Abstract
MUC1 is a glycoprotein that plays an important role in cancer pathogenesis. In order to study the effect of glycosylation on the conformational propensities of the tandem repeat domain of MUC1, we have determined the structure of the MUC1 tandem repeat peptide AHGVTSAPDTRPAPGSTAPP, O-glycosylated with the trisaccharide (α-Glc-1,4-β-Glc-1,4-α-GalNAc-) at Thr5. This glycopeptide was synthesized to model a heavily Oglycosylated threonine residue in the tandem repeat domain. The NMR experiments used in this study included TOCSY, NOESY, ROESY, DQF-COSY, HSQC and 1D NMR. The peak volumes determined using the program SPARKY were converted into distance constraints using the program CALIBA. The programs FiSiNOE and HABAS were used to generate angle constraints. Using conformational restraints obtained from NMR, the program DYANA was used to determine the structures of the peptide. Finally, structural refinement was performed within the SYBYL software package using GLYCAM parameters and Kollman-all atom types. The presence of strong sequential αN connectivities suggested an extended conformation of the peptide backbone. Strong sequential αδ connectivities were indicative of a trans conformation of the Ala-Pro peptide bonds. In addition, presence of sequential NN connectivities in the peptide segments Gly3-Val4-Thr5-Ser6, Asp9-Thr10-Arg11 and Gly-Ser16 were indicative of twist-like conformations of the peptide backbone in these peptide segments.
Highlights
MUC1 [1] is a glycoprotein expressed on the cell surface of normal and cancer cells
In order to characterize the conformational changes on acceptor substrates that take place following initial glycosylation, previous studies were focused on MUC1-based tandem repeat peptides monoglycosylated with a single GalNAc residue at Thr5 [11]
Using NMR, we have investigated the structure of a MUC1-based tandem repeat peptide O-glycosylated with the trisaccharide (α-Glc-1,4-β-Glc-1,4-α-GalNAc-) at Thr5
Summary
MUC1 [1] is a glycoprotein expressed on the cell surface of normal and cancer cells. The large extracellular fragment of MUC1 consists of tandem repeats of a 20 amino acid motif. In order to characterize the conformational changes on acceptor substrates that take place following initial glycosylation, previous studies were focused on MUC1-based tandem repeat peptides monoglycosylated with a single GalNAc residue at Thr5 [11]. Using NMR, we have investigated the structure of a MUC1-based tandem repeat peptide O-glycosylated with the trisaccharide (α-Glc-1,4-β-Glc-1,4-α-GalNAc-) at Thr5.
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