Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
Glycobiology. 2010 Mar;20(3):381-94. doi: 10.1093/glycob/cwp189. Epub 2009 Dec 7.
Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essential components of the mammalian dystroglycan-mediated pathway; however, the glycosylation of Drosophila Dystroglycan (DG) has not yet been explored. In this study, we characterized the glycosylation of Drosophila DG using a combination of glycosidase treatments, lectin blots, trypsin digestion, and mass spectrometry analyses. Our results demonstrated that DG extracellular domain is O-mannosylated in vivo. We found that the concurrent in vivo activity of the two Drosophila protein O-mannosyltransferases, Rotated Abdomen and Twisted, is required for O-mannosylation of DG. While our experiments unambiguously determined some O-mannose sites far outside of the mucin-type domain of DG, they also provided evidence that DG bears a significant amount of O-mannosylation within its central region including the mucin-type domain, and that O-mannose can compete with O-GalNAc glycosylation of DG. We found that Rotated Abdomen and Twisted could potentiate in vivo the dominant-negative effect of DG extracellular domain expression on crossvein development, which suggests that O-mannosylation can modulate the ligand-binding activity of DG. Taken together these results demonstrated that O-mannosylation of Dystroglycan is an evolutionarily ancient mechanism conserved between Drosophila and humans, suggesting that Drosophila can be a suitable model system for studying molecular and genetic mechanisms underlying human dystroglycanopathies.
最近的研究强调了一种利用果蝇作为模型系统来研究人类先天性肌肉萎缩症（称为 dystroglycanopathies）机制的新可能性，这些疾病是由于α- dystroglycan 的异常糖基化引起的。其中一些疾病与 O-糖基化缺陷有关，这是由两种蛋白质 O-甘露糖基转移酶介导的最突出的α-dystroglycan 糖基化类型之一。果蝇似乎拥有哺乳动物 dystroglycan 介导途径的所有必需成分的同源物；然而，果蝇 Dystroglycan (DG) 的糖基化尚未得到探索。在这项研究中，我们使用糖苷酶处理、凝集素印迹、胰蛋白酶消化和质谱分析相结合的方法来表征果蝇 DG 的糖基化。我们的结果表明，DG 细胞外结构域在体内被 O-甘露糖化。我们发现，两种果蝇蛋白 O-甘露糖基转移酶 Rotated Abdomen 和 Twisted 的体内同时活性是 DG 的 O-甘露糖化所必需的。虽然我们的实验明确确定了 DG 远在其粘液型结构域之外的一些 O-甘露糖位点，但它们也提供了证据表明 DG 在其中央区域包括粘液型结构域内存在大量的 O-甘露糖化，并且 O-甘露糖可以与 DG 的 O-GalNAc 糖基化竞争。我们发现 Rotated Abdomen 和 Twisted 可以在体内增强 DG 细胞外结构域表达对横静脉发育的显性负效应，这表明 O-甘露糖化可以调节 DG 的配体结合活性。总之，这些结果表明，Dystroglycan 的 O-甘露糖化是果蝇和人类之间保守的一种古老的进化机制，这表明果蝇可以成为研究人类 dystroglycanopathies 分子和遗传机制的合适模型系统。
