Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage.
The alpha-anomeric Galbeta1-3GalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosylation. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reversible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-binding specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than Gal, since ABL recognizes GalNAc more than Gal in TFD. The synthesis was accomplished by C-6 oxidation of Gal from TFD or its alpha-benzyl derivative (BzlalphaTFD), followed by reductive amination between the C-6 aldehyde yielded and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or BzlalphaTFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens. Both immunogens raised anti-KLH antibody titers; however, TFD-KLH did not raise anti-TFD antibodies showing low TFD immunogenicity. In contrast, BzlalphaTFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps improve anti-carbohydrate immune response. When IgG and IgM anti-TFD antibodies were analyzed by competitive ELISA using TFD-related carbohydrates as inhibitors, a high specificity to TFD as well as an enhanced binding to GalNAc over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activity of GlcNAc in contrast to GalNAc. These findings indicate that the anti-TFD antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithelial cell lines.
Irazoqui
F J
FJ
Departamento de Química Biológica-CIQUIBIC-CONICET, 5000 Córdoba, Argentina.
Lopez
P H
PH
Vides
M A
MA
Nores
G A
GA
D000818:Animals; D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000276:immunology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006001:Glycoconjugates / Q000138:chemical synthesis; D051379:Mice
D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D006001:Glycoconjugates; C016467:Thomsen-Friedenreich antigen