PUB - Publikationen an der Universität Bielefeld

Dystroglycan (DG) is a cell adhesion complex that is widely expressed in tissues. It is composed by two subunits, a-DG, a highly glycosylated protein that interacts with several extracellular matrix proteins, and transmembrane 13-DG whose, cytodomain binds to the actin cytoskeleton. Glycosylation of a-DG is crucial for functioning as a receptor for its multiple extracellular binding partners. Perturbation of a-DG glycosylation is the central event in the pathogenesis of severe pathologies such as muscular dystrophy and cancer. 13-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 13-DG, HEK-293 cells were transiently transfected with a plasmid carrying the 13-DG subunit with GFP fused at its C-terminus. With this strategy, we aimed at forcing 13-DG to occupy multiple intracellular locations instead of sitting tightly at its canonical plasma membrane milieu, where it is commonly found in association with a-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 13-DG binding. A series of already known 13-DG interactors have been found, including ezrin and emerin, whilst significant new matches, which include potential novel 13-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 13-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 13-DG for a proper nuclear localization. These results expand the role of 13-DG as a nuclear scaffolding protein and provide novel evidence of a possible link between dystroglycanopathies and nuclear envelopathies displaying with muscular dystrophy.