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 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.
Kirnarsky
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L
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Prakash
O
O
Vogen
S M
SM
Nomoto
M
M
Hollingsworth
M A
MA
Sherman
S
S
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
D006020:Glycopeptides; D018396:Mucin-1; D010446:Peptide Fragments; D000116:Acetylgalactosamine