Abstract：
Dystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, α-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane β-DG whose, cytodomain binds to the actin cytoskeleton. Glycosylation of α-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of α-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. β-DG acts as a scaffold for several cytoskeletal and nuclear proteins and very little is known about the fine regulation of some of these intracellular interactions and how they are perturbed in diseases. To start filling this gap by identifying uncharacterized intracellular networks preferentially associated with β-DG, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing β-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with α-DG. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis led to the identification of an interactome formed by 313 exclusive protein matches for β-DG binding. A series of already known β-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments, such as cytoskeleton, endoplasmic reticulum/Golgi, mitochondria, nuclear membrane and the nucleus itself. Of particular interest amongst the novel identified matches, Lamina-Associated Polypeptide-1B (LAP1B), an inner nuclear membrane protein, whose mutations are known to cause nuclear envelopathies characterized by muscular dystrophy, was found to interact with β-DG in HEK-293 cells. This evidence was confirmed by immunoprecipitation, Western blotting and immunofluorescence experiments. We also found by immunofluorescence experiments that LAP1B looses its nuclear envelope localization in C2C12 DG-knock-out cells, suggesting that LAP1B requires β-DG for a proper nuclear localization. These results expand the role of β-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy.
摘要：
营养不良聚糖(DG)是在组织中广泛表达的细胞粘附复合物。它由两个子单元组成，α-DG,一种高度糖基化的蛋白质，与几种细胞外基质蛋白相互作用，和跨膜β-DG，细胞结构域与肌动蛋白细胞骨架结合。α-DG的糖基化对于作为其多个细胞外结合配偶体的受体起作用是至关重要的。α-DG糖基化的扰动是严重病理如肌营养不良和癌症的发病机理中的中心事件。β-DG充当几种细胞骨架和核蛋白的支架，对这些细胞内相互作用中的一些的精细调节以及它们在疾病中如何受到干扰知之甚少。为了通过识别优先与β-DG相关的未表征的细胞内网络来开始填补这一空白，用携带β-DG亚基的质粒瞬时转染HEK-293细胞,其中GFP在其C-末端融合。有了这个策略,我们的目标是迫使β-DG占据多个细胞内位置，而不是紧紧地坐在其典型的质膜环境，通常与α-DG相关。通过抗GFP抗体的免疫沉淀，然后进行shot弹枪蛋白质组分析，从而鉴定了由313个与β-DG结合的专有蛋白质匹配形成的相互作用组。已经发现了一系列已知的β-DG相互作用物，包括ezrin和emerin,虽然重要的新比赛，其中包括潜在的新型β-DG相互作用者及其相关网络，在不同的亚细胞区室中被鉴定出来，如细胞骨架，内质网/高尔基,线粒体,核膜和细胞核本身。特别感兴趣的是新颖的识别匹配，层相关多肽-1B（LAP1B），一种内核膜蛋白，已知其突变会导致以肌营养不良为特征的核包膜病，在HEK-293细胞中发现与β-DG相互作用。免疫沉淀证实了这一证据，蛋白质印迹和免疫荧光实验。我们还通过免疫荧光实验发现，LAP1B在C2C12DG敲除细胞中失去其核包膜定位，这表明LAP1B需要β-DG才能进行适当的核定位。这些结果扩展了β-DG作为核支架蛋白的作用，并提供了新的证据，证明了肌营养不良症与核包膜病之间可能存在联系。
