Conformational consequences of protein glycosylation: Preparation of O‐mannosyl serine and threonine building blocks, and their incorporation into glycopeptide sequences derived from α‐dystroglycan

Abstract

With the goal to investigate the structural impact of O‐mannosyl glycosylation on α‐dystroglycan, a glycoprotein that has an important role in the extracellular organization of muscle, glycopeptides derived from its mucin‐like sequence have been prepared by solid‐phase peptide synthesis. Two approaches have been explored to obtain needed mannosylated serine and threonine building blocks. With the α‐carboxyl group unprotected, and with tetraaceto‐1‐fluoro‐α‐D‐mannose as the sugar donor, the desired α‐O‐mannosyl‐Fmoc‐Ser/Thr formed, along with mannosyl ester isomers and the species with mannose attached to both hydroxyl and carboxyl functions. Relevant mechanistic questions and stability issues were elucidated. Alternatively, building blocks were made with the α‐carboxyl protected/activated as the pentafluorophenyl (Pfp) ester. Glycopeptides synthesized herein contained 5–9 residues, and featured one, two, and four consecutive Ser and/or Thr residues O‐glycosylated with mannose. Circular dichroism (CD) spectra for Man‐containing glycopeptides recorded in water show them to be not well ordered. For one of the α‐dystroglycan‐derived sequences, the comparative conformational consequences of glycosylation by either Man or GalNAc have been examined by CD and NMR, with both methods showing a more organized structure when GalNAc is present. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 358–368, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Glycosylation of α-Dystroglycan

“…Our (33,34). How these changes in glycosylation may affect the functional properties of ␣-DG in vivo remains to be determined.…”

“…The synthesis of constructs in the Ac-PPTTTTKKP-NH 2 series has been reported (33). Assay of Enzyme Activity on Defined Peptide and Glycopeptide Substrates-Initially we tested the ability of various members (ppGalNAc-T1, -T2, -T3, -T4, -T5, -T7, -T10, -T11, and -T16) to glycosylate each set of peptides and glycopeptides derived from ␣-DG, along with appropriate positive controls (Figs.…”

“…Furthermore, O-GalNAc addition is required for viability in Drosophila (26 -28) and is known to affect ECM protein secretion (29) and protease sensitivity (30). Additionally, the presence of O-GalNAc glycosylation can influence peptide and protein structural properties, stabilizing more extended structures in regions where these glycans are present (31,32), in contrast to the effects of O-Man modifications that have more modest conformational effects (33,34). The molecular morphology of ␣-DG as revealed by electron microscopy (35,36) indicates N-and C-terminal globular domains separated by a central extended region.…”

“…Interestingly, two members of the ppGalNAc-T family that require pre-existing O-GalNAc modifications on glycopeptide substrates, ppGalNAc-T7 and -T10 (40,48,49), showed no activity on any of the O-mannosylated substrates used in the present study. It is anticipated that O-Man and O-GalNAc glycopeptides would interact differently with the enzymes, given the differences in the structure of the sugar residue and the conformational properties of the glycopeptides (33,34).…”

Mannosylated lipopetides as model vaccines for targeting the mannose receptor

“…Both acetylated and deacetylated delivery systems that contained mannose successfully reduced tumour size in mice when compared to glucose delivery systems. (acetylated mannosyl donor) to Fmoc-Ser-OH using BF3.Et2O in DCM at RT for 4 h generated 2 (Fmoc-serine mannosylated amino acid building blocks) as a mixture of α and β due to attack of the Lewis acid from both below (β, 25.44%) and above (α, 74.55%) [230]. For synthesis details see …”