Synthetic, Structural, and Biosynthetic Studies of an Unusual Phospho-Glycopeptide Derived from α-Dystroglycan

Abstract

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid phase glycopeptide synthesis using hyper-acid sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.

Protein O-Linked Mannose β-1,4-N-Acetylglucosaminyl-transferase 2 (POMGNT2) Is a Gatekeeper Enzyme for Functional Glycosylation of α-Dystroglycan

“…Man414 is only seven amino acids in length and lacks the TPT motif (Table 1). However, the kinetics of Man414 with POMGNT1 have been studied previously (26), and the homologous residue in rabbit (Oryctolagus cuniculus) has been site-mapped with mannose (21), making it a useful predicted core M1 glycopeptide for this study.…”

Effects of prednisolone on the dystrophin-associated proteins in the blood–brain barrier and skeletal muscle of dystrophic mdx mice

“…11 induced a total recovery of a-DG mRNA increment, but a partial recovery of the functional protein level. Many posttranslational modifications of newly synthesized a-DG have been described, including glycosylation, 81,82 and the glucocorticoids are known to modulate the glycosylation of many proteins. 83 Our results suggest that the a-DG alteration in mdx animals may involve post-translational events rather than gene expression.…”

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.…”

“…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).…”