title: The precursor to B-type natriuretic peptide is an O-linked glycoprotein.
abstract: 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.
authors.affiliation: Scios Inc., 6500 Paseo Padre Parkway, Fremont, CA 94555, USA.
authors.forename: Ute
authors.initials: U
authors.lastname: Schellenberger
authors.forename: Jessica
authors.initials: J
authors.lastname: O'Rear
authors.forename: Andrew
authors.initials: A
authors.lastname: Guzzetta
authors.forename: Rodney A
authors.initials: RA
authors.lastname: Jue
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Protter
authors.forename: N Stephen
authors.initials: NS
authors.lastname: Pollitt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006031:Glycosylation; D006333:Heart Failure / Q000097:blood; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020097:Natriuretic Peptide, Brain / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014357:Trypsin / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; D020097:Natriuretic Peptide, Brain; D009439:Neuraminidase; D014357:Trypsin