To study the effect of O-glycosylation on the conformational propensities of a
peptide backbone, the 15-residue
peptide PPAHGVTSAPDTRPA (PPA15) from the MUC1
protein core and its analogue PPA15(T7), glycosylated with alpha-N-
acetylgalactosamine on Thr7, were prepared and investigated by NMR spectroscopy. The
peptide contains both the GVTSAP sequence, which is an effective substrate for
GalNAc-T1 and -T3
transferases, and the PDTRP fragment, which is a well-known
immunodominant epitope recognized by several anti-MUC1
monoclonal antibodies. Useful structural results were obtained in water upon decreasing the temperature to 5-10 degrees C. The
sugar attachment slightly affected the conformational equilibrium of the
peptide backbone near the glycosylated Thr7 residue. The clustering of low-energy conformations for both PPA15 and PPA15(T7) within the GVTSAP and APDTRP fragments revealed structural similarities between glycosylated and nonglycosylated
peptides. For the GVTSAP region, minor but distinct clusters formed by either PPA15 or PPA15(T7) conformers showed distinct structural propensities of the
peptide backbone specific for either the nonglycosylated or the glycosylated
peptide. The
peptide backbone of the APDTRP fragment, which is a well-known
immunodominant region, resembled an S-shaped bend. A similar structural motif was found in the GVTSAP fragment. The S-shaped structure of the
peptide backbone is formed by consecutive inverse gamma-turn conformations partially stabilized by hydrogen bonding. A comparison of the
solution structure of the APDTRP 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.