The precursor to B-type natriuretic peptide is an O-linked glycoprotein

• Published in: Archives of biochemistry and biophysics 2006-07, Vol.451 (2), p.160-166
• Main Authors: Schellenberger, Ute, O’Rear, Jessica, Guzzetta, Andrew, Jue, Rodney A., Protter, Andrew A., Stephen Pollitt, N.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; B-Type natriuretic peptide (BNP); Carbohydrate Sequence; CHO Cells; Cricetinae; Enzymatic deglycosylation; Glycosylation; Heart Failure - blood; Humans; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Natriuretic Peptide, Brain - blood; Natriuretic Peptide, Brain - chemistry; Natriuretic Peptide, Brain - metabolism; Neuraminidase - pharmacology; O-linked glycosylation; Peptide Fragments - blood; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Peptide Mapping; Promoter Regions, Genetic; Propeptide (proBNP); Protein Precursors - blood; Protein Precursors - chemistry; Protein Precursors - genetics; Protein Precursors - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis, Protein; Trypsin - pharmacology
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2006.03.028

Human pro-B-type natriuretic peptide (proBNP), the precursor for B-type natriuretic peptide (BNP), was expressed in Chinese hamster ovary cells (CHO) and compared by Western blot analysis to BNP cross-reacting material immunoprecipitated from the plasma of heart failure patients. Both recombinant and native forms co-migrated as a diffuse band centered around 25 kDa and were reduced to a 12 kDa species by treatment with a mixture of O-link deglycosylation enzymes. The 108-amino acid CHO-expressed protein was examined by tryptic mapping and LC–MS and found to be an O-linked glycoprotein. Determination of the sites of O-glycosyl addition by blank cycle sequencing of tryptic and Glu-C ( Staphylococcus aureus V8 protease) peptides showed that there are seven sites of glycosylation confined to a 36-amino acid residue stretch within the center of the propeptide region. This data is consistent with previous observations of higher molecular weight isoforms of BNP.