title: The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.
abstract: MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.
authors.affiliation: Research Group of Peptide Chemistry, Eötvös L. University, Hungarian Academy of Sciences, Budapest, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Uray
authors.forename: Mamoru
authors.initials: M
authors.lastname: Mizuno
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Inazu
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Goto
authors.forename: Ferenc
authors.initials: F
authors.lastname: Hudecz
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002942:Circular Dichroism; D000939:Epitopes / Q000276:immunology*; D006031:Glycosylation; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D055262:Mucin-2 / Q000737:chemistry / Q000276:immunology*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D014867:Water / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D055262:Mucin-2; D010455:Peptides; D014867:Water
keywords: MUC2 mucin; antibody binding of epitope; glycosylated peptides