The epitope recognized by the unique anti-MUC1 monoclonal antibody MY.1E12 involves sialyl alpha 2-3galactosyl beta 1-3N-acetylgalactosaminide linked to a distinct threonine residue in the MUC1 tandem repeat.
The specificity of the MY.1E12 mAb that was generated by immunizing mice with human milk fat globule (HMFG) was investigated. Fluorescein isothiocyanate (FITC)-conjugated peptides corresponding to a portion of the MUC1 tandem repeat were enzymatically glycosylated with N-acetylgalactosamine, galactose, and then sialic acid. The MY.1E12 mAb was examined for its affinity to the resulting glycopeptides by fluorescence polarization. Its affinity for the peptide whose Thr within the VTS sequence bears a Neu5Ac alpha 2-3Gal beta 1-3GalNAc trisaccharide (K(d)=1.4 x 10(-7) M) was significantly higher than for the same peptide whose Thr bears an unsialylated disaccharide (K(d)=3.9 x 10(-6) M). The MY.1E12 mAb also bound strongly to a purified recombinant MUC1 fusion protein with six tandem repeats that was expressed by transfected MCF-7 breast cancer cells. The removal of sialic acids from the fusion protein significantly decreased MY.1E12 mAb reactivity, much more so than the MUC1-specific 115D8 antibody, whose epitope is known to be destroyed by desialylation. Thus, the attachment of the sialyl alpha 2-3Gal beta 1-3 beta 1-3GalNAc trisaccharide onto the Thr within the VTS motif significantly increases the binding of the MY.1E12 antibody to the MUC1 repeat sequence.
Takeuchi
Hideyuki
H
Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
Kato
Kentaro
K
Denda-Nagai
Kaori
K
Hanisch
Franz-Georg
FG
Clausen
Henrik
H
Irimura
Tatsuro
T
D000116:Acetylgalactosamine / Q000276:immunology*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005690:Galactose / Q000276:immunology*; D006017:Glycolipids / Q000276:immunology*; D006020:Glycopeptides / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D066292:Lipid Droplets; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology*; D019158:N-Acetylneuraminic Acid / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D020080:Tandem Repeat Sequences; D013912:Threonine; D014312:Trisaccharides / Q000138:chemical synthesis
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D005456:Fluorescent Dyes; D006017:Glycolipids; D006020:Glycopeptides; D006023:Glycoproteins; D018396:Mucin-1; D011993:Recombinant Fusion Proteins; D014312:Trisaccharides; C110367:milk fat globule; D013912:Threonine; D019158:N-Acetylneuraminic Acid; D016650:Fluorescein-5-isothiocyanate; D000116:Acetylgalactosamine; D005690:Galactose