Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification (PTM) that fulfills important biological roles during embryonic development. Three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knock-out (KO) of O-Man glycosyltransferase genes. We established a human cell library for analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man glycosylated adhesion molecules and receptors.

Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.

The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells (PCs) in mice. Using a combination of a fluorescent reporter line (Ai14) to indicate Cre-mediated recombination and a floxed Dystroglycan line (Dag1flox ), we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2Cre line exhibits a gradual mosaic pattern of Cre recombination in PCs from Postnatal Day 7 (P7) to P14, while loss of Dag1 protein is not complete until P30. Ptf1aCre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including PCs and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1aCre results in stochastic loss of Dag1 protein in these neurons. NestinCre , which is often described as a \"pan-neuronal\" Cre line for the central nervous system, does not drive Cre-mediated recombination in PCs. We identify a Calb1Cre line that drives efficient and complete recombination in embryonic PCs, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8-mediated delivery of Cre at P0 results in gradual transduction of PCs during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar PCs at different developmental stages and illustrate the importance of validating the loss of protein following recombination.

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.

Dystroglycan (DG) is an extracellular matrix receptor consisting of an α- and a β-DG subunit encoded by the DAG1 gene. The homozygous mutation (c.2006G>T, p.Cys669Phe) in β-DG causes muscle-eye-brain disease with multicystic leukodystrophy in humans. In a mouse model of this primary dystroglycanopathy, approximately two-thirds of homozygous embryos fail to develop to term. Mutant mice that are born undergo a normal postnatal development but show a late-onset myopathy with partially penetrant histopathological changes and an impaired performance on an activity wheel. Their brains and eyes are structurally normal, but the localization of mutant β-DG is altered in the glial perivascular end-feet, resulting in a perturbed protein composition of the blood-brain and blood-retina barrier. In addition, α- and β-DG protein levels are significantly reduced in muscle and brain of mutant mice. Owing to the partially penetrant developmental phenotype of the C669F β-DG mice, they represent a novel and highly valuable mouse model with which to study the molecular effects of β-DG functional alterations both during embryogenesis and in mature muscle, brain and eye, and to gain insight into the pathogenesis of primary dystroglycanopathies.

[This corrects the article DOI: 10.3389/fmolb.2023.1325284.].

Dystroglycan is a ubiquitously expressed heterodimeric cell-surface laminin receptor with roles in cell adhesion, signalling, and membrane stabilisation. More recently, the transmembrane β-subunit of dystroglycan has been shown to localise to both the nuclear envelope and the nucleoplasm. This has led to the hypothesis that dystroglycan may have a structural role at the nuclear envelope analogous to its role at the plasma membrane. The biochemical fraction of myoblast cells clearly supports the presence of dystroglycan in the nucleus. Deletion of the dystroglycan protein by disruption of the DAG1 locus using CRISPR/Cas9 leads to changes in nuclear size but not overall morphology; moreover, the Young\'s modulus of dystroglycan-deleted nuclei, as determined by atomic force microscopy, is unaltered. Dystroglycan-disrupted myoblasts are also no more susceptible to nuclear stresses including chemical and mechanical, than normal myoblasts. Re-expression of dystroglycan in DAG1-disrupted myoblasts restores nuclear size without affecting other nuclear parameters.

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.

Dystroglycan (Dag1) is a transmembrane glycoprotein that links the extracellular matrix to the actin cytoskeleton. Mutations in Dag1 or the genes required for its glycosylation result in dystroglycanopathy, a type of congenital muscular dystrophy characterized by a wide range of phenotypes including muscle weakness, brain defects, and cognitive impairment. We investigated interneuron (IN) development, synaptic function, and associated seizure susceptibility in multiple mouse models that reflect the wide phenotypic range of dystroglycanopathy neuropathology. Mice that model severe dystroglycanopathy due to forebrain deletion of Dag1 or Pomt2, which is required for Dystroglycan glycosylation, show significant impairment of CCK+/CB1R+ IN development. CCK+/CB1R+ IN axons failed to properly target the somatodendritic compartment of pyramidal neurons in the hippocampus, resulting in synaptic defects and increased seizure susceptibility. Mice lacking the intracellular domain of Dystroglycan have milder defects in CCK+/CB1R+ IN axon targeting, but exhibit dramatic changes in inhibitory synaptic function, indicating a critical postsynaptic role of this domain. In contrast, CCK+/CB1R+ IN synaptic function and seizure susceptibility was normal in mice that model mild dystroglycanopathy due to partially reduced Dystroglycan glycosylation. Collectively, these data show that inhibitory synaptic defects and elevated seizure susceptibility are hallmarks of severe dystroglycanopathy, and show that Dystroglycan plays an important role in organizing functional inhibitory synapse assembly.

Dystroglycan (DG) is a transmembrane protein widely expressed in multiple cells and tissues. It is formed by two subunits, α- and β-DG, and represents a molecular bridge between the outside and the inside of the cell, which is essential for the mechanical and structural stability of the plasma membrane. The α-subunit is a cell-surface protein that binds to the extracellular matrix (ECM) and is tightly associated with the plasma membrane via a non-covalent interaction with the β-subunit, which, in turn, is a transmembrane protein that binds to the cytoskeletal actin. DG is a versatile molecule acting not only as a mechanical building block but also as a modulator of outside-inside signaling events. The cytoplasmic domain of β-DG interacts with different adaptor and cytoskeletal proteins that function as molecular switches for the transmission of ECM signals inside the cells. These interactions can modulate the involvement of DG in different biological processes, ranging from cell growth and survival to differentiation and proliferation/regeneration. Although the molecular events that characterize signaling through the ECM-DG-cytoskeleton axis are still largely unknown, in recent years, a growing list of evidence has started to fill the gaps in our understanding of the role of DG in signal transduction. This mini-review represents an update of recent developments, uncovering the dual role of DG as an adhesion and signaling molecule that might inspire new ideas for the design of novel therapeutic strategies for pathologies such as muscular dystrophy, cardiomyopathy, and cancer, where the DG signaling hub plays important roles.