is not susceptible to proteolysis.nnnMETHODSnMice were immunized with an immune complex consisting of monoclonal antibody (MAb) 24C5 (specific for BNP peptide 11-22) and the entire BNP molecule. The MAb used in our assay (Ab-BNP2) recognizes the immune complex but neither free BNP nor MAb 24C5.nnnRESULTSnWe used MAbs 24C5 and Ab-BNP2 to develop a new type of sandwich BNP assay (the single-epitope sandwich assay), which requires only a short BNP fragment (fragment 11-22) for immunodetection. This assay recognizes both BNP and proBNP with the same efficiency and sensitivity and demonstrates both considerably less susceptibility to antigen degradation and greater stability of the measured antigen than conventional sandwich BNP immunoassays.nnnCONCLUSIONSnWe have developed this sensitive single-epitope sandwich assay for detecting BNP, proBNP, and their fragments in human blood. This assay appears promising for use in clinical studies to assist in triage, management, and outcomes assessment in heart failure patients.

N-terminal and C-terminal fragments of IGFBP-4 as novel biomarkers for short-term risk assessment of major adverse cardiac events in patients presenting with ischemia.

OBJECTIVESnPregnancy Associated Plasma Protein A (PAPP-A)-derived N- and C-terminal fragments of IGF-binding protein-4 (NT- and CT-IGFBP-4) released from vulnerable atherosclerotic plaques are proposed to be used for cardiovascular risk assessment.nnnDESIGN AND METHODSnNT- and CT-IGFBP-4 were measured by novel immunoassays in EDTA-plasma of 180 patients admitted to the emergency department with symptoms of myocardial ischemia but without ST-segment elevation. Six-month incidence of major adverse cardiac events (MACE), including myocardial infarction, cardiac death, percutaneous coronary interventions, and coronary artery bypass grafting was recorded.nnnRESULTSnSixteen patients met the endpoint. NT- and CT-IGFBP-4 were strong predictors of MACE: area under ROC curve (AUC) 0.856 and 0.809, respectively. NT-IGFBP-4 concentrations≥214μg/L and CT-IGFBP-4 concentrations≥124μg/L were associated with increased risk of future MACE: adjusted hazard ratio 13.79 and 7.93, respectively.nnnCONCLUSIONSnIGFBP-4 fragments can be utilized as biomarkers for MACE prediction in patients with suspected myocardial ischemia.

Different Susceptibility of B-Type Natriuretic Peptide (BNP) and BNP Precursor (proBNP) to Cleavage by Neprilysin: The N-Terminal Part Does Matter

BACKGROUNDnProtease neprilysin is known to be responsible for the degradation of natriuretic peptides. A recent heart failure (HF) drug, LCZ696 (Entresto(TM)), that combines a neprilysin inhibitor and an angiotensin II receptor inhibitor was suggested to augment circulating B-type natriuretic peptide (BNP) concentrations, making the results of BNP measurements diagnostically ambiguous. Because the main form of measured BNP in HF patients is represented by its uncleaved precursor, proBNP, it is important to know the susceptibility of proBNP to cleavage by neprilysin.nnnMETHODSnBNP 1-32 and nonglycosylated and glycosylated forms of proBNP 1-108 were incubated with neprilysin for different time periods. BNP immunoreactivity was analyzed using 2 sandwich immunoassays: one utilizing monoclonal antibody (mAb) KY-BNP-II (epitope 14-21) as capture with mAb 50E1 (epitope 26-32) for detection and a single-epitope sandwich BNP (SES-BNP) immunoassay specific to the epitope 11-17. Mass-spectrometry was applied to determine the sites of BNP cleavage.nnnRESULTSnIn contrast to BNP, both forms of proBNP were resistant to degradation by neprilysin. The SES-BNP assay was much less susceptible to the BNP cleavage by neprilysin compared with the immunoassay utilizing antibodies specific to the region 14-21, comprising the site Arg17-Ile18, known as the site of BNP cleavage by neprilysin.nnnCONCLUSIONSnThese findings suggest that modulation of neprilysin activity by specific inhibitors may not greatly influence the circulating concentrations of immunoreactive BNP, mostly represented in HF by proBNP, which is not susceptible to neprilysin. The different susceptibility of the BNP regions to neprilysin-dependent degradation highlights the importance of the choice of epitopes for reliable BNP immunodetection.

Clinical differences between total PAPP-A and measurements specific for the products of free PAPP-A activity in patients with stable cardiovascular disease

OBJECTIVESnWe have previously reported that increases in total pregnancy-associated plasma protein-A (PAPP-A) which are thought to be indicative of vulnerable plaques and thus poor outcomes predict outcomes in patients with stable coronary artery disease. We hypothesized that the determination of CT- and NT-fragments of insulin-like growth factor binding protein 4 (CT- and NT-IGFBP4) which should be indicative of free PAPP-A would result in better performance.nnnMETHODSnIn 229 stable cardiovascular patients with indication for heart catheterization after performance of a stress test and an echocardiogram, CT- and NT-IGFBP4 were measured. Their values were investigated in relation to clinical characteristics, findings of noninvasive investigations, laboratory data and coronary angiography as well as to outcomes after a follow-up of 1094±307days.nnnRESULTSnCT-IGFBP4 values were independently predicted by patients with B-type (p=0.0069) or complex coronary lesions (p=0.0445). B-type and vulnerable coronary lesions were independently predicted by levels of CT-IGFBP4≥a cutoff of 31.55ng/mL derived from ROC analysis (p=0.0090 and 0.0480). NT-IGFBP4 was not predictive of coronary characteristics. Both IGFBP4 fragments were strongly dependent on age and renal function and were not predictive of outcomes.nnnCONCLUSIONnDespite the relation of CT-IGFBP4 to a more severe coronary artery disease, CT- and NT-IGFBP4, in contrast to our report based on total PAPP-A, failed to predict any long-term outcomes in patients with stable cardiovascular disease. Further knowledge about the interaction of the PAPP-A-insulin-like growth factor system is needed to explain values of IGFBP4 fragments in these patients.

Measurement of B-type natriuretic peptide by two assays utilizing antibodies with different epitope specificity.

OBJECTIVESnTo compare plasma BNP values determined by a conventional-type and a novel-type of BNP assays.nnnDESIGN AND METHODSnPlasma samples (n=94) from HF patients were analyzed by the novel-type Single Epitope Sandwich (SES assay prototype) and the conventional-type Siemens ADVIA Centaur BNP assays. Both assays were calibrated using recombinant proBNP (expressed in E. coli).nnnRESULTSnThe SES assay measured 1.2- to 7.2-fold (2.1±0.9; mean, SD) greater BNP concentrations compared to the Siemens assay. A subset of six samples (6.4%) demonstrated the largest (3- to 7.2-fold higher) difference.nnnCONCLUSIONSnThe SES prototype assay appears to more accurately measure the absolute concentrations of BNP immunoreactive forms.

Thrombin-Mediated Degradation of Human Cardiac Troponin T

BACKGROUNDnCardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation.nnnMETHODSncTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis.nnnRESULTSnThe comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69.nnnCONCLUSIONSnThe results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.