Unraveling the Complexity of Circulating Forms of Brain Natriuretic Peptide

Abstract

Determining the circulating form of a biomarker is critical for the development of a highly sensitive and specific immunoassay. The hormone brain natriuretic peptide (BNP) is produced in the atria as a preprohormone, and the cleavage of its signal sequence produces the circulating prohormone proBNP (amino acid residues 1–108, human sequence). The prohormone is subsequently cut to yield a 76-amino acid N-terminal (NT) fragment (NT-proBNP) and the 33-amino acid active hormone BNP (comprising residues 77–108 of proBNP). The 2 commercially available immunoassays detect either the circulating NT fragment or BNP and, potentially, could detect proBNP. However, whether these assays detect all possible circulating forms of BNP remains unclear. The potential circulating forms of BNP have different intrinsic physical characteristics that can dictate antigenicity, extent of nonspecific binding during isolation, endogenous clearance kinetics, circulating half-life, and/or affinity for other proteins including cellular receptors and putative serum/plasma carrier proteins. Consequently, characterization of the physical status of each circulating form of BNP is key to assay development. It is critical to determine whether the circulating BNP forms have posttranslational modifications (PTM), such as phosphorylation or N- and O-linked glycosylation, and/or bind to other protein(s). In a recent issue of Clinical Chemistry , an article by Seferian et al. (1) described 2 approaches to shedding light on the circulating forms of BNP. The 2 strategies examined differences in ( a ) antigenicity, ( b ) antibody binding, and ( c ) the theoretical and observed MWs of the unmodified recombinant form and the circulating endogenous form(s). The data presented indicated that the chemistry of circulating BNP is complex. This complexity needs to be taken into account during immunoassay development. Is there a potential PTM? Seferian et al. (1) produced a large number of peptides and fragments of BNP (pro, NT-pro, and BNP) …