Structural Effects of O-Glycosylation on a 15-Residue Peptide from the Mucin (MUC1) Core Protein

Abstract

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.

Synthesis and Structural Model of an α(2,6)‐Sialyl‐T Glycosylated MUC1 Eicosapeptide under Physiological Conditions

“…[8,26] The drawback of "insufficient structure" under physiological conditions was circumvented by lowering the temperature, and/or addition of solvents and adjustment of pH to low values. [27][28][29][30][31] As an example, Kirnarsky et al [32,33] studied glycosylated 15-mers of MUC1 at low temperatures (5 and 10 8C) in water, and 9-mers in DMSO.…”

“…[27,32,43] All ROE values used for structure calculation are listed in Table S1 of the Supporting Information.…”

“…Coltart et al [30] found a similar methyl group association in their study of the glycosylated N-terminal fragment STTAV of the cell surface glycoprotein CD43. It was suggested by Kirnarsky et al [32] that the N-acetyl group of the GalNAc moiety interacts directly with the peptide backbone, possibly through hydrogen bonding. An intramolecular hydrogen bonding between the amide proton of GalNAc and the carbonyl oxygen of the O-linked threonine residue was also proposed by Naganagowda et al [44] as the key stabilizing element, opposed to the O-linked serine analogue for which this interaction seemed to be missing.…”

Label-free sensing of the binding state of MUC1 peptide and anti-MUC1 aptamer solution in fluidic chip by terahertz spectroscopy

“…It comprises an extracellular domain composed of a region including nearly identical 20-amino-acid-long repeats, a cytoplasmic domain of 69 amino acids, and a hydrophobic membrane-spanning domain of 31 amino acids [1]. In particular, the 9-amino-acid-long peptide sequence APDTRPAP often belongs to the variable tandem repeat (VTR) domain from the epitope within a highly immunogenic region of MUC1 [2]. As a well-known tumor biomarker associated with disease severity, MUC1 is up-regulated and aberrantly glycosylated in a variety of human epithelial cancer cells, and often free-floats in the bloodstream [3], thus making liquid biopsy for MUC1 potentially helpful in cancer diagnosis.…”

NMR-based Structural Studies of the Glycosylated MUC1 Tandem Repeat Peptide

“…It has been hypothesized that the GalNAc transferases following initial glycosylation, bind to the GalNAc residue through their lectin domain, which results in conformational changes in its catalytic domain or in the acceptor substrate peptide backbone that facilitate glycosylation at proximal and distant sites [9]. 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]. We extended those studies to glycosylation of tandem repeat peptides with bulky carbohydrate moieties.…”