title: Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells.
abstract: CD175 or Tn antigen is a carbohydrate moiety of N-acetylgalactosamine (GalNAc)α1-O- linked to the residue of amino acid serine or threonine in a polypeptide chain. Despite the chemical simplicity of the Tn antigen, its antigenic structure is considered to be complex and the clear determinants of Tn antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal GalNAc residue of the Tn antigen irrespective of the peptide context or with low selectivity to the glycoproteins. In contrast, IgG mAbs recognized the Tn antigen in the context of a specific peptide motif. Particularly, JA3 mAb reacted to the GSPP or GSPAPP, and JA5 mAb recognized specifically the GSP motif (glycosylation sites are underlined). The major O-glycan carrier proteins CD43 and CD162 and isoforms of CD45 expressed on Jurkat cells were precipitated by anti-Tn mAbs with different affinities. In summary, our data suggest that Tn antigen-Ab binding capacity is determined by the peptide context of the Tn antigen, antigenic specificity of the Ab and class of the immunoglobulin. The newly generated anti-Tn IgG mAbs with the strong specificity to glycoprotein CD43 can be particularly interesting for the application in leukemia diagnostics and therapy.
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine,  University of Copenhagen, Blegdamsvej 3, dept. 24.6.48, DK-2200 N Copenhagen,  Denmark.
authors.forename: Ola
authors.initials: O
authors.lastname: Blixt
authors.forename: Olga I
authors.initials: OI
authors.lastname: Lavrova
authors.forename: Dmitriy V
authors.initials: DV
authors.lastname: Mazurov
authors.forename: Emiliano
authors.initials: E
authors.lastname: Cló
authors.forename: Stjepan K
authors.initials: SK
authors.lastname: Kracun
authors.forename: Nicolai V
authors.initials: NV
authors.lastname: Bovin
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Filatov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis / Q000737:chemistry*; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology*; D001667:Binding, Competitive; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry*; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000737:chemistry*; D019169:Jurkat Cells; D007938:Leukemia; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D018832:Molecular Chaperones / Q000096:biosynthesis; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D015703:Antigens, CD; D015295:Antigens, Tumor-Associated, Carbohydrate; C505369:C1GALT1C1 protein, human; D007074:Immunoglobulin G; D007075:Immunoglobulin M; C060192:MUC1 protein, human; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D018396:Mucin-1; D010446:Peptide Fragments; D011994:Recombinant Proteins; C040286:Tn antigen