tailieunhanh - Báo cáo Y học: NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylatedmucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and thebindingepitopeof peptideandglycopeptideantigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-a-D-GalNAc)RP are known lig-ands of the monoclonal antibody. | Eur. J. Biochem. 269 1444-1455 2002 FEBS 2002 NMR-based determination of the binding epitope and conformational analysis of MUC-1 glycopeptides and peptides bound to the breast cancer-selective monoclonal antibody SM3 Heiko Moller1 Nida Serttas1 Hans Paulsen1 Jov M. Burchell2 Jovce Tavlor-Panadimitriou2 B and Bernd Mever1 1 Institute of Organic Chemistry University of Hamburg Germany imperial Cancer Research Fund Breast Cancer Biology Group Guy s Hospital London UK Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 MUC-1 structures are recognized by the monoclonal antibody SM3 which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT O-a-D-GalNAc RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference STD NMR spectroscopy. The peptide s epitope is predominantly located in the N-terminal PDT segment whereas the C-ter-minal RP segment has fewer interactions with the protein. In contrast the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well. Keywords glycopeptide antibody .