B-type Natriuretic Peptide circulating forms: Analytical and bioactivity issues

    B-Type Natriuretic Peptide circulating forms: Analytical and bioactivity issues Tim G. Yandle, A. Mark Richards PII: DOI: Reference:

S0009-8981(15)00329-0 doi: 10.1016/j.cca.2015.07.004 CCA 14030

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Clinica Chimica Acta

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19 December 2014 4 July 2015 5 July 2015

Please cite this article as: Yandle Tim G., Richards A. Mark, B-Type Natriuretic Peptide circulating forms: Analytical and bioactivity issues, Clinica Chimica Acta (2015), doi: 10.1016/j.cca.2015.07.004

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ACCEPTED MANUSCRIPT B-Type Natriuretic Peptide circulating forms: analytical and bioactivity issues a*

Tim G. Yandle , A. Mark Richards

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Department of Medicine, Christchurch Heart Institute, University of Otago, Christchurch, 2 Riccarton b Avenue, Christchurch 8140, New Zealand; Cardiac Department, Cardiovascular Research Institute, National University Heart Centre, National University of Singapore, 1E Kent Ridge road, NUHS Tower Block, Level 9, Singapore 119228, Singapore.

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*Corresponding author at: Department of Medicine, Christchurch Heart Institute, University of Otago, Christchurch, 2 Riccarton Avenue, Christchurch 8140, New Zealand. Tel.: +64 3 3640895. E-mail address [email protected]

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Abstract

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B-type Natriuretic Peptide (BNP), A-type and C-type Natriuretic Peptides (ANP and CNP) comprise a family of peptides that retain a common ring structure and conserved amino acid sequences. All are present in the heart, but only BNP and ANP are regarded as primarily cardiac secretory products. BNP and ANP, acting through a guanylyl cyclase receptor, increase sodium and water excretion by the kidney, induce vasodilation, reduce blood pressure, counteract the bioactivity of the reninangiotensin-aldosterone and sympathetic nervous systems and possess anti-hypertrophic and antifibrotic properties. BNP is synthesised in cardiomyocytes first as the precursor peptide preproBNP. Removal of the signal peptide from preproBNP produces proBNP which is cleaved to produce the biologically active carboxy-terminal BNP peptide and the inactive N-terminal fragment, NT-proBNP. BNP, NT-proBNP, proBNP and the C-terminal portion of the BNP signal peptide have been detected in human plasma as well as multiple sub-forms including truncated forms of BNP and NT-proBNP, as well as variable glycosylation of NT-proBNP and proBNP. The origin of these circulating forms, their potential bioactivity and their detection by current analytical methods are presented in this review.

Keywords: BNP, proBNP, NT-proBNP, glycosylation, truncation, natriuretic peptide

Abbreviations: ANP, A-Type Natriuretic Peptide (atrial natriuretic peptide): BNP, B-Type Natriuretic Peptide (brain natriuretic peptide); CNP, C-Type Natriuretic Peptide; proBNP, pro B-Type Natriuretic Peptide; NT-proBNP, amino-terminal proBNP; IDE, Insulin Degrading Enzyme; DPP-IV, Dipeptidyl Peptidase IV; GLP-1, Glucagon-Like Peptide-1; GIP, Glucose-dependent Insulinotropic Polypeptide; MS, Mass Spectrometry; NPR-A, NPR-B, NPR-C, natriuretic peptide receptors A, B, and C respectively: N-terminal or C-terminal, amino-terminal or carboxy-terminal end of a peptide.

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1 Introduction

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B-Type Natriuretic (BNP) - originally called Brain Natriuretic Peptide, together with two other related

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peptides A-Type natriuretic peptide (ANP, Atrial Natriuretic Peptide) and C-Type natriuretic peptide (CNP) comprise a family of peptides. They share a 17 amino acid ring structure within which six

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amino acids are conserved across the family (Figure 1). Apart from amino acid sequence differences, each member is distinguished by the length of the amino-terminal (N-terminal) and carboxy-terminal (C-terminal) arms extending from each ring. BNP and ANP are synthesised in both the atria and

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ventricles of the heart in response to increased transmural pressure or stretch of cardiomyocytes [1, 2]. BNP is predominantly synthesised and secreted from ventricular tissue while ANP is mainly

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secreted from atrial tissue in healthy subjects with an increasing proportion of ANP derived from ventricular tissue in heart failure where secretion rates are high.

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The natriuretic peptides (NP) elicit their actions through two membrane bound receptors, Natriuretic

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Peptide Receptor-A (NPR-A), which binds both ANP and BNP, and Natriuretic Peptide Receptor-B (NPR-B) which binds CNP. Binding of the appropriate peptides to these receptors activates their

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intracellular guanylyl cyclase domain to produce the second messenger cGMP which transduces their actions. A third receptor lacking an intracellular domain - Natriuretic Peptide Receptor-C (NPR-C) receptor, often called the natriuretic peptide “clearance” receptor, is able to bind all three members of

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the family, ANP, BNP and CNP. As inferred by its name, this receptor binds and internalises the NP, which are then subjected to intracellular degradation [3, 4]. Circulating NP are also eliminated by enzymatic degradation in circulation. Studies in sheep, for example, have shown that the NPR-C receptor and neprilysin play equivalent roles in clearing NP [5]. Similarly, in humans with left ventricular impairment, administration of a neprilysin inhibitor results in a clear twenty five percent reduction in the metabolic clearance rate of infused BNP [6]. BNP has qualitatively similar physiological actions to those of ANP in promoting natriuresis and diuresis through its action on the kidney and its antagonism of the salt and fluid retaining reninangiotensin-aldosterone system. These actions of BNP to eliminate salt and water, together with its direct action on the vascular system to induce vasodilatation, culminate in a reduction of blood

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ACCEPTED MANUSCRIPT pressure [7-9]. Further beneficial effects accrue from its local action within the heart where it exerts antifibrotic and antihypertrophic actions [10, 11].

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The increased circulating concentrations of BNP and amino-terminal proBNP (NT-proBNP) in heart

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failure and their dynamic correlation with its severity and left ventricular dysfunction [12, 13] have driven the extensive use of these peptides, now endorsed in all authoritative clinical guidelines for the

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diagnosis and management of heart failure. Evaluation of these tests in multicentre observational studies and in randomized therapeutic trials [14-16] have confirmed the utility of both BNP and NTproBNP in (i) the diagnosis of acute heart failure in patients with dyspnoea, (ii) risk stratification in all

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grades of acute and chronic heart failure and in (iii) serial monitoring of cardiac status and titration of anti-heart failure treatment. In particular, the American Guideline for the Management of Heart Failure

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strongly supports the measurement of BNP and NTproBNP for the diagnosis of heart failure and acutely decompensated heart failure (ADHF). Their use for establishing prognosis or disease severity

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in patients with chronic heart failure or ADHF is also strongly supported [17]. However, the

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performance of these immunoassay-based tests is dependent on their recognition of BNP or NTproBNP in the circulation. The multitude of recently identified BNP and NT-proBNP circulating forms

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has exposed some limitations in current assays, but may also represent an opportunity to develop improved methods.

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1.1 Synthesis and metabolism of BNP The bioactive form of human BNP was initially identified through its purification from human atrial tissue and shown by sequencing to be the 32 amino acid form of BNP shown in Figures 1 and 2 [18, 19]. The high molecular weight BNP precursor proBNP was also identified in the same atrial extracts by a combination of amino acid sequencing and comparison with the established proBNP cDNA sequence [18, 20]. Both peptides were quickly shown to circulate in normal subjects and in patients with heart failure where circulating BNP concentrations were markedly raised. Further, the proportion of proBNP was generally greater than BNP in heart failure patients [21-23]. These early studies typically resolved immunoreactive BNP into high and low MWt BNP (proBNP and BNP) forms with gel chromatography or size exclusion HPLC that did not have the ability to resolve BNP metabolite forms from BNP itself. However at least one early study using reverse phase HPLC noted the presence in plasma of low molecular weight BNP forms other than the established 32 amino acid form [23].

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ACCEPTED MANUSCRIPT Identification of BNP and proBNP in the heart suggested that BNP is cleaved from the proBNP precursor by enzymatic processing between amino acid residues 76 and 77, but the fate of the aminoterminal portion of proBNP (residues 1-76) was unknown. This amino-terminal fragment of proBNP

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(NT-proBNP,) was later identified in human plasma, shown to be secreted by the heart and to have

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raised concentrations in subjects with heart failure [12, 24]. Subsequent studies have established that

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cleavage of proBNP between residues 76 and 77 is most likely undertaken by the enzymes furin and/or corin [25].

These foundations, completed 10 years ago, laid the basis for the BNP synthesis scheme shown in

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Figure 2 where the 108 amino acid precursor proBNP, produced in cardiomyocytes, is cleaved between residues 76 and 77 by the processing enzymes corin or furin to produce the biologically

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active 32 amino acid BNP peptide plus the 76 amino acid NT-proBNP peptide. All three peptides proBNP, BNP and NT-proBNP are secreted by the heart and circulate in humans [1, 12, 21, 26]. This

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scheme has been greatly enhanced over the last decade with; (i) the introduction of sensitive mass

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spectrometry methods providing identification of many new low molecular weight circulating forms of BNP, and the absence or near-absence of the 32 amino acid form in some subjects; (ii) detection of

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proBNP and NT-proBNP glycosylation at multiple sites; (iii) identification of truncated forms of NTproBNP and proBNP in circulation; and (iv) identification of a BNP signal peptide fragment in circulation. These recent developments will be discussed separately below.

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The numerous circulating proBNP, NT-proBNP and BNP circulating forms and the nomenclature used to describe them can be confusing. The nomenclature and sequence numbering in current use is based on the three main products of the BNP gene – proBNP, NT-proBNP and BNP (Figure 2). The first amino acid in each of these peptides is designated as amino acid number 1. Each peptide or fragment is named and followed the amino acid sequence numbers in brackets. For example, the full name for the complete 108 amino acid proBNP peptide is proBNP(1-108). Likewise the mature 76 amino acid NT-proBNP peptide is NT-proBNP(1-76) and that for the mature 32 amino acid BNP peptide is BNP(1-32). Fragments derived from these peptides are similarly named. For instance, proBNP and BNP peptides that have lost the first two amino acids are named as proBNP(3-108) and BNP(3-32) respectively. Unfortunately this system does not provide unique names. As can be seen in Figure 2, BNP is cleaved from the carboxy-terminal end of proBNP and contains proBNP amino acids

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ACCEPTED MANUSCRIPT 77 through to 108. It is equally valid to name this mature BNP peptide as BNP(1-32), which is in common use, or to name it as a proBNP fragment i.e. proBNP(77-108) (see Figure 2). In this review we have followed current practice in naming each peptide fragment based on its derivation from

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proBNP(1-108), NT-proBNP(1-76) or BNP(1-32).

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1.2 Enzymes Degrading BNP

Many of the circulating forms of BNP described below have been produced by the action of circulating or cell surface enzymes. It is likely that many enzymes participate in this process, however four

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enzymes in particular are known to cleave BNP at sites that would produce many of the circulating BNP forms. These four enzymes, Neprilysin, Dipeptidyl Peptidase IV, Insulin Degrading Enzyme and

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Meprin, are described below.

Neprilysin (EC.3.4.24.11) has dominated the literature on natriuretic peptide degradation since it was

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identified as an enzyme responsible for the degradation of ANP. This enzyme, also known as neutral

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endopeptidase, endopeptidase-24.11 and enkephlinase A, inactivates ANP by a primary cleavage between Cys-7 and Phe-8 within the ring structure. Cleavage at this position produces an inactive ring

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opened metabolite that is present in the circulation [27-32]. As shown in Figure 3, neprilysin cleaves BNP at two main positions [33], of which cleavage between Met-4 and Val-5 is the primary cleavage site with slower cleavage within the ring structure between Arg-17 and Ile-18. Cleavage at these

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positions would give rise to the truncated metabolite BNP(5-32), and the ring opened metabolites BNP(1-17:18-32) and BNP(5-17:18-32) – all of which have been identified after BNP incubation with purified neprilysin [34-36]. However Incubation of human BNP with purified human, porcine or mouse neprilysin shows its degradation rate is tenfold slower than that of ANP or CNP, probably due to its much higher Km and lower catalytic rate constant [34-37]. These effects, together with a lower affinity of human BNP for the NPR-C clearance receptor compared to ANP, contribute to the longer half-life of BNP in humans (22 minutes) compared to ANP (3 minutes) [38]. Hence human BNP is relatively resistant to the action of neprilysin despite retention of the Cys-Phe bond that is readily cleaved in ANP [4, 35, 37, 38]. The serine protease dipeptidyl-peptidase IV (DPP-IV) removes dipeptides from the N-terminal end of proteins or peptides, commonly cleaving after X-Pro or X-Ala residues where X represents any amino acid. The requirement for Pro or Ala at these positions however is not strict because they can be

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ACCEPTED MANUSCRIPT replaced by at least five other amino acids. DPP IV is present in many tissues and can truncate a large number of peptides in vitro [39, 40], but is best known for its inactivation of the incretin peptides Glucagon-Like Peptide-1 (GLP-1) and Glucose-dependent Insulinotropic Polypeptide (GIP) that

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stimulate insulin secretion [41], and for the potentiation of insulin secretion by administration of DPP

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IV inhibitors [42]. BNP contains a preferred N-terminal Ser-Pro sequence that is rapidly removed by

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DPP IV to produce the metabolite BNP(3-32) [36] (Figure 3). Likewise, proBNP and NT-proBNP possess a similar N-terminal His-Pro sequence that is probably removed by DPP IV giving rise to proBNP(3-108) and NT-proBNP(3-76) derivatives [43].

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Insulin degrading enzyme (IDE) is a metallopeptidase that regulates insulin and cerebral amyloid beta peptide levels [4, 44]. Ralat et al have recently shown that this enzyme degrades all three members of

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the natriuretic peptide family [4, 45] and, as shown in Figure 3, initially cleaves BNP near its Cterminal end between Leu-29 and Arg-30 and also between Arg-30 and Arg-31. A secondary site with

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slower cleavage is located near the N-terminal end between Lys-3 and Met-4. Cleavage at these sites

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could result in the formation of BNP metabolites truncated after positions 4, 29 or 30. A further three minor cleavage sites within the BNP ring could also result in multiple ring opened or deleted forms

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[45].

The enzyme meprin is widely distributed in tissues including the brush border membranes of the kidney and has been shown to cleave mouse, rat and pig BNP between amino acids 6 and 7 to

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produce their corresponding BNP(7-32) forms [46]. However, two opposing claims have been made for Meprin action on human BNP(1-32). In the first, human BNP(1-32) was shown to be cleaved between Gly-7 and Ser-8 to produce BNP(8-32) [47], however a recent study has not found any evidence for Meprin cleavage of human BNP [48].

2 BNP circulating forms The work of Shimazu et al first drew attention to the possibility that the 32 amino acid BNP(1-32) form may not be the major BNP form in plasma. Their study, using HPLC coupled to immunoassay, found that BNP(1-32) is at most a very minor constituent of peripheral plasma being rapidly truncated to BNP(3-32) upon incubation in plasma [49] –a process now known to be catalysed by the enzyme

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ACCEPTED MANUSCRIPT DPP IV that rapidly removes the amino-terminal Ser-Pro dipeptide from BNP [36]. Subsequent progress on the identification of this and other BNP metabolites has been facilitated by the availability and improved sensitivity of mass spectrometry (MS) instruments. Even so, the low concentrations of

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individual circulating BNP metabolites has precluded their analysis in healthy individuals instead

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restricting analysis to patients with heart failure where immunoreactive BNP concentrations are high.

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These investigations, often using an immunoaffinity purification/concentration step combined with specific inhibitors to prevent BNP degradation, have identified a substantial number of truncated BNP forms in plasma and have confirmed BNP(1-32) to be absent or present only in very low

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concentrations. Initially, BNP(1-32) was not detected in blood from three patients with heart failure [50]. However it was later found at low concentrations in eleven out of twelve patients with NYHA

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class III or IV heart failure where its mean concentration was 37 pg/ml compared to 3342 pg/ml of immunoreactive BNP measured in the same samples by the Biosite Triage immunoassay [51]. Similarly low concentrations of MS measured BNP(1-32) were also found in thirty nine out of seventy

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class I to IV heart failure patients at 19pg/ml compared to 722 pg/ml of immunoreactive BNP

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measured with the Beckman DXI assay [52]. The respective 90 and 40 fold differences between MS measured BNP(1-32) and immunoreactive BNP concentrations in these studies is substantial and

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remains largely unexplained. Even the sums of all MS measured fragments in the first study [51] did not come close to the BNP concentrations measured by immunoassay. However, proBNP cross-

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reactivity in these assays could explain some, but not all, of the discrepancy in results between measurement techniques. At least six BNP peptides truncated at their N- or C- terminal ends have been identified in patients with heart failure (Figure 4A) of which the N-terminal truncated forms BNP(3-32), BNP(4-32) and BNP(5-32) are more prevalent, but smaller amounts of the N- and C-terminal truncated forms BNP(531), BNP(5-27) and BNP(5-26) also circulate [51-53]. Individually, these fragments circulate at similar concentrations to BNP(1-32) ranging from 7 pg/mL to 228 pg/mL but on average BNP(1-32) still represents only 10% of total MS measured BNP forms, suggesting substantial degradation of BNP in circulation [51]. As shown in Figure 4A the cleavage sites required to produce these truncated BNP forms fit within the activity profiles of IDE, neprilysin or DPP IV shown in Figure 3, but as yet there is no direct proof of their in vivo action on circulating BNP(1-32).

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ACCEPTED MANUSCRIPT Taken together, it is clear that BNP(1-32) is metabolised to smaller metabolites, at least in patients with heart failure. It is cleaved near its N- and C- terminal ends to such an extent that very little, if any, BNP (1-32) remains in circulation, and instead its truncated metabolites predominate. The extremely

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low circulating concentrations of MS measured BNP(1-32), and even its sum with all other MS

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measured fragments, is in stark contrast to the many fold higher immunoreactive BNP concentrations

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measured in the same samples with the Triage immunoassay [51]. These large differences are difficult to reconcile and at best can be only partly explained by the cross-reactivities that affect BNP

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immunoassays described below.

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2.1 Effect of BNP Truncation on BNP Immunoassays

Little is known about the cross-reactivities of truncated BNP forms in BNP assays apart from BNP(3-

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32), which has close to full cross-reactivity in five different immunoassays [49, 54, 55]. Similarly,

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BNP(4-32), had close to 160% cross-reactivity in three immunoassays (Access 2, ADVIA Centaur, and AxSYM), while BNP(1-31) cross-reacted only in the Access 2 immunoassay but not the ADVIA

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Centaur and AxSYM assays [49, 55]. These cross-reactivity patterns reflect the use of inner arm (e.g. amino acids 5-13) or ring-directed antibodies that avoid the truncated N-terminal end, combined with C-terminal directed antibodies that are sensitive to C-terminal truncation. The Shionogi BC203

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antibody (used in Abbott AxSYM, Siemens Dimension, Siemens Advia and Shionoria assays) for example, requires the presence of the C-terminal His-32 on BNP [49, 56] and should not recognise any circulating C-terminal truncated forms. In contrast, their N-terminal directed antibodies include one from Scios that requires BNP residues 5-13 or one from Shionogi (KY-BNP-II) that requires amino acids 14-21. Both antibodies should recognise the main N-terminal truncated forms as they contain these BNP 5-13 or 14-21 epitopes. So the Abbott, Siemens and Shionoria assays that employ either of the two N-terminal antibodies in conjunction with the BC203 antibody should recognise the main truncated forms including BNP(1-32), BNP(3-32), BNP(4-32) and BNP(5-32), but not BNP(5-26), BNP(5-27) and BNP(5-31). A table of BNP and NT-proBNP diagnostic assays and antibodies used in them is available on the International Federation of Clinical Chemistry website [57]. BNP assays also cross-react with proBNP because it carries the entire BNP(1-32) molecule at its Cterminal end. Most BNP results therefore carry some contribution from cross-reactivity to proBNP.

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ACCEPTED MANUSCRIPT However, estimates of proBNP cross-reactivities vary considerably in the few instances where they have been measured. For example the Biosite Triage point of care assay has been reported as having <2%, 5-19% and close to 100% cross reactivity to proBNP [54, 58, 59] while proBNP cross-

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reacted 70-80% in the Shionogi assay [60]. In other assays (Architect, AxSYM, Centaur, Access,

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Triage) it had 6-14% recovery while its glycosylated form had greater recovery at 17-38% in the same

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assays [58]. This variability in cross-reactivity seen with proBNP may be related to difficulties in measuring its cross-reactivity with peptide standards. Recombinant peptides used as standards may have tags attached to them and are often not supplied with purity data such as net peptide content

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from amino acid analysis. Similarly, recombinant glycosylated proBNP supplied by HyTest is not provided with a molecular weight presumably due to a variable degree of glycosylation in the product.

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A more practical approach to assessing the contribution of endogenous proBNP to BNP immunoassay results has been to compare BNP results on plasma samples before and after proBNP

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depletion using an antibody specific for proBNP. Comparison of BNP concentrations in 180 patients

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with chronic heart failure revealed a 53% decrease in apparent BNP concentration as measured on the Beckman Access 2 analyser following depletion of proBNP [61]. Such a large decrease suggests

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the influence of proBNP on BNP assays in general may be even greater than that indicated by the cross-reactivity data above.

Based on current knowledge, epitopes for diagnostic assays would ideally be located between BNP

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residues 6 and 26 to avoid the variable effects arising from truncation of BNP peptides in circulation (Figure 4). Assays using these epitopes should measure most truncated forms of BNP and give an integrated measure of BNP secretion. As described above, current diagnostic immunoassays largely meet these criteria in employing at least one ring directed antibody [49, 57] and cross react with proBNP, but are unlikely to detect the C-terminal truncated forms. However, the latter forms are relatively rare being reported at very low concentrations in only 3 out of 12 heart failure patients [51] and seem unlikely to influence diagnostic decisions based on the assays. The cross-reactivity of proBNP in these assays can also be regarded as an advantage as their combined measurement of BNP and proBNP will reflect “total” BNP secretion. This could contribute to an improved diagnostic performance relative to measurement of BNP alone. Assays designed to measure specific BNP forms may also find a use. As discussed in section 3, changes in proteolytic enzyme expression in the heart

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ACCEPTED MANUSCRIPT during remodelling or the development of hypertrophy could increase production of some BNP metabolites secreted by the heart. Specific measurement of these metabolites in the peripheral circulation could reflect early changes in cardiac tissue enzyme expression and lead to earlier

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diagnosis. Similarly, changes in proBNP glycosylation at residue Thr-71 between acute and chronic

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2.2 Bioactivity of Truncated BNP Forms

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heart failure patients [62] justify further exploration with mass spectrometry based assays.

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The secreted and circulating forms of BNP mediate its bioactivity in both the heart and peripheral tissues. This activity is usually assessed by incubation of BNP or its forms with cell lines that express the NPR-A and/or NPR-B receptors, or with mammalian cell lines expressing recombinant forms of

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these receptors. Upon activation by BNP the intracellular portion of the receptors generate the second

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messenger cGMP whose response can be measured. Using these methods, Niederkofler et al (unpublished results in [51]) measured cGMP responses to the truncated BNP forms found in their

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MS study. All of these forms, including the N-terminal truncated forms BNP(3-32), BNP(4-32), BNP(532); the N- and C- terminal truncated forms BNP(5-31); the C-terminal truncated forms BNP(1-25) and BNP(1-26), retained bioactivity giving similar responses to BNP(1-32) [51]. Heublin et al have also

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shown that the stimulation of cGMP production by the DPP IV product BNP(3-32) is similar to that of BNP(1-32) when tested on human cardiac fibroblasts or cardiomyocytes [54]. Similarly BNP(8-32), a possible product of Meprin action on human BNP(1-32), and mouse BNP(7-32) retained full bioactivity when tested in appropriate systems [46, 63]. When combined, these in vitro data suggest that N- or C-terminal truncation does not lead to a drastic loss of BNP receptor binding and subsequent decreased cGMP production. However, differences between the intact and truncated forms do appear when tested in vivo. Infusion of human BNP(3-32) or BNP(8-32) in dogs showed that BNP(3-32) elicited a reduced natriuresis and diuresis, lacked vasodilatory actions and increased urinary cGMP to a lesser extent than BNP(1-32) [64]. In contrast, BNP(8-32) with its longer truncation retained the vasodilating actions of BNP(1-32), but like BNP(3-32) had reduced natriuretic, diuretic and urinary cGMP responses compared to BNP(1-32) [63]. These reduced in vivo responses compared to those

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ACCEPTED MANUSCRIPT measured in vitro could result from more rapid degradation in vivo if the truncated forms were more

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susceptible to degradation than BNP(1-32) [64].

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3 Secreted Forms of BNP

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The measurement and characterization of secreted BNP forms can be achieved by sampling blood from the coronary sinus of the heart, which contains venous blood draining atrial and ventricular tissues. This blood containing newly secreted BNP can be sampled by placing a catheter into the

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coronary sinus during right heart catheterization. Net secretion by the heart can be calculated from the difference between the coronary sinus BNP concentration and that in a simultaneously drawn

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arterial sample. We and others have used these techniques coupled with immunoassay measurement to show that immunoreactive BNP, NT-proBNP and proBNP are all secreted by the heart [1, 12, 21,

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26]. The identity of these major forms has been established by size exclusion HPLC, or by reverse

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phase HPLC with relatively steep gradients that do not resolve truncated peptides from their parent forms. However, the full identity of secreted BNP forms has received little attention apart from an early

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study in which we found BNP metabolites comprised one third of the immunoreactive BNP in coronary sinus plasma [23]. We have recently used highly specific antisera to the N- and C- terminal ends of BNP combined with reverse phase HPLC to characterize the BNP forms present in matching coronary

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sinus and arterial plasma samples. The main immunoreactive BNP peak in the coronary sinus samples consistently eluted ahead of BNP(1-32) standards indicating this material was unlikely to be intact BNP(1-32). However, a considerable proportion of the immunoreactive BNP eluted at the position of BNP(3-32) suggestive of DPP IV action, and at other positions consistent with truncated and ring cleaved metabolites [65]. These peaks observed in coronary sinus plasma were larger than those in matching arterial plasma indicative of their cardiac secretion. While these results need confirmation with more specific mass spectrometry analysis, they indicate that much of the BNP secreted by cardiac tissues is converted to other forms before or during its exit from the heart. Several enzymes currently known to degrade BNP, namely DPP IV, IDE and neprilysin, are located within the heart and may be in a position to mediate this conversion [39, 66-70]. Of these, DPP IV is ideally situated in cardiac capillaries [39, 71] with high levels of expression in cardiac microvascular endothelial cells [72] that could degrade BNP during its transport out of cardiac tissues. In a similar

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ACCEPTED MANUSCRIPT manner, DPP IV lining gut capillaries degrades a substantial portion of newly secreted glucagon-like peptide-1 before it leaves the local venous drainage [73]. Neprilysin is also well positioned within the heart to degrade BNP after cellular secretion. It is located on ventricular myocytes and in non-

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myocyte cardiac tissue where its expression and activity are increased fourfold in patients with heart

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failure [68], although its levels are not elevated in ventricular tissue membranes from heart failure

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patients [69]. Changes in cardiac expression of these enzymes due to remodelling and development of hypertrophy could alter the pattern of BNP metabolite forms entering the circulation. Analysis of these metabolite forms in coronary sinus plasma could identify those forms that reflect these early

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changes. While routine diagnostic sampling from the coronary sinus is not practicable, knowledge of BNP forms with diagnostic potential that enter the general circulation and how they are subsequently

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modified may help identify diagnostically useful forms in peripheral plasma. The recent observation that the ratio of BNP(5-32) to BNP(3-32) may be useful as a rule out test for restenosis [53] suggests

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that measurement of such peripheral BNP fragments might be useful.

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4 ProBNP and NT-proBNP Circulating Forms ProBNP is now recognised as a major circulating form of BNP that is present in healthy subjects as well as those with heart failure. Confirmation that the high molecular weight form identified in early

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studies [21-23] was indeed proBNP has come from its immunologic characterisation in two-site immunoassays, a specific immunoassay recognising the processing site unique to proBNP and by MS identification of its fragments following tryptic digestion [74-76]. Recent studies using a proBNPspecific assay have established that proBNP is secreted by the human heart [26] and circulates in humans. In healthy individuals, its median concentrations in two studies were 14 and 54 pg/mL and were raised to 271pg/mL in patients with NYHA class III or IV heart failure [77] and 150pg/mL in class C heart failure [78]. In general, median proBNP concentrations are either similar to those of BNP [75, 78, 79] or two fold higher [77], but are less than those of NT-proBNP [78]. NT-proBNP was initially identified in plasma from patients with heart failure as a high molecular weight 8.6KDa peptide recognised by antisera specific to its N-terminal end [24]. It has subsequently been further characterised with many antisera in single and two-site immunoassays [80] and by MS after tryptic digestion [76]. This 76 amino acid peptide is produced from its precursor peptide proBNP

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ACCEPTED MANUSCRIPT by the processing enzymes furin or corin that cleave between amino acids 76 and 77 to produce the bioactive peptide BNP(1-32) and NT-proBNP(1-76) (Figure 2). It is secreted by the heart [12, 81] and cleared by multiple tissues including the kidneys [81], has a longer half-life (in sheep) of 70 minutes

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than the 22.6 minute half-life of BNP in humans [9, 82] and consequently circulates at higher

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concentrations than BNP [12]. Its raised plasma concentrations in patients with heart failure and its

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equivalent diagnostic performance to BNP have encouraged its widespread use in diagnosis and

4.1 Truncated proBNP and NT-proBNP Forms

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management of heart failure [83].

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As described in section 1.2, DPP IV rapidly cleaves the N-terminal Ser-Pro dipeptide from BNP to produce the truncated BNP(3-32) form. It also cleaves a similar His-Pro dipeptide off the N-terminal

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end of proBNP, although at a fourfold slower rate, to produce the truncated proBNP(3-108) form [43].

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This peptide circulates at relatively low concentrations in normal subjects, but although proBNP(3108) concentrations are raised in patients with asymptomatic left ventricular dysfunction or heart th

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failure (50 percentiles 8, 18, and 43 pmol/L respectively) they are inferior to BNP and NT-proBNP for detection of asymptomatic left ventricular dysfunction or heart failure in these groups [43]. Since the entire sequence of NT-proBNP is identical to the N-terminal end of proBNP it is not surprising that

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truncation has also been observed in circulating NT-proBNP. Chromatographic analysis of NTproBNP using immunoassays specific to its N- and C- terminal ends shows these assays detect less immunoreactive NT-proBNP compared to an assay recognising a more internal NT-proBNP region indicating that circulating NT-proBNP is truncated at it N- and C- terminal ends [84]. These indirect observations have recently been extended by MS analysis after tryptic digestion of immunopurified NT-proBNP. Foo et al identified several non-tryptic NT-proBNP fragments in the digest that revealed truncation of the endogenous peptide at its N-terminal and C-terminal ends [85]. Cleavage points within NT-proBNP that would give rise to these fragments are shown in Figure 4. These include the DPP-IV cleavage site between Pro-2 and Leu-3 plus other sites near the N- and C- terminal ends of NT-proBNP. Together, these observations strongly point to circulating NT-proBNP being comprised of some intact NT-proBNP (1-76) peptide accompanied by a substantial content of N- and C- terminal truncated forms.

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4.2 Glycosylation of ProBNP and NT-proBNP

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Schellenberger et al first established that proBNP was glycosylated after finding that human

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recombinant proBNP synthesised in Chinese Hamster Ovary (CHO) cells had a molecular weight

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twice that of its theoretical molecular weight. Characterisation of this recombinant peptide by mass spectrometry revealed glycosylation at seven Threonine or Serine sites on the proBNP sequence (Figure 5). These were located within the N-terminal region of proBNP and included full glycosylation

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at amino acids Ser-37, Ser-44, Thr-48, Ser-53 and Thr-71 and partial glycosylation at amino acids Thr-36 and Thr-58. Glycosylation of the endogenous human proBNP and NT-proBNP forms were also

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demonstrated through a decrease in their molecular weights after deglycosylation [59, 86]. Subsequent work by others has added much detail to these early findings. Analysis of immunoreactivity before and after deglycosylation at epitopes spread along the first 76 residues of the

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proBNP/NT-proBNP sequence reveals that endogenous human NT-proBNP and proBNP are

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glycosylated between residues 28 and 56 in agreement with those found in the recombinant peptide [87, 88]. Immunoassays directed to this region for example, show a 3 to 50 fold increase in

samples [87].

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immunoreactive NT-proBNP after deglycosylation of NT-proBNP prepared from individual plasma

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In contrast, there is a major difference in the degree of glycosylation at Thr-71 between circulating proBNP and NT-proBNP with proBNP being more heavily glycosylated in this position compared to NT-proBNP. Indeed only 30% of circulating proBNP in patients with heart failure is not glycosylated at this position [25] while circulating NT-proBNP is largely free of glycosylation at this position [88]. This is largely due to glycosylation on Thr-71 blocking the action of furin and corin to cleave proBNP at residue 76 nearby [25, 88]. Together these processes result in circulating NT-proBNP forms that are predominantly glycosylated in their central region (amino acids 36-58), but enriched in forms lacking glycosylation on Thr-71 [88]. On the other hand, proBNP that escapes processing and enters the circulation is enriched in forms glycosylated at Thr-71 with only 30% of circulating proBNP lacking glycosylation at this position (Figure 5) [25]. The influence of Thr-71 glycosylation on in vivo proBNP processing has recently been evaluated by Vodovar et al [62] in patients with acutely decompensated heart failure (ADHF), chronic heart failure

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ACCEPTED MANUSCRIPT (CHF) or non-cardiac dyspnoea (non-ADHF). Their findings show reduced glycosylation of proBNP at Thr-71 in ADHF and non-ADHF patients compared to CHF and that the degree of glycosylation in heart failure is inversely related to circulating NT-proBNP concentrations. Further, the rate of proBNP

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processing, as measured by the ratio of NT-proBNP to proBNP in plasma, is very closely (inversely)

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correlated to the degree of proBNP T-71 glycosylation. Plasma furin activity is also strongly correlated

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with plasma NT-proBNP concentrations. These results suggest that as proBNP synthesis increases, glycosylation at Thr-71 may decrease while at the same time the plasma concentration of the processing enzyme furin increases resulting in a synergistic increase in processing capability and

circulation that is discussed in section 4.4 below.

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BNP concentrations. These results also lend support to the possibility of proBNP processing in

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Figure 5 summarises these details in the overall processing scheme for BNP peptides. ProBNP is synthesised in cardiomyocytes as the 134 amino acid preproBNP peptide that comprises a 108 amino

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acid proBNP segment plus a 26 amino acid signal peptide added at its N-terminal end. The signal

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peptide is removed during preproBNP synthesis producing the 108 amino acid precursor proBNP. ProBNP is then glycosylated during its passage through the Golgi, including glycosylation at residue

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Thr-71 (large black peg on proBNP right side of Figure 5) which inhibits furin/corin processing between nearby residues 76 and 77. This unprocessed proBNP is exported to plasma by an unknown process (bottom line of Figure 5). Some proBNP that is not glycosylated at Thr-71 is also exported

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because 30% of circulating proBNP is not glycosylated on Thr-71. ProBNP that does not carry glycosylation on Thr-71 (left side of Figure 5) is processed by furin or corin to BNP and NT-proBNP, and exported to plasma (bottom line of Figure 5). Circulating NT-proBNP is greatly enriched in forms devoid of glycosylation on Thr-71 because of this process. Not shown in Figure 5 is the subsequent truncation of BNP and NT-proBNP to produce the circulating forms shown in Figure 4.

4.3 Effects of Glycosylation and Truncation on NT-proBNP and proBNP Measurement 4.3.1 NT-proBNP assays The complex mix of circulating glycosylated, non-glycosylated and truncated proBNP and NT-proBNP peptides plus BNP and its metabolites creates significant complexity in their measurement by

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ACCEPTED MANUSCRIPT immunoassay. Existing NT-proBNP assays usually cross-react with proBNP, but not with BNP [54, 58, 59], resulting in most NT-proBNP measurements containing a contribution from proBNP. The extent of this interference depends on each assay’s cross-reactivity with proBNP, which seems to

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vary considerably between common diagnostic assays and even between different publications that

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have evaluated the same assay [54, 58, 59]. These relationships are further complicated by the effect

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of glycosylation in the central region of NT-proBNP (Section 4.2) that inhibits antibody binding to this region. The Roche NT-proBNP assay and other analysers using its reagents are the dominant diagnostic method for NT-proBNP measurement. An early version of this method, the Roche proBNP-

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I assay, employs a detection antibody that cross-reacts poorly with glycosylated NT-proBNP because it recognises amino acids 39-50 within the NT-proBNP central glycosylated region. The resulting

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effect of glycosylation on the assay is substantial with up to ten fold increases in concentration appearing after deglycosylation of NT-proBNP in plasma samples [87, 89]. These observations and those of Nishikimi et al [89] suggest the Roche proBNP-I assay measures only 20% of circulating NT-

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proBNP, reflecting the small proportion of non-glycosylated NT-proBNP in circulation. The cross-reactivity of proBNP in these NT-proBNP assays is similarly complicated by the presence

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or absence of glycosylation. Cross-reactivities to non-glycosylated and glycosylated proBNP measured in the Roche NT-proBNP assay and three other assays based on the same reagents, range from 29 to 249 percent for non-glycosylated proBNP. The glycosylated forms however, exhibit

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much lower cross-reactivities at <1 to 2 percent [54, 58, 59] as would be expected from the inhibitory effect of glycosylation on these assays. The newer Roche NT-proBNP-II assay also uses antibodies directed to the central glycosylated region (residues 42-46) [57] and is likely to be similarly affected by glycosylation. Our own unpublished observations suggest this assay has <5% cross-reactivity to glycosylated proBNP when calculated on a weight basis. Indeed nearly all NT-proBNP diagnostic assays employ Roche NT-proBNP I or NT-proBNP II antibodies or others that also recognise residues 39-50 or 42-46 in the central glycosylated region [57]. These alternative assays, which will presumably suffer the same interference from glycosylation as the Roche assays, include those produced by Siemens, Ortho Clinical Diagnostics, Alere, bioMerieux, Mitsubishi Chemical, Radiometer and Response Biomedical.

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ACCEPTED MANUSCRIPT Roubille et al have recently taken advantage of their unique antibody to the hinge region of proBNP to affinity-deplete plasma of proBNP and observe its effect on NT-proBNP and BNP results. Interestingly, depletion of plasma from 180 patients with chronic heart failure resulted in only a 5%

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decrease in NT-proBNP measured by the Roche assay while BNP results decreased by 50 percent

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[61]. The reason for the small decline in NT-proBNP results is unclear, but may reflect the very low

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cross-reactivity of glycosylated proBNP in the assay as discussed above. The large decrease in BNP results however points to significant interference by proBNP in BNP assays as discussed earlier in section 2.1.

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The data above suggests that optimal responses in NT-proBNP assays would be gained by use of epitopes located away from the glycosylated or truncated regions. Indeed Seferian et al have

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evaluated numerous antisera to residues along the length of the length of NT-proBNP for their recognition of endogenous NT-proBNP [87]. From their studies, it is clear that antibodies raised

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against peptides in the 13-24 and 63-73 regions respond much better to endogenous NT-proBNP

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than those recognising the truncated 1-12 and glycosylated 36-58 regions. In particular, their optimised 2-site immunoassay using antibodies raised against amino acids 13-20 and 63-71

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produced results that were fourteen fold higher than a similar assay directed to the glycosylated region and six fold greater than those from the Roche assay when measured on the same samples [87]. Whether these assays directed to non-truncated and non-glycosylated sites merely register

be seen.

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higher NT-proBNP concentrations or deliver improved diagnostic sensitivity and specificity remains to

4.3.2 ProBNP assays

Very specific 2-site immunoassays to proBNP have been established through use of antibodies that recognise epitopes near the N- and C- terminal ends of proBNP. These assays usually include an antibody recognising the C-terminal end of proBNP, which contains the BNP sequence, plus a second antibody recognising the N-terminal end of proBNP. However, careful selection of antisera and the epitopes they recognise is needed to avoid the truncated and glycosylated regions present in circulating proBNP. These regions include truncation at the N-terminus (section 4.1) and probably the C-terminus, glycosylation in the central region between amino acids 36 and 58 and the substantial glycosylation of amino acid Thr-71 (section 4.2). Potentially useful epitopes based on these criteria

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ACCEPTED MANUSCRIPT include amino acids 13-24 as described for NT-proBNP section 4.3.1, plus sites within the BNP ring structure at the other end of proBNP as in section 2.1. The few published 2-site immunoassays for proBNP tend to follow these principles, but most employ antisera to the very C-terminal end of

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proBNP [74, 90, 91] that could miss C-terminal truncated forms.

An alternative single site immunoassay for proBNP is marketed by Bio-Rad [75]. The antiserum in this

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assay recognises the proBNP processing site – the “hinge” region – that is unique to proBNP and which confers very low cross-reactivity to BNP and NT-proBNP on the assay [26, 75]. However, its cross-reactivity with glycosylated proBNP does not appear to have been measured. As discussed

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above, glycosylation of proBNP at amino acid Thr-71 blocks processing of proBNP at the nearby residues 76 and 77 through steric hindrance. The antibody used in this assay is directed to the same

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site, requiring residues 75 to 80 for binding. It is equally likely that glycosylation at Thr-71 will also inhibit binding of antisera to this hinge region to some extent. If this were the case, the assay would

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under report the proBNP concentration and predominantly measure the 30% of circulating proBNP

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that is free of glycosylation at Thr-71. Nevertheless as described above the assay has provided useful information on the secretion of proBNP and its diagnostic potential. These show that proBNP has

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results [77, 78].

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similar sensitivity and specificity to BNP and may improve risk stratification when combined with BNP

4.4 Bioactivity of proBNP and its Processing in the Periphery ProBNP circulates at concentrations equivalent to or higher than those of BNP, but despite its relatively large contribution to total circulating immunoreactive BNP very little has been done to assess its in vivo or in vitro biological activity. It is 12 to 16 fold less potent than BNP when their potency is measured by the cGMP response in cell lines expressing recombinant NPR-A receptor [59, 92]. It is also 6 to 8 fold less potent when tested on cultured human vascular endothelial or smooth muscle cells [59], and does not stimulate cGMP production in cultured cardiac fibroblasts and cardiomyocytes that respond to BNP [54]. As described below, the human form of proBNP has been administered to rats as an intravenous bolus dose to evaluate the processing of proBNP to BNP in circulation, but the plasma cGMP and other physiological responses such as vasodilation and natriuresis have not been measured.

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ACCEPTED MANUSCRIPT The relatively high circulating concentrations of proBNP in patients with heart failure have led to speculation that proBNP may be processed in blood or tissues to provide an additional supply of circulating or tissue BNP [78, 93, 94]. This speculation is supported by results from two studies. First,

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Semenov et al have used a rat model to evaluate in vivo processing of human glycosylated and non-

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glycosylated proBNP. They obtained clear evidence for processing of non-glycosylated proBNP from

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the appearance of circulating human BNP within six minutes of intravenous proBNP injection. In contrast, glycosylated proBNP was not processed as would be expected from the inhibitory effect of glycosylation on the processing enzymes [94]. However further studies infusing proBNP combined

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with measurement of physiological parameters and plasma cGMP are needed to evaluate whether this peripheral production of BNP is physiologically relevant. There are of course substantial

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differences between BNP processing in humans and rats, including sequence differences at the processing sites and production of the 45 amino acid BNP form in rats versus the 32 amino acid BNP form in humans. However MS analysis of the processing products in these studies showed human

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proBNP had been processed at the correct site and the BNP products included BNP(1-32) plus a

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range of truncated BNP metabolites identical to those found in human plasma [94]. Second, Ichiki et al have shown that non-glycosylated recombinant proBNP is processed in vitro by human plasma to

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produce BNP, and that proBNP processing in plasma samples correlates with the plasma concentration of the processing enzyme corin [93]. These early studies offer interesting prospects of a

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role for proBNP in the delivery of a protected form of BNP to target tissues where it could be processed to the active form. A similar system has been proposed for transport of the natriuretic peptide uroguanylin from the gut to the kidney in the form of its degradation resistant precursor prouroguanylin [95, 96]. This analogy with prouroguanylin would be complete if proBNP was also processed in the kidney to produce a local source of BNP.

5 BNP Signal Peptide Signal peptides direct the transfer into the endoplasmic reticulum of nascent peptides or proteins destined for expression on the cell surface or for export. They are cleaved off the peptide/protein during synthesis by a signal peptidase and have not usually been regarded as an exported product, although a few publications have indicated their existence at cell surfaces or attached to exported

19

ACCEPTED MANUSCRIPT proteins. Our recent identification of BNP signal peptide, along with several other signal peptides, in human plasma has revealed that signal peptides can circulate independently in humans [97, 98]. BNP is first synthesised as the 134 amino acid preproBNP peptide including a 26 amino acid signal peptide

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at its amino-terminal end, which is removed during preproBNP synthesis (Figure 5). The subsequent

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fate of the signal peptide is unknown apart from the recent discovery of its C-terminal portion

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circulating in blood from healthy volunteers and in patients following acute myocardial infarction. This newly discovered circulating peptide, fully characterised by mass spectrometry, is a 10 amino acid carboxy-terminal fragment from the BNP signal peptide comprising residues 17 to 26, the N-terminal

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leucine of which carries formyl, glyoxal or methyl glyoxal modifications on its amino group [98]. Measurements of trans-organ arteriovenous gradients of BNP signal peptide indicate that, like BNP,

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this new peptide is secreted by the heart, and may also be produced across the head, kidney and limb. It circulates in healthy individuals at concentrations ranging from 7 to 25 pmol/L and its plasma

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concentrations rise rapidly in evolving acute myocardial infarction peaking just 4 to 5 hours after onset

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of symptoms. The timing of post–infarction peak plasma concentrations of BNP signal peptide is

6 Conclusion

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similar to that for myoglobin but much earlier than those of CKMB and Troponin I [98].

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Understanding of the synthesis and generation of BNP circulating forms has become increasingly detailed and complex leading to transition from the simple scheme known ten years ago (Figure 2) to the far more complicated scheme shown in Figure 5. Greater complexity still is likely as further details are unearthed. BNP is truncated to multiple forms variably trimmed at each end of the molecule and while the major products of these reactions have been identified in plasma, there is scope for more to be identified. In contrast, proBNP seems to be more resistant to degradation by enzymes and may be processed to BNP in circulation offering interesting possibilities for its local conversion in tissues other than the heart. The discovery of variable glycosylation of proBNP and NT-proBNP, its effect on proBNP processing and the downstream effects on circulating glycosylated forms of proBNP and NTproBNP, has exposed limitations in some current diagnostic assays. However the clear message arising from these studies, that glycosylated regions (epitopes) of proBNP and NT-proBNP and the Nterminal and C-terminal truncated regions of all three peptides should be avoided by immunoassays

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ACCEPTED MANUSCRIPT provides a rational basis for new assay development. At the same time the glycosylation and truncation of these peptides offers considerable scope for new BNP form-specific assays that may

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better reflect the presence, progression or prediction of heart failure and its outcome.

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ACCEPTED MANUSCRIPT Figure Legends

Figure 1. Structures of the Natriuretic Peptide Family. Each member contains a 17 amino acid ring

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lengths of the amino and carboxy-terminal arms extending from the ring.

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structure. ANP, BNP and CNP are also distinguished by their amino acid sequences and by the

Figure 2. Synthesis of BNP and NT-proBNP. The 108 amino acid precursor proBNP synthesised in

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cardiomyocytes is cleaved by the processing enzymes furin or corin between amino acids 76 and 77 to produce the 32 amino acid peptide BNP and the 76 amino acid N-terminal fragment NT-proBNP.

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All three peptides are present in the heart and circulate in humans.

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Figure 3. Known BNP cleavage sites due to the actions of Neprilysin, Dipeptidyl Peptidase IV and

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Insulin degrading enzyme. Bold black arrows indicate primary cleavage points while hollow arrows

Enzyme.

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indicate secondary points. Thin arrows point to minor cleavage points due to Insulin Degrading

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Figure 4. Circulating forms of BNP and cleavage points in BNP and NT-proBNP required to produce fragments that have been identified. Each cleavage point (Black Arrows) marking the beginning or end of an identified fragment could be achieved by a single endopeptidase cleavage or by sequential action of aminopeptidases or both. Removal of the dipeptide from the amino-terminal ends of BNP and proBNP is most likely due to DPP IV (Figure 3). A: Circulating forms of BNP identified by Niederkofler et al [51] and cleavage sites within BNP required to produce them. B: Cleavage points in NT-proBNP required to produce non-tryptic peptides identified by Foo et al [85].

22

ACCEPTED MANUSCRIPT Figure 5. Synthesis, glycosylation and processing of BNP peptides. Glycosylation sites observed in recombinant proBNP [86] are marked by black pegs on proBNP and NT-proBNP. Pegs with unfilled squares represent sites on recombinant proBNP that were partially glycosylated. Glycosylation at

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residue Thr-71 is represented by a large black peg adjacent to the furin/corin cleavage site. ProBNP

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is synthesised in cardiomyocytes as the 134 amino acid preproBNP peptide that comprises a 108

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proBNP segment with a 26 amino acid signal peptide. The signal peptide is removed during preproBNP synthesis and later cleaved to produce BNP Signal Peptide(17-26) that is found in plasma (top left). The 108 amino acid proBNP peptide is then glycosylated in its central region, but with

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variable glycosylation on Thr-71. Glycosylation at Thr-71 inhibits furin/corin processing between residues 76 and 77 nearby (right side of figure). This unprocessed proBNP is exported to plasma

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(bottom line) by an unknown process. Some proBNP that is not glycosylated at Thr-71 is also exported because 30% of circulating proBNP is not glycosylated on Thr-71. ProBNP that does not carry glycosylation on Thr-71 (left side of figure) is processed by furin or corin to BNP and NT-

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proBNP, which are exported to plasma (bottom line of figure). Circulating NT-proBNP is greatly

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enriched in forms devoid of glycosylation on Thr-71 as a result of this process. Not shown is the

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(Figure 4).

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subsequent truncation of BNP and NT-proBNP to produce additional truncated forms shown in

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References

[7] [8] [9]

[10]

[11] [12]

[13] [14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

T

IP

SC R

NU

[6]

MA

[5]

D

[4]

TE

[3]

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[2]

Yasue H, Yoshimura M, Sumida H, et al. Localization and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Circulation 1994; 90:195-203. LaPointe MC. Molecular regulation of the brain natriuretic peptide gene. Peptides 2005; 26:944-956. Potter LR. Guanylyl cyclase structure, function and regulation. Cellular Signalling 2011; 23:1921-1926. Potter LR. Natriuretic peptide metabolism, clearance and degradation. FEBS J 2011; 278:1808-1817. Charles CJ, Espiner EA, Nicholls MG, et al. Clearance receptors and endopeptidase 24.11: equal role in natriuretic peptide metabolism in conscious sheep. Am J Physiol 1996; 271:R373-380. Lainchbury JG, Richards AM, Nicholls MG, Espiner EA, Yandle TG. Brain natriuretic peptide and neutral endopeptidase inhibition in left ventricular impairment. J Clin Endocrinol Metab 1999; 84:723-729. Boerrigter G, Costello-Boerrigter LC, Burnett JC, Jr. Natriuretic peptides in the diagnosis and management of chronic heart failure. Heart Fail Clin 2009; 5:501-514. Espiner EA. Physiology of natriuretic peptides. J Intern Med 1994; 235:527-541. Holmes SJ, Espiner EA, Richards AM, Yandle TG, Frampton C. Renal, endocrine, and hemodynamic effects of human brain natriuretic peptide in normal man. J Clin Endocrinol Metab 1993; 76:91-96. Cataliotti A, Chen HH, Redfield MM, Burnett JC, Jr. Natriuretic peptides as regulators of myocardial structure and function: pathophysiologic and therapeutic implications. Heart Fail Clin 2006; 2:269-276. Calvieri C, Rubattu S, Volpe M. Molecular mechanisms underlying cardiac antihypertrophic and antifibrotic effects of natriuretic peptides. J Mol Med 2012; 90:5-13. Hunt PJ, Richards AM, Nicholls MG, Yandle TG, Doughty RN, Espiner EA. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol 1997; 47:287-296. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet 1994; 343:440-444. Maisel AS, Krishnaswamy P, Nowak RM, et al. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. The New England journal of medicine 2002; 347:161-167. Maisel AS, Daniels LB. Breathing not properly 10 years later: what we have learned and what we still need to learn. J Am Coll Cardiol 2012; 60:277-282. Januzzi JL, van Kimmenade R, Lainchbury J, et al. NT-proBNP testing for diagnosis and short-term prognosis in acute destabilized heart failure: an international pooled analysis of 1256 patients: the International Collaborative of NT-proBNP Study. Eur Heart J 2006; 27:330337. Yancy CW, Jessup M, Bozkurt B, et al. 2013 ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation 2013; 128:e240-327. Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human brain natriuretic peptides in cardiac atrium. Biochem Biophys Res Commun 1990; 167:693-700. Tateyama H, Hino J, Minamino N, Kangawa K, Ogihara T, Matsuo H. Characterization of immunoreactive brain natriuretic peptide in human cardiac atrium. Biochem Biophys Res Commun 1990; 166:1080-1087. Sudoh T, Maekawa K, Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide. Biochem Biophys Res Commun 1989; 159:1427-1434. Mukoyama M, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans - evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest 1991; 87:1402-1412.

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[29] [30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40] [41] [42] [43]

T

IP

SC R

[28]

NU

[27]

MA

[26]

D

[25]

TE

[24]

CE P

[23]

Tateyama H, Hino J, Minamino N, et al. Concentrations and molecular forms of human brain natriuretic peptide in plasma. Biochem Biophys Res Commun 1992; 185:760-767. Yandle TG, Richards AM, Gilbert A, Fisher S, Holmes S, Espiner EA. Assay of brain natriuretic peptide (BNP) in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab 1993; 76:832-838. Hunt PJ, Yandle TG, Nicholls MG, Richards AM, Espiner EA. The amino-terminal portion of pro-brain natriuretic peptide (Pro-BNP) circulates in human plasma. Biochem Biophys Res Commun 1995; 214:1175-1183. Semenov AG, Tamm NN, Seferian KR, et al. Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases. Clin Chem 2010; 56:1166-1176. Costello-Boerrigter LC, Lapp H, Boerrigter G, et al. Secretion of prohormone of B-type natriuretic peptide, proBNP1-108, is increased in heart failure. JACC Heart Fail 2013; 1:207212. Stephenson SL, Kenny AJ. The hydrolysis of a-human atrial natriuretic peptide by pig kidney microvillar membranes is initiated by endopeptidase-24.11. Biochem J 1987; 243:183-187. Vanneste Y, Michel A, Dimaline R, Najdovski T, Deschodt-Lanckman M. Hydrolysis of alphahuman atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site. The Biochemical journal 1988; 254:531-537. Kenny AJ, Stephenson SL. Role of endopeptidase-24.11 in the inactivation of atrial natriuretic peptide. FEBS Lett 1988; 232:1-8. Seymour AA, Swerdel JN, Fennell SA, Delaney NG. Atrial natriuretic peptides cleaved by endopeptidase are inactive in conscious spontaneously hypertensive rats. Life Sci 1988; 43:2265-2274. Charles CJ, Espiner EA, Yandle TG, Cameron VA, Richards AM. Biological actions of cleaved atrial natriuretic factor (ANF101-105/106-126) in conscious sheep. J Cardiovasc Pharmacol 1991; 17:403-410. Yandle T, Crozier I, Nicholls G, Espiner E, Carne A, Brennan S. Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma. Biochem Biophys Res Commun 1987; 146:832-839. Norman JA, Little D, Bolgar M, Di Donato G. Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass spectrometry. Biochem Biophys Res Commun 1991; 175:22-30. Kenny AJ, Bourne A, Ingram J. Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11. Biochem J 1993; 291:83-88. Watanabe Y, Nakajima K, Shimamori Y, Fujimoto Y. Comparison of the hydrolysis of the three types of natriuretic peptides by human kidney neutral endopeptidase 24.11. Biochemical and Molecular Medicine 1997; 61:47-51. Brandt I, Lambeir AM, Ketelslegers JM, Vanderheyden M, Scharpe S, De Meester I. Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin Chem 2006; 52:82-87. Walther T, Stepan H, Pankow K, Becker M, Schultheiss HP, Siems WE. Biochemical analysis of neutral endopeptidase activity reveals independent catabolism of atrial and brain natriuretic peptide. Biol Chem 2004; 385:179-184. Smith MW, Espiner EA, Yandle TG, Charles CJ, Richards AM. Delayed metabolism of human brain natriuretic peptide reflects resistance to neutral endopeptidase. J Endocrinol 2000; 167:239-246. Lambeir AM, Durinx C, Scharpe S, De Meester I. Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev Clin Lab Sci 2003; 40:209-294. Vanderheyden M, Bartunek J, Goethals M, et al. Dipeptidyl-peptidase IV and B-type natriuretic peptide. From bench to bedside. Clin Chem Lab Med 2009; 47:248-252. Deacon CF. Circulation and degradation of GIP and GLP-1. Horm Metab Res 2004; 36:761765. Deacon CF, Carr RD, Holst JJ. DPP-4 inhibitor therapy: new directions in the treatment of type 2 diabetes. Front Biosci 2008; 13:1780-1794. Lam CS, Burnett JC, Jr., Costello-Boerrigter L, Rodeheffer RJ, Redfield MM. Alternate circulating pro-B-type natriuretic peptide and B-type natriuretic peptide forms in the general population. J Am Coll Cardiol 2007; 49:1193-1202.

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ACCEPTED MANUSCRIPT

[51]

[52]

[53] [54]

[55] [56]

[57]

[58]

[59] [60]

[61] [62] [63]

[64]

[65]

[66]

T

IP

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NU

[49]

MA

[48]

D

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[46]

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Fernandez-Gamba A, Leal MC, Morelli L, Castano EM. Insulin-degrading enzyme: structurefunction relationship and its possible roles in health and disease. Curr Pharm Des 2009; 15:3644-3655. Ralat LA, Guo Q, Ren M, et al. Insulin-degrading enzyme modulates the natriuretic peptidemediated signaling response. J Biol Chem 2011; 286:4670-4679. Pankow K, Wang Y, Gembardt F, et al. Successive action of meprin A and neprilysin catabolizes B-type natriuretic peptide. Circ Res 2007; 101:875-882. Walther T, Screening methods for inhibitors of the metalloprotease meprin. In: Organization WIP, editor, 2007. Dickey DM, Potter LR. Human B-type natriuretic peptide is not degraded by meprin A. Biochem Pharmacol 2010; 80:1007-1011. Shimizu H, Masuta K, Aono K, et al. Molecular forms of human brain natriuretic peptide in plasma. Clin Chim Acta 2002; 316:129-135. Hawkridge AM, Heublein DM, Bergen HR, 3rd, Cataliotti A, Burnett JC, Jr., Muddiman DC. Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Natl Acad Sci U S A 2005; 102:17442-17447. Niederkofler EE, Kiernan UA, O'Rear J, et al. Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure. Circ Heart Fail 2008; 1:258264. Miller WL, Phelps MA, Wood CM, et al. Comparison of mass spectrometry and clinical assay measurements of circulating fragments of B-type natriuretic peptide in patients with chronic heart failure. Circ Heart Fail 2011; 4:355-360. Fujimoto H, Suzuki T, Aizawa K, et al. Processed B-type natriuretic peptide is a biomarker of postinterventional restenosis in ischemic heart disease. Clin Chem 2013; 59:1330-1337. Heublein DM, Huntley BK, Boerrigter G, et al. Immunoreactivity and guanosine 3',5'-cyclic monophosphate activating actions of various molecular forms of human B-type natriuretic peptide. Hypertension 2007; 49:1114-1119. Rawlins ML, Owen WE, Roberts WL. Performance characteristics of four automated natriuretic peptide assays. Am J Clin Pathol 2005; 123:439-445. Tetin SY, Ruan Q, Saldana SC, et al. Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy. Biochemistry 2006; 45:14155-14165. A table of BNP and NTproBNP diagnostic assays and antibodies used in them is available on the International Federation of Clinical Chemistry website, http://www.ifcc.org/ifcc-scientificdivision/documents-of-the-sd/. Luckenbill KN, Christenson RH, Jaffe AS, et al. Cross-reactivity of BNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC Committee for Standardization of Markers of Cardiac Damage. Clin Chem 2008; 54:619-621. Liang F, O'Rear J, Schellenberger U, et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 2007; 49:1071-1078. Nishikimi T, Minamino N, Horii K, Matsuoka H. Do commercially available assay kits for Btype natriuretic peptide measure Pro-BNP1-108, as well as BNP1-32? Hypertension 2007; 50:e163; author reply e164. Roubille F, Delseny D, Cristol JP, et al. Depletion of proBNP1-108 in patients with heart failure prevents cross-reactivity with natriuretic peptides. PloS one 2013; 8:e75174. Vodovar N, Seronde MF, Laribi S, et al. Post-translational modifications enhance NT-proBNP and BNP production in acute decompensated heart failure. Eur Heart J 2014. Boerrigter G, Costello-Boerrigter LC, Harty GJ, et al. B-type natriuretic peptide 8-32, which is produced from mature BNP 1-32 by the metalloprotease meprin A, has reduced bioactivity. Am J Physiol Regul Integr Comp Physiol 2009; 296:R1744-1750. Boerrigter G, Costello-Boerrigter LC, Harty GJ, Lapp H, Burnett JC, Jr. Des-serine-proline brain natriuretic peptide 3-32 in cardiorenal regulation. Am J Physiol Regul Integr Comp Physiol 2007; 292:R897-901. Mahagamasekera PG, Ruygrok PN, Palmer SC, et al. B-type natriuretic peptide forms within the heart, coronary sinus, and peripheral circulation in humans: evidence for degradation before secretion. Clin Chem 2014; 60:549-558. Kuo WL, Montag AG, Rosner MR. Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues. Endocrinology 1993; 132:604-611.

AC

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26

ACCEPTED MANUSCRIPT

[74]

[75] [76] [77]

[78]

[79] [80]

[81]

[82] [83]

[84]

[85] [86]

[87] [88]

[89]

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[69]

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[68]

Linardi A, Panunto PC, Ferro ES, Hyslop S. Peptidase activities in rats treated chronically with N(omega)-nitro-L-arginine methyl ester (L-NAME). Biochem Pharmacol 2004; 68:205214. Fielitz J, Dendorfer A, Pregla R, et al. Neutral endopeptidase is activated in cardiomyocytes in human aortic valve stenosis and heart failure. Circulation 2002; 105:286-289. Kokkonen JO, Kuoppala A, Saarinen J, Lindstedt KA, Kovanen PT. Kallidin- and bradykinindegrading pathways in human heart: degradation of kallidin by aminopeptidase M-like activity and bradykinin by neutral endopeptidase. Circulation 1999; 99:1984-1990. Raizada V, Luo W, Skipper BJ, McGuire PG. Intracardiac expression of neutral endopeptidase. Mol Cell Biochem 2002; 232:129-131. Hartel S, Gossrau R, Hanski C, Reutter W. Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry. Histochemistry 1988; 89:151-161. Matheeussen V, Baerts L, De Meyer G, et al. Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries. Biol Chem 2011; 392:189-198. Hansen L, Deacon CF, Orskov C, Holst JJ. Glucagon-like peptide-1-(7-36)amide is transformed to glucagon-like peptide-1-(9-36)amide by dipeptidyl peptidase IV in the capillaries supplying the L cells of the porcine intestine. Endocrinology 1999; 140:5356-5363. Shimizu H, Masuta K, Asada H, Sugita K, Sairenji T. Characterization of molecular forms of probrain natriuretic peptide in human plasma. Clinica chimica acta; international journal of clinical chemistry 2003; 334:233-239. Giuliani I, Rieunier F, Larue C, et al. Assay for Measurement of Intact B-Type Natriuretic Peptide Prohormone in Blood. Clin Chem 2006. Hammerer-Lercher A, Halfinger B, Sarg B, et al. Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin Chem 2008; 54:858-865. Dries DL, Ky B, Wu AH, Rame JE, Putt ME, Cappola TP. Simultaneous assessment of unprocessed ProBNP1-108 in addition to processed BNP32 improves identification of highrisk ambulatory patients with heart failure. Circulation Heart failure 2010; 3:220-227. Macheret F, Boerrigter G, McKie P, et al. Pro-B-type natriuretic peptide(1-108) circulates in the general community: plasma determinants and detection of left ventricular dysfunction. J Am Coll Cardiol 2011; 57:1386-1395. Miller WL, Grill DE, Jaffe AS. Comparison of novel pro-BNP(1-108) and standard BNP assays in heart failure patients. Clin Chim Acta 2012; 413:920-926. Ala-Kopsala M, Moilanen AM, Rysa J, Ruskoaho H, Vuolteenaho O. Characterization of molecular forms of N-terminal B-type natriuretic peptide in vitro. Clin Chem 2010; 56:18221829. Palmer SC, Yandle TG, Nicholls MG, Frampton CM, Richards AM. Regional clearance of amino-terminal pro-brain natriuretic peptide from human plasma. Eur J Heart Fail 2009; 11:832-839. Pemberton CJ, Johnson ML, Yandle TG, Espiner EA. Deconvolution analysis of cardiac natriuretic peptides during acute volume overload. Hypertension 2000; 36:355-359. Januzzi JL, Jr., Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. The American journal of cardiology 2005; 95:948-954. Ala-Kopsala M, Magga J, Peuhkurinen K, et al. Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides. Clin Chem 2004; 50:1576-1588. Foo JY, Wan Y, Schulz BL, et al. Circulating fragments of N-terminal pro-B-type natriuretic peptides in plasma of heart failure patients. Clin Chem 2013; 59:1523-1531. Schellenberger U, O'Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to Btype natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 2006; 451:160166. Seferian KR, Tamm NN, Semenov AG, et al. Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem 2008; 54:866-873. Semenov AG, Postnikov AB, Tamm NN, et al. Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 2009; 55:489-498. Nishikimi T, Ikeda M, Takeda Y, et al. The effect of glycosylation on plasma N-terminal proBNP-76 levels in patients with heart or renal failure. Heart 2012; 98:152-161.

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Seferian KR, Tamm NN, Semenov AG, et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 2007; 53:866873. Nishikimi T, Okamoto H, Nakamura M, et al. Direct immunochemiluminescent assay for proBNP and total BNP in human plasma proBNP and total BNP levels in normal and heart failure. PloS one 2013; 8:e53233. Dickey DM, Potter LR. ProBNP(1-108) is resistant to degradation and activates guanylyl cyclase-A with reduced potency. Clin Chem 2011; 57:1272-1278. Ichiki T, Huntley BK, Heublein DM, et al. Corin is present in the normal human heart, kidney, and blood, with pro-B-type natriuretic peptide processing in the circulation. Clin Chem 2011; 57:40-47. Semenov AG, Seferian KR, Tamm NN, et al. Human pro-B-type natriuretic peptide is processed in the circulation in a rat model. Clin Chem 2011; 57:883-890. Moss NG, Fellner RC, Qian X, et al. Uroguanylin, an intestinal natriuretic peptide, is delivered to the kidney as an unprocessed propeptide. Endocrinology 2008; 149:4486-4498. Qian X, Moss NG, Fellner RC, Goy MF. Circulating prouroguanylin is processed to its active natriuretic form exclusively within the renal tubules. Endocrinology 2008; 149:4499-4509. Pemberton CJ. Signal peptides: new markers in cardiovascular disease? Biomark Med 2014; 8:1013-1019. Siriwardena M, Kleffmann T, Ruygrok P, et al. B-type natriuretic peptide signal peptide circulates in human blood: evaluation as a potential biomarker of cardiac ischemia. Circulation 2010; 122:255-264.

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ACCEPTED MANUSCRIPT Highlights The peptides BNP and NTproBNP are cleaved from their precursor proBNP in the heart. All three peptides circulate along with their N- and C- terminal truncated forms.

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proBNP and NTproBNP forms with variable glycosylation also circulate.

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Immunoassays should avoid truncated and glysosylated regions for maximum detection.

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proBNP has reduced bioactivity, but may be processed to bioactive BNP in circulation.

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