The precursor to B-type natriuretic peptide is an O-linked glycoprotein |
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Authors: | Schellenberger Ute O'Rear Jessica Guzzetta Andrew Jue Rodney A Protter Andrew A Pollitt N Stephen |
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Institution: | Scios Inc., 6500 Paseo Padre Parkway, Fremont, CA 94555, USA. |
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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. |
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Keywords: | B-Type natriuretic peptide (BNP) Propeptide (proBNP) O-linked glycosylation Mass spectrometry Enzymatic deglycosylation |
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