Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation.
The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines.
Karsten
U
U
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Diotel
C
C
Klich
G
G
Paulsen
H
H
Goletz
S
S
Müller
S
S
Hanisch
F G
FG
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000378:metabolism; D006020:Glycopeptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018396:Mucin-1 / Q000276:immunology* / Q000378:metabolism; D010504:Periodic Acid / Q000737:chemistry; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid / Q000276:immunology*
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D006020:Glycopeptides; D018396:Mucin-1; D010504:Periodic Acid; C009288:metaperiodate