In Vitro Molecular Structure of N-Terminal B-Type Natriuretic Peptide: Monomer or Oligomer?

To the Editor: In the circulation, the A-type natriuretic peptide and the B-type natriuretic peptide (BNP) regulate cardiovascular homeostasis. Each hormone is derived from biosynthetic precursors that are processed to the active C-terminal hormones and the N-terminal fragments (1). Measurements of both BNP and N-terminal proBNP in plasma are recommended as diagnostic tools for heart failure. The peptides are measured by immunoassays that use antibodies directed against specific epitopes. A basic understanding of the native peptide structure in terms of posttranslational modifications is thus paramount, because changes in posttranslational processing will affect assay measurement and clinical interpretation drastically. A recent report in Clinical Chemistry addressed the possibility of natriuretic peptide oligomerization in a study that used a recombinant N-terminal proBNP peptide (possessing a glycine–serine N-terminal extension) spiked into 3 matrices (2). Buffer, plasma, or serum was incubated for various times and temperatures; the material was then applied to a reversed-phase column, a gel-filtration column, and a reversed-phase column, respectively. All 3 columns were eluted with acidic acetonitrile. Antibodies directed against linear epitopes (residues 1–22, 10–29, and 57–76) were used to assay fractions for N-terminal proBNP immunoreactivity with the intent of delineating the peptide sequences. We note that the immunoreactivity of endogenous N-terminal proBNP from unspiked plasma does not yield the same response with the different assays [see Fig. 1A of (2)], suggesting that the antibodies possess different affinities for full-length N-terminal proBNP. We believe this possibility is worth considering, given that immunoreactivity differences are a primary means the authors used to infer analyte structure. In other words, how are the different affinities taken into account in the elution profiles? An experiment showing the relative affinities for full-length N-terminal proBNP in buffer alone seems warranted. The authors also note from Fig. 1 in their report that chemical deglycosylation decreases the immunoreactivity for all of the studied epitopes, as well as for N-terminal pro–A-type natriuretic peptide. We suggest this result is due to N- to O-acyl migration in the presence of the harsh acid they used, which likely could be reversed by treatment with base. Alternatively, enzymatic deglycosylation may mitigate these deleterious effects.