Significance of Proline Residue on Short Mucin Peptide Interactions with Mouse MUC1 Monoclonal Antibody Studied by Saturation Transfer Difference NMR Spectroscopy.

In this study we investigated to see whether or not a shortened MUC1 mucin peptide epitope with the sequence GVTSAPD containing a single prolyl residue would still bind specific monoclonal antibody as its native sequence (e.g., PDTRP), known to be the specific recognition site on the Variable Number Tandem Repeat (VNTR) region of MUC1 mucin by the immune system. The affinity of GVTSAPD peptide to a mouse Muc1 mucin specific monoclonal antibody (clone 6A4, IgG1 isotype) was investigated by Saturation Transfer Difference NMR spectroscopy (STD NMR). Results showed that the shortened mucin epitope GVTSAPD still retained affinity to Muc1 specific monoclonal antibody (mAb) while one that lacks the prolyl residue at position 6 lost its affinity, which suggests that P6 is necessay for antibody binding. The interactions observed by STD NMR occurred strongest at the P6 side chain 1H's (βH and γH); the P6Hα showed lower degree of saturation transfer effect. Minor interactions also occurred at the methyl groups of V2' T3 and A5. Mucin peptides derived from the VNTR region have been the target of cancer vaccine research, thus properties associated with mucin peptide structure, conformation and antibody interaction are central to peptide design or engineering towards that end.