Clinical Significance of Pro-B-Type Natriuretic Peptide Glycosylation and Processing

Since the principal discovery of the cardiac natriuretic peptide hormones about 25 years ago, a large amount of research has identified 2 peptides derived from pro-B-type natriuretic peptide (proBNP),1 BNP and N-terminal-proBNP (NT-proBNP), as useful plasma markers in cases of heart failure (HF) and other cardiac diseases. Our present understanding of proBNP processing and the structural biochemistry and metabolism of circulating proBNP-derived peptides, by contrast, are still far from complete. In particular, posttranslational maturation and metabolism are still poorly characterized. In this issue of Clinical Chemistry, Semenov et al. describe important results that may substantially improve our current understanding of the regulation of proBNP processing (1). Like many other hormones, BNP is derived from a prohormone. The precursor proBNP hormone is encoded by a separate gene, NPPB (natriuretic peptide precursor B), which has been assigned to chromosome 1 in humans. Its transcription has been shown to be regulated by cardiac-specific gene regulators, such as GATA, the muscle-CAT binding site, and activating protein 1/cyclic adenosine monophosphate response element-like elements (2). These cis elements have since been found to be the molecular targets of many different clinically relevant stimuli that lead to basal and inducible regulation of the NPPB gene. Transcription of the human NPPB gene can be activated through various proinflammatory and hypertrophic stimuli, such as mechanical stretch, ischemic injury/hypoxia, endothelin-1, angiotensin II, interleukins, and adrenergic agonists (2). The 5′ flanking region of the NPPB gene contains acute-phase regulatory elements, and the expression of this gene is induced with the rapid kinetics of an early- response gene. In contrast to A-type natriuretic peptide (ANP), it is believed that most BNP regulation is carried out during gene expression, with most BNP being synthesized in bursts of activation from physiological and pathophysiological stimuli, when peptide secretion occurs. Only limited amounts of proBNP and processed BNP coexist in the secretory granules of the human atrial myocardium (3). The available information on posttranslational proBNP processing is partially based on indirect observations (see Fig. 1 ). Translation of the NPPB gene produces an initial gene product, precursor proBNP 1–134, which undergoes rapid removal of a 26-amino acid signal peptide in the sarcoplasmic reticulum during translation before synthesis of the C-terminal part of the prohormone sequence has been completed. This cleavage yields a prohormone of 108 amino acid residues, proBNP 1–108. Prohormone convertases such as furin and corin (4)(5) may subsequently cleave proBNP to release 2 portions: the 76-residue amino-terminal portion (NT-proBNP 1–76) and the biologically active 32-residue molecule, BNP 1–32. The cleavage site is located between amino acid residues 76 and 77 (-Leu72-Arg73-Ala74-Pro75-Arg76↓-Ser77-). In vitro experiments and cell-based assays have demonstrated that furin and corin are able to process proBNP. The significance of each protease for in vivo proBNP processing is still controversial, and other convertases may be involved as well. Furin is a membrane-associated calcium-dependent serine endopeptidase in the yeast Kex2 family. It is localized in the trans-Golgi networks and recycles between the trans-Golgi networks and plasma membranes. Whereas corin is uniquely distributed in the myocardium, furin is found in various tissues; however, the furin gene is highly expressed in hypertrophic cardiomyocytes. proBNP possesses a furin-cleavable sequence at its processing site (-Arg73-Ala74-Pro75-Arg76↓-Ser77-). Corin is a membrane-bound type II transmembrane serine peptidase that is produced at high concentrations in the heart and is up-regulated in hypertrophic cardiomyocytes and failing myocardium. In the presence of adequate stimuli, corin cleaves proANP into the N-terminal proANP 1–98 and the C-terminal ANP 99–126, which are then released into the blood; however, corin has been shown to be capable of processing proBNP as well. Cardiac secretion of BNP and NT-proBNP has been demonstrated by sampling blood from the coronary sinus, and the circulation of intact proBNP in the blood has also been described (6). In addition, BNP and NT-proBNP have been recognized to be modified into a mixture of various fragments (7)(8). A substantial portion of the immunoreactive BNP circulates in humans as protease-degraded fragments, and its N-terminal end seems to be particularly susceptible to enzymatic degradation. Proteolytic fragmentation of NT-proBNP 1–76 at both the C- and N-terminal ends of the molecule to produce smaller immunoreactive molecules in the circulation has also been reported. Commercial assays for BNP and NT-proBNP measure a mixture of cleaved and uncleaved analytes, as well as proBNP to varying extents (9). Initial studies based on fractionating of plasma samples by chromatography and testing the fractions for BNP immunoreactivity, as well as later studies that used western blot analysis of plasma samples from HF patients, have revealed the presence of both low molecular weight and high molecular weight forms of immunoreactive BNP and NT-proBNP (7)(10). Immunoreactive NT-proBNP and BNP in plasma were shown to elute at a position corresponding to a much higher molecular weight than expected by chromatography of the synthetic proBNP and NT-proBNP standards. This result was confirmed in western-blotting studies. The presence of a leucine zipper-like motif in the proBNP sequence led to speculations that proBNP and its N-terminal fragments form oligomers. This process is unlikely to occur in vivo, however, and proBNP has been shown to circulate as a monomer (11). Recently, in vivo glycosylation of proBNP and NT-proBNP was identified as an explanation for this phenomenon, because the synthetic standards used in these experiments were nonglycosylated (12). proBNP may be posttranslationally glycosylated to a varying degree at several sites (Thr36, Ser37, Ser44, Thr48, Ser53, Thr58, Thr71) in its N-terminal region (12). The central portion of human circulating NT-proBNP (amino acid residues 28–56) is also glycosylated, but the C-terminal end (residues 61–76) of the molecule is almost free of O-linked glycans (13). When plasma samples were deglycosylated, western-blotting bands were found at regions corresponding to the nonglycosylated proBNP and NT-proBNP standards (10)(12).