Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 133, Issue 36, Pages 14418-14430Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja205473q
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Funding
- National Institute of General Medicine (NIGMS) of the National Institutes of Health [R01GM090269]
- Center for Research Resources [P41RR018502, P41RR005351]
- National institute of Health [R21AR056055]
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Aberrant glycosylation of alpha-dystroglycan (alpha-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of alpha-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from alpha-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 beta(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 alpha-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated alpha-DG.
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