Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site.
BACKGROUND
Processing of the brain natriuretic peptide (BNP) precursor, proBNP, is a convertase-dependent reaction that produces 2 molecules--the active BNP hormone and the N-terminal part of proBNP (NT-proBNP). Although proBNP was first described more than 15 years ago, very little is known about the cellular mechanism of its processing. The study of proBNP processing mechanisms is important, because processing impairments could be associated with the development of heart failure (HF).


METHODS
The biochemical properties of recombinant proBNP and NT-proBNP and the same molecules derived from the blood of HF patients were analyzed by gel-filtration chromatography, site-directed mutagenesis, and different immunochemical methods with a panel of monoclonal antibodies (MAbs).


RESULTS
Part of the proBNP molecule (amino acid residues 61-76) located near the cleavage site was inaccessible to specific MAbs because of the presence of O-glycans, whereas the same region in NT-proBNP was completely accessible. We demonstrated that a convertase (furin) could effectively cleave deglycosylated (but not intact) proBNP. Of several mutant proBNP forms produced in a HEK 293 cell line, only the T71A variant was effectively processed in the cell.


CONCLUSIONS
Only proBNP that was not glycosylated in the region of the cleavage site could effectively be processed into BNP and NT-proBNP. Site-directed mutagenesis enabled us to ascertain the unique suppressing role of T71-bound O-glycan in proBNP processing.
Semenov
Alexander G
AG
HyTest Ltd., Turku, Finland. alexander.semenov@hytest.fi
Postnikov
Alexander B
AB
Tamm
Natalia N
NN
Seferian
Karina R
KR
Karpova
Natalia S
NS
Bloshchitsyna
Marina N
MN
Koshkina
Ekaterina V
EV
Krasnoselsky
Mihail I
MI
Serebryanaya
Daria V
DV
Katrukha
Alexey G
AG
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D045683:Furin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D020097:Natriuretic Peptide, Brain / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D011498:Protein Precursors; D020097:Natriuretic Peptide, Brain; D045683:Furin