title: Epitope structure of the carbohydrate recognition domain of asialoglycoprotein receptor to a monoclonal antibody revealed by high-resolution proteolytic excision mass spectrometry.
abstract: Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.
authors.affiliation: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Raluca
authors.initials: R
authors.lastname: Stefanescu
authors.forename: Rita
authors.initials: R
authors.lastname: Born
authors.forename: Adrian
authors.initials: A
authors.lastname: Moise
authors.forename: Beat
authors.initials: B
authors.lastname: Ernst
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D037263:Asialoglycoprotein Receptor / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D037263:Asialoglycoprotein Receptor; D004220:Disulfides; D000939:Epitopes; D014357:Trypsin