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Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated ?-DG and the transmembrane ?-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of ?-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of ?-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the ?-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the ?-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of ?-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the ?-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.

Dystroglycan (DG), composed of α and β subunits, belongs to the dystrophin-associated glycoprotein complex. α-DG is an extracellular matrix protein that undergoes a complex post-translational glycosylation process. The bifunctional glycosyltransferase like-acetylglucosaminyltransferase (LARGE) plays a crucial role in the maturation of α-DG, enabling its binding to laminin. We have already structurally analyzed the N-terminal region of murine α-DG (α-DG-Nt) and of a pathological single point mutant that may affect recognition of LARGE, although the structural features of the potential interaction between LARGE and DG remain elusive. We now report on the crystal structure of the wild-type human α-DG-Nt that has allowed us to assess the reliability of our murine crystallographic structure as a α-DG-Nt general model. Moreover, we address for the first time both structures in solution. Interestingly, small-angle X-ray scattering (SAXS) reveals the existence of two main protein conformations ensembles. The predominant species is reminiscent of the crystal structure, while the less populated one assumes a more extended fold. A comparative analysis of the human and murine α-DG-Nt solution structures reveals that the two proteins share a common interdomain flexibility and population distribution of the two conformers. This is confirmed by the very similar stability displayed by the two orthologs as assessed by biochemical and biophysical experiments. These results highlight the need to take into account the molecular plasticity of α-DG-Nt in solution, as it can play an important role in the functional interactions with other binding partners.

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Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.

Dystroglycan (DG) is a receptor for several extracellular matrix proteins, including laminin, perlecan and agrin, that provides structural stability to the plasma membrane [1]. In skeletal muscle cells, DG is the central component of the dystrophin-glycoprotein-complex (DGC), a group of peripheral and membrane proteins that connects the extracellular matrix to dystrophin. Mutations in any of the DGC components are linked to distinct forms of muscular dystrophies characterized by disassembling of the whole complex followed by degeneration of muscle fibers [2]. DG is formed by two subunits, alpha and beta: _-DG is a highly glycosylated peripheral membrane protein that binds extracellular molecules while _-DG is a transmembrane protein whose cytosolic domain interacts with dystrophin linking DG to the actin cytoskeleton. The two DG subunits arise from a post-translational cleavage of a single DG precursor and remain associated via a non-covalent interaction. The molecular details of DG processing are still largely unknown. Recently, we showed that the correct formation of the _/_-DG interface is likely to stabilize the precursor and it is essential for its cleavage. Moreover, mutations hitting the _/_-DG binding epitopes inhibit the membrane targeting of mutant proteins that are retained in subcellular compartments [3]. At this stage, we widened the analysis of DG processing and maturation with the help of confocal microscopy to determine how single and multiple missense mutations located at the _/_-DG interface affect the assembly, targeting and function of the entire DG complex. We also analyzed the processing of wild-type and mutant DG precursors in the presence of different inhibitors of protein trafficking and glycosylation. In addition, a novel targeting vector for introducing multiple mutations of the _/_ interface in transgenic mice has been produced and it is currently used for preparing knock-in mice.Although no natural mutations are associated to the DG gene, a finest biochemical analysis of DG processing and maturation might be helpful to give new insights into congenital muscular dystrophies characterized by secondary defects of the _-DG glycosylation. References1. Ibraghimov-Beskrovnaya O., et al. (1992) Nature 355, 696-702.2.Barresi R. and Campbell K.P. (2006) J. Cell. Sci. 119, 199-207.3. Sciandra F. et al. (2009) FEBS J., 276, 4933-4945.

During the last 15 years, following its identification and first detailed molecular characterization, the dystroglycan (DG) complex has taken centre stage in biology and biomedicine. Functions in different cells and tissues have been identified for this complex, ranging from its typical role in skeletal muscle as a sarcolemmal stabilizer, highlighted by the recently identified "secondary dystroglycanopathies", to a variety of very diverse functions including embryogenesis, cancer progression, virus particle entry and cell signalling. Such functional promiscuity can be in part explained when considering the multiple domain organization of the two DG subunits, the extracellular alpha-DG and the transmembrane beta-DG, that has been largely scrutinized, but only in part unraveled, exploiting a variety of recombinant and transgenic approaches. Herein, while rapidly recapitulating some of the functions that nowadays can be assigned safely to each DG domain, we also try to envisage a sort of worry list featuring and dwelling on some of the most compelling "mysteries" that should be solved to finally understand DG's functional diversity.

The interaction between alpha-dystroglycan (alpha-DG) and beta-dystroglycan (beta-DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin-glycoprotein complex. The importance of the alpha-beta interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the alpha-beta interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C-terminus of alpha-DG and the ectodomain of beta-DG. As no ultimate structural data are yet available on the alpha-beta interface, site-directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine-scanning analysis of the recombinant beta-DG ectodomain allowed the identification of two phenylalanines important for alpha-DG binding, namely F692 and F718. In this article, similar experiments performed on the alpha-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two beta-DG phenylalanines. In 293-Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates alpha- and beta-DG, generating a pre-DG of about 160 kDa. This uncleaved pre-DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pulldown assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.