title: An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding.
abstract: The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea [2] Department of Cancer Biology, Seoul National University College of Medicine, Seoul, Korea [3] Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Yujean
authors.initials: Y
authors.lastname: Lee
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea [2] Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Hyori
authors.initials: H
authors.lastname: Kim
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Korea [2] Department of Cancer Biology, Seoul National University College of Medicine, Seoul, Korea [3] Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Junho
authors.initials: J
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006031:Glycosylation; D057809:HEK293 Cells; D006333:Heart Failure / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020097:Natriuretic Peptide, Brain / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain