title: Glycosylation and processing of pro-B-type natriuretic peptide in cardiomyocytes.
abstract: B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79.
authors.affiliation: Molecular Cardiology, Nephrology and Hypertension, Cleveland Clinic, Cleveland, OH, United States.
authors.forename: Jianhao
authors.initials: J
authors.lastname: Peng
authors.forename: Jingjing
authors.initials: J
authors.lastname: Jiang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Qi
authors.forename: Xue-Long
authors.initials: XL
authors.lastname: Sun
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Wu
mesh_terms: D045683:Furin / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism*; D020097:Natriuretic Peptide, Brain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
chemical_list: D020097:Natriuretic Peptide, Brain; D006026:Glycoside Hydrolases; C119812:CORIN protein, human; D012697:Serine Endopeptidases; D045683:Furin