Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, USA.
J Am Chem Soc. 2011 Sep 14;133(36):14418-30. doi: 10.1021/ja205473q. Epub 2011 Aug 22.
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.
α- 连接的岩藻糖基化聚糖酶（α-DG）的糖基化异常会导致与细胞外基质的相互作用丧失，这是几种疾病发病机制的核心。为了研究α-DG 的蛋白质糖基化，我们开发了一种简便的合成方法，用于制备源自α-DG 的异常磷酸化 O-甘露糖基糖肽。该方法采用适当保护的磷酸甘露糖苷与 Fmoc 保护的苏氨酸衍生物偶联，然后在使用超酸敏感的 Sieber 酰胺树脂的自动化固相糖肽合成中使用所得衍生物。合成工作还提供了一种还原的磷酸三糖，该衍生物的 NMR 数据证实了异常磷酸聚糖结构的正确结构分配。糖肽的合成使得有可能探索调节关键聚糖结构的因素。已确定具有 6-磷酸-O-甘露糖基残基的糖肽不是将β（1,2）-GlcNAc 连接到 O-甘露糖基部分的酶 POMGnT1 的作用的受体，而未磷酸化的衍生物则很容易被该酶延伸。这一发现意味着在确定 O-聚糖结构命运时存在特定的事件顺序。还发现 POMGnT1 的活性取决于在多肽/糖肽序列的背景下接受部位的位置。通过 NMR 进行的构象分析表明，O-甘露糖基修饰不会对肽骨架产生主要的构象影响。然而，据推测，这些残基在糖蛋白糖基化的早期引入，具有调节随后的 O-GalNAc 添加位点的能力，这些添加确实会产生构象效应。这些研究表明，通过获得离散的糖肽结构，有可能揭示对α-DG 的 O-聚糖加工的复杂调节，这对其正常的翻译后成熟以及与低聚糖基化α-DG 相关的病理学机制都具有重要意义。
