Structural effects of O-glycosylation on a 15-residue peptide from the mucin (MUC1) core protein

To study the effect of O-glycosylation on the conformational propensities o f a peptide backbone, the 15-residue peptide PPAHGVTSAPDTRPA (PPA15) from t he MUC1 protein core and its analogue PPA15(T7), glycosylated with alpha-N- acetylgalactosamine on Thr7, were prepared and investigated by NMR spectros copy. The peptide contains both the GVTSAP sequence, which is an effective substrate for GalNAc-Tl and -T3 transferases, and the PDTRP fragment, which is a well-known immunodominant epitope recognized by several anti-MUC1 mon oclonal antibodies. Useful structural results were obtained in water upon d ecreasing the temperature to 5-10 degrees C, The sugar attachment slightly affected the conformational equilibrium of the peptide backbone near the gl ycosylated Thr7 residue. The clustering of low-energy conformations for bot h PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed struc tural similarities between glycosylated and nonglycosylated peptides. For t he GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA 15(T7) conformers showed distinct structural propensities of the peptide ba ckbone specific for either the nonglycosylated or the glycosylated peptide. The peptide backbone of the APDTRP fragment, which is a well-known immunod ominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the peptide backbon e is formed by consecutive inverse gamma-turn conformations partially stabi lized by hydrogen bonding. A comparison of the solution structure of the AP DTRP fragment with a crystal structure of the MUC1 peptide antigen bound to the breast tumor-specific antibody SM3 demonstrated significant structural similarities in the general shape.