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Different susceptibility of B-Type natriuretic peptide (BNP) and BNP precursor (proBNP) to cleavage by neprilysin: the N-terminal part does matter.

BACKGROUND: Protease 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.

METHODS: BNP 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.

RESULTS: In contrast to BNP, both forms of proBNP were resistant to degradation by neprilysin. The SESBNP 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.

CONCLUSIONS: These 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. B-type natriuretic peptide (BNP) [3] is a circulating peptide hormone involved in maintaining cardiorenal homeostasis. BNP is synthesized in the form of a prohormone, BNP precursor (proBNP), which consists of 108 amino acid residues (aar) (1). The enzyme-mediated processing of proBNP gives rise to the active BNP hormone (32 aar) and the N-terminal fragment of proBNP (NT-proBNP, 76 aar) (2).

The increased production of proBNP is associated with cardiac pathologies caused by pressure or volume overload. Both products of proBNP processing, BNP and NT-proBNP, are currently established as biomarkers of heart failure (HF) and included in international guidelines (3). Nonprocessed intact proBNP is also present in the circulation and represents a substantial part of the BNP immunoreactivity found in the blood of healthy individuals and HF patients (4-6). Both the exogenous administration and augmentation of the endogenous BNP have been considered as potential therapeutic strategies in HF. A recombinant form of BNP (nesiritide) was introduced as a therapeutic agent for the treatment of this disease. However, this approach is currently considered to be inefficient (7).

It is thought that BNP clearance is, at least partially, associated with cleavage by neprilysin, a ubiquitous protease that is especially abundant in the kidney. Neprilysin cleaves numerous vasoactive peptides, including NPs. All 3 members of the NP family, A-type NP (ANP), BNP, and C-type NP (CNP), are substrates of neprilysin (8). Thus, preventing the breakdown of endogenous NPs by neprilysin inhibition represents an alternative strategy to exogenous BNP administration.

Interest in neprilysin has recently been greatly stimulated by the release of the new HF drug LCZ696 (EntrestoTM), developed by Novartis and approved by the US Food and Drug Administration (9, 10). LCZ696 consists of 2 active components, the neprilysin inhibitor AHU377 and the angiotensin receptor inhibitor valsartan. Because LCZ696 is expected to augment circulating BNP concentrations via inhibition of BNP degradation by neprilysin, it was suggested that treatment with this drug may interfere with the use of BNP measurements for HF diagnosis/prognosis or treatment monitoring by making such measurements diagnostically ambiguous and misleading (9-11).

In clinical practice, BNP concentrations are determined by means of sandwich immunoassays. Because all conventional BNP immunoassays cross-react with proBNP (12), BNP measurements actually reflect the total concentration of both immunoreactive forms, the bioactive BNP and the intact prohormone, proBNP. Notably, the main form of plasma BNP immunoreactivity in HF is represented by proBNP (5, 6). Given the suggested influence of neprilysin inhibition on BNP measurements in patient plasma samples, it is important to know whether proBNP, the major form of BNP immunoreactivity, is as susceptible to neprilysin as BNP is. The goal of the present study was to compare the susceptibility of BNP and proBNP to proteolysis by neprilysin.

Materials and Methods

The following materials were used in this study: synthetic BNP 1-32 from Bachem (5); BNP-specific monoclonal antibody (mAb) KY-BNP-II (epitope 14-21) from Shionogi (13); BNP-specific mAb 24C5 (epitope 11-17), 50E1 (epitope 26-32), Ab-BNP2 (specific for immune complex of BNP/proBNP with mAb 24C5), human recombinant proBNP (expressed in Escherichia colt), and human recombinant glycosylated proBNP (expressed in mammalian cells) from HyTest (5, 14); and recombinant human neprilysin [expressed in CHO (Chinese hamster ovary) cells] from R&D systems (15).

The sandwich 2-step immunofluorescence assay was performed as described previously (16). The mAbs KYBNP-II and 24C5 were used for capture and 50E1 and Ab-BNP2 for detection and were labeled with stable europium chelate (17). The single epitope sandwich (SES)BNP immunoassay has been described previously (14). This assay uses a capture mAb, 24C5, which is specific for the BNP peptide 11-17, and a detection mAb, AbBNP2, which recognizes only the immune complex of mAb 24C5 with BNP/proBNP. The lower limit of detection for the assay is 0.12 pmol/L and the total imprecision is 9.2% (18).

In the proteolysis studies, BNP or proBNP were incubated with neprilysin in a final volume of 0.15 mL containing 300 nmol/L substrate (BNP or proBNP), 40 nmol/L neprilysin, 150 mmol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl, and 0.05% Triton X-100. The control samples, without neprilysin, were incubated under the same conditions. The reaction time, buffer composition, and amount of substrate and neprilysin were similar to those previously used in studies of in vitro BNP cleavage by neprilysin (8, 19). Three repeats were performed for each sample.

The immunoreactivity of synthetic BNP 1-32, nonglycosylated proBNP 1-108, and glycosylated proBNP 1-108 in samples either treated or nontreated with neprilysin was tested using an immunoassay based on antibodies specific to the region 14-21 (mAb KY-BNP-II) in combination with an mAb specific to the region 27-32 of BNP (mAb 50E1). Epitope 14-21 of the mAb KYBNP-II comprises the known BNP cleavage site of neprilysin, [Arg.sub.17]-[Ile.sub.18]. The lower limit of detection for the assay is 4.0 pmol/L and the total imprecision is 9.6%. The SES-BNP assay was used to test the sensitivity of epitope 11-17 to neprilysin cleavage. Values were averaged (n = 3) and the SD was calculated.

Mass spectrometry (MS) was performed on an Ultraflex II MALDI-TOF mass spectrometer (Bruker Daltonik). Before the MS analysis, the samples were lyophilized and then desalted with ZipTip C18 micro tips according to the manufacturer's protocol. We interpreted correspondence of the found masses in MALDITOF experiments to the BNP and peptides using GPMAW 8.0 software (Lighthouse Data) with a precision criterion of 0.01%-0.02%. For calculations, we assumed an intact disulfide bond between Cys-10 and Cys-26 of BNP.

Results

As shown in Fig. 1, in contrast to BNP 1-32, which was cleaved by neprilysin, neither form of proBNP, nonglycosylated or glycosylated, was degraded by neprilysin when measured by the KY-BNP-II (14-21)-50E1 (26-32) assay. The SES-BNP assay (epitope 11-17) was found to be much less susceptible to BNP cleavage by neprilysin compared with the immunoassay utilizing the mAb Ky-BNP-II (epitope 14-21). The SES-BNP assay detected 62.4% of immunoreactive BNP after 4 h of incubation with neprilysin, whereas the immunoassay utilizing mAb Ky-BNP-II detected only 7.4%. Both proBNP forms were resistant to proteolysis as measured by the SES-BNP assay (Fig. 1). The sites of in vitro BNP cleavage by neprilysin as determined by MS are shown in Table 1. ProBNP remained intact even after 6 h of incubation with neprilysin.

Discussion

HF is defined as a clinical syndrome in which the heart is unable to maintain cardiac output to meet metabolic demands. The production of NPs is considered to be a regulatory mechanism aimed to counteract the effects of HF (20-22). The recently introduced HF drug LCZ696 represents a new strategy in HF treatment by inhibiting neprilysin for the prevention of NP breakdown (10). The implementation of LCZ696 in clinical practice may generate new challenges for the use of BNP as a biomarker. It is thought that increasing BNP concentrations in a patient's blood by neprilysin inhibition can make BNP measurements irrelevant for monitoring purposes because the resulting BNP concentrations might not accurately reflect cardiac function in this case (10, 23). However, due to the very complex nature of the NP system, the effect of neprilysin inhibition on the concentrations of proBNP-derived peptides might not be so straightforward. Apart from BNP, the intact precursor proBNP is present in the circulation at concentrations even higher than BNP, and in chronic HF proBNP is the predominant form. All conventional BNP immunoassays are known to substantially cross-react with proBNP (12). Thus, the susceptibility of proBNP to neprilysin is a crucial factor that should be considered in the analysis of the influence of neprilysin inhibition on measured concentrations of BNP.

In the current study, we observed that in contrast to BNP, proBNP, which differs from BNP by the presence of a 76-aar N-terminal extension, was not degraded by neprilysin. This suggests that proBNP is not a substrate for neprilysin. This finding is in accordance with previously published data regarding the influence of the length of N-terminal extensions of NPs on neprilysin activity (24, 25). This influence is well represented by ANP and its N-terminal prolonged analog urodilatin, in that ANP is degraded much faster than urodilatin. Additionally, D-type NP, with the longest extension among all of the NPs, is not degraded by neprilysin (25). Neither the glycosylated nor nonglycosylated forms of proBNP were susceptible to proteolysis by neprilysin, suggesting that glycosylation does not play a role in the resistance of proBNP to neprilysin.

Neprilysin-mediated inactivation of BNP is considered to be one of the clearance mechanisms, but at the same time some authors have reported that BNP is a relatively poor substrate of neprilysin compared with ANP (26-28). So, presently, the available data on the role of neprilysin in the degradation of BNP is not consistent. The reason for this inconsistency is not definitively known. The experimental conditions may have a marked effect on whether neprilysin does or does not degrade BNP. Also, cleavage of BNP by neprilysin is clearly species dependent, which may be the cause of this discrepancy.

There are data showing that, in mice, meprin A, a protease expressed in kidney, may increase the effect of neprilysin by cleaving the first 6 N-terminal residues of mouse BNP (29). The simultaneous action of meprin A and neprilysin together on degradation of human BNP and proBNP appears also to be important to test, although the ability of meprin A to degrade human BNP is rather questionable (28). One may also suggest that the cleavage of the aminoterminal dipeptide from human BNP 1-32 by dipeptidylpeptidase IV may increase the degradation rate of BNP by neprilysin. However, it was shown to have no effect on the resistance of human BNP to human neprilysin, suggesting that the synergistic effect of successive action of several proteases is likely species-specific (19).

BNP is cleaved by neprilysin at several sites. The initial neprilysin cleavage site is in the N-terminus of BNP, [Met.sub.4]-[Phe.sub.5] (30). There are currently no commercial BNP immunoassays that utilize antibodies with epitopes comprising this site. Another known neprilysin cleavage site is located within the ring structure of BNP, i.e., [Arg.sub.17]-[Ile.sub.18] (8), and it is reasonable to suggest that all BNP immunoassays utilizing antibodies with epitopes comprising this site will be sensitive to the proteolytic activity of neprilysin. There are 3 commercially available BNP immunoassays that utilize mAb KYBNP-II specific to the region 14-21: 2 from Siemens (formerly Bayer and Dade Behring) and one from Shionogi (31). Some other commercially available BNP immunoassays (manufactured by Alere and Beckman Coulter) utilize a polyclonal antibody with an undetermined epitope, which may also comprise the site [Arg.sub.17]-[Ile.sub.17]. We found that the epitope 11-17, which is applied in the SES-BNP immunoassay (32), was much less sensitive to BNP degradation by neprilysin than the immunoassay based on the mAb Ky BNP-II (epitope 14-21). We explain the different susceptibilities of the assays to BNP neprilysin cleavage by the observation that different sites within the BNP sequence are not cleaved by neprilysin simultaneously, but instead are cleaved consecutively. As shown by our MS studies, the site [Arg.sub.17]-[Ile.sub.18] is attacked before the other sites, i.e., [Lys.sub.14]-[Met.sub.15], [Gly.sub.23]-[Leu.sub.24], and [Val.sub.28]-[Leu.sub.29].

Our current findings may have several important consequences from a physiological and clinical prospective. First, the lack of susceptibility of proBNP to neprilysin activity suggests that the effect of neprilysin inhibitors on the measured BNP immunoreactivity should not be as significant as might be expected, because a major circulating BNP immunoreactive form, proBNP, is not degraded by neprilysin. Second, we suggest that the beneficial effect of the inhibition of neprilysin activity in HF is likely realized through the augmentation of the concentrations of ANP and CNP, rather than BNP. However, considering the presence of some amount of active BNP hormone in the circulation and its sensitivity to neprilysin, modest increases in BNP concentration might be also responsible for the findings in the PARADIGM-HF (Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure) trial, which demonstrated a marked improvement in outcomes with LCZ696 (neprilysin inhibitor and angiotensin receptor inhibitor) compared with enalapril (inhibitor of angiotensin-converting enzyme) alone in HF patients (9). Third, one may also speculate that proBNP represents an "enhanced BNP" that is not affected by proteolytic degradation in the circulation. Unprocessed proBNP, however, exhibits markedly reduced physiological activity compared with BNP and is currently considered to be insufficient to promote an adequate physiological natriuretic hormone response in HF patients (6). Our previous studies in rats have revealed that processing of proBNP in the circulation is possible (33). Given this, the influence of LCZ696 on the efficiency of proBNP processing, giving rise to bioactive BNP, appears also to be important to evaluate. Finally, BNP immunoassays based on antibodies with epitopes comprising the site [Arg.sub.17]-[Ile.sub.17] are more sensitive to the modulation of neprilysin activity than immunoassays with antibodies that do not have epitopes comprising this site.

In conclusion, the present data suggest that the effect of neprilysin inhibition on the level of immunoreactive BNP in patients treated with LCZ696 may be assay dependent. In patients under LCZ696 therapy, BNP testing with immunoassays utilizing antibodies with epitopes comprising the site [Arg.sub.17]-[Ile.sub.17] may be compromised because of the sensitivity of such assays to the neprilysin-mediated BNP cleavage and, consequently, the results of BNP measurements obtained with this type of assay may not well reflect the cardiac function. One may suggest that NT-proBNP, which is considered to be insensitive to neprilysin cleavage, should be used as HF biomarker, rather than BNP, along with LCZ696 therapy. However, this hasty suggestion is based on an overly simplified model of a complex biological phenomenon. Undoubtedly, more clinical data are needed. Considering the complexity of the NP system and the diversity of HF states, the effect of neprilysin inhibition on the level of BNP and NT-proBNP as well as the BNP/NT-proBNP ratio should be studied in future clinical trials using several immunoassays based on different antibodies to fully utilize the diagnostic and prognostic value of these biomarkers.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: A.G. Semenov, HyTest Ltd.; A.G. Ka trukha, HyTest Ltd.

Consultant or Advisory Role: None declared.

Stock Ownership: A.G. Katrukha, HyTest Ltd.

Honoraria: None declared.

Research Funding: HyTest Ltd., Turku, Finland.

Expert Testimony: None declared.

Patents: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, and final approval of manuscript.

Acknowledgments: We are grateful to Dr. Alexander B. Postnikov for the constructive criticism and helpful comments in the preparation of this manuscript.

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Alexander G. Semenov [1] * and Alexey G. Katrukha [1,2]

[1] HyTest Ltd., Turku, Finland; [2] School of Biology, Moscow State University, Moscow, Russia.

* Address correspondence to this author at: HyTest Ltd., Intelligate, 6th floor, Joukahaisenkatu 6, Turku, Finland 20520. Fax ?2-5120909; e-mail alexander.semenov@hytest.fi.

Received January 8, 2016; accepted January 28, 2016.

Previously published online at DOI: 10.1373/clinchem.2016.254524

[3] Nonstandard abbreviations: BNP, B-type natriuretic peptide; proBNP, BNP precursor; aar, amino acid residue; NT-proBNP, N-terminal fragment of proBNP; HF, heart failure; ANP, A-type NP; CNP, C-type NP; mAb, monoclonal antibody; SES, single epitope sandwich; MS, mass spectrometry.

Caption: Fig. 1. Degradation of BNP 1-32, nonglycosylated (expressed in E. coli) and glycosylated (expressed in mammalian cells) proBNP 1-108 by the action of neprilysin.

The immunoreactivity of BNP, nonglycosylated and glycosylated proBNP was measured by the KY-BNP-II (14-21)-50E1 (26-32) assay and the SES-BNP assay (24C5 (11-17) Ab-BNP2). The immunoreactivity was calculated as (immunoreactivity in neprilysin-treated sample/immunoreactivity in nontreated sample) x 100%. The data are presented as the mean (n = 3)(SD).
Table 1. Sites of BNP cleavage by neprilysin detected
by MS.

Incubation time           Bonds hydrolyzed

0.5 h               [Met.sub.4]-[Val.sub.5]
2 h                 [Met.sub.4[-[Val.sub.5]; [Arg.sub.17]
                    -[Ile.sub.18]
4 h                 [Met.sub.4]-[Val.sub.5], [Arg.sub.17]-
                    [Ile.sub.18], [Lys.sub.14]-[Met.sub.15],
                    [Gly.sub.23]-[Leu.sub.24], [Val.sub.28]-
                    [Leu.sub.29]
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Title Annotation:Proteomics and Protein Markers
Author:Semenov, Alexander G.; Katrukha, Alexey G.
Publication:Clinical Chemistry
Article Type:Report
Date:Apr 1, 2016
Words:4175
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