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In skeletal muscle, dystroglycan (DG) is the central component of the dystrophin-glycoprotein complex (DGC), a multimeric protein complex that ensures a strong mechanical link between the extracellular matrix and the cytoskeleton. Several muscular dystrophies arise from mutations hitting most of the components of the DGC. Mutations within the DG gene (DAG1) have been recently associated with two forms of muscular dystrophy, one displaying a milder and one a more severe phenotype. This review focuses specifically on the animal (murine and others) model systems that have been developed with the aim of directly engineering DAG1 in order to study the DG function in skeletal muscle as well as in other tissues. In the last years, conditional animal models overcoming the embryonic lethality of the DG knock-out in mouse have been generated and helped clarifying the crucial role of DG in skeletal muscle, while an increasing number of studies on knock-in mice are aimed at understanding the contribution of single amino acids to the stability of DG and to the possible development of muscular dystrophy. [ABSTRACT FROM AUTHOR]

A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies. [ABSTRACT FROM AUTHOR]

Background: α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-Dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein. Results: We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ∼40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min. Conclusions: Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization. [ABSTRACT FROM AUTHOR]

Dystroglycan (DG) is a membrane-associated protein complex formed by two noncovalently linked subunits, α-DG, a highly glycosylated extracellular protein, and β-DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C-terminal domain of α-DG and the N-terminal extracellular domain of β-DG. It is well known that under both, physiological and pathological conditions, gelatinases ( i.e. MMP-9 and/or MMP-2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β-DG ectodomain by gelatinases, identifying a main cleavage site on the β-DG ectodomain produced by MMP-9. In this article, we have deepened the pattern of the β-DG ectodomain digestion by MMP-2 by using a combined approach based on SDS-PAGE, Orbitrap, and HPLC-ESI-IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β-DG ectodomain mutants by gelatinases. © 2012 IUBMB IUBMB Life, 64(12): 988-994, 2012 [ABSTRACT FROM AUTHOR]

Background: Epitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells. Results: We have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane. Conclusions: Further characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies. [ABSTRACT FROM AUTHOR]

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, α-DG, a highly glycosylated extracellular matrix protein, and β-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of α-DG and the N-terminal extracellular domain of β-DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP-9 and/or MMP-2) that removes a portion or the whole β-DG ectodomain producing a 30 kDa truncated form of β-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of β-DG, β-DG(654-750) with human MMP-9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS-PAGE, MALDI-TOF and HPLC-ESI-IT mass spectrometry, we were able to identify one main MMP-9 cleavage site that is localized between the amino acids His-715 and Leu-716 of β-DG, and we analysed the proteolytic fragments of β-DG(654-750) produced by MMP-9 enzymatic activity. © 2009 IUBMB IUBMB Life, 61: 1143–1152, 2009 [ABSTRACT FROM AUTHOR]

The interaction between α-dystroglycan (α-DG) and β-dystroglycan (β-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 α–β interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the α–β 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 α-DG and the ectodomain of β-DG. As no ultimate structural data are yet available on the α–β 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 β-DG ectodomain allowed the identification of two phenylalanines important for α-DG binding, namely F692 and F718. In this article, similar experiments performed on the α-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two β-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 α- and β-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 pull-down 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. Structured digital abstract • : alpha DG (uniprotkb: ) binds ( ) to beta DG (uniprotkb: ) by solid phase assay ( ) • : laminin (uniprotkb: ) binds ( ) to beta DG (uniprotkb: ) by pull down ( ) [ABSTRACT FROM AUTHOR]

The dystroglycan adhesion complex consists of two noncovalently interacting proteins: α-dystroglycan, a peripheral extracellular subunit that is extensively glycosylated, and the transmembrane β-dystroglycan, whose cytosolic tail interacts with dystrophin, thus linking the F-actin cytoskeleton to the extracellular matrix. Dystroglycan is thought to play a crucial role in the stability of the plasmalemma, and forms strong contacts between the extracellular matrix and the cytoskeleton in a wide variety of tissues. Abnormal membrane targeting of dystroglycan subunits and/or their aberrant post-translational modification are often associated with several pathologic conditions, ranging from neuromuscular disorders to carcinomas. A putative functional hotspot of dystroglycan is represented by its intersubunit surface, which is contributed by two amino acid stretches: approximately 30 amino acids of β-dystroglycan (691–719), and approximately 15 amino acids of α-dystroglycan (550–565). Exploiting alanine scanning, we have produced a panel of site-directed mutants of our two consolidated recombinant peptides β-dystroglycan (654–750), corresponding to the ectodomain of β-dystroglycan, and α-dystroglycan (485–630), spanning the C-terminal domain of α-dystroglycan. By solid-phase binding assays and surface plasmon resonance, we have determined the binding affinities of mutated peptides in comparison to those of wild-type α-dystroglycan and β-dystroglycan, and shown the crucial role of two β-dystroglycan phenylalanines, namely Phe692 and Phe718, for the α–β interaction. Substitution of the α-dystroglycan residues Trp551, Phe554 and Asn555 by Ala does not affect the interaction between dystroglycan subunits in vitro. As a preliminary analysis of the possible effects of the aforementioned mutations in vivo, detection through immunofluorescence and western blot of the two dystroglycan subunits was pursued in dystroglycan-transfected 293-Ebna cells. [ABSTRACT FROM AUTHOR]

Abstract: The α/β dystroglycan (DG) complex links the extracellular matrix to the actin cytoskeleton. The extensive glycosylation of α-DG is believed to be crucial for the interaction with its extracellular matrix-binding partners. We characterized a monoclonal antibody, directed against the β-DG-binding epitope (≈positions 550–565), which recognizes preferentially hypoglycosylated α-DG. In Western blot, the antibody was able to detect a number of partially glycosylated α-DG isoforms from rat brain and chicken skeletal muscle tissue samples. In addition, we demonstrated its inhibitory effect on the interaction between α- and β-DG in vitro and preliminary immunostaining experiments suggest that such hypoglycosylated α-DG isoforms could play a role within cells. [Copyright &y& Elsevier]

Dystroglycan (DG) is an adhesion complex, expressed in a wide variety of tissues, formed by an extracellular and a transmembrane subunit, α-DG and β-DG, respectively, interacting noncovalently. Recently, we have shown that the recombinant ectodomain of β-DG, β-DG(654-750), behaves as a natively unfolded protein, as it is able to bind the C-terminal domain of α-DG, while not displaying a defined structural organization. We monitored the effect of a commonly used denaturing agent, the anionic detergent sodium dodecylsulphate (SDS), on β-DG(654-750) using a number of biophysical techniques. Very low concentrations of SDS (≤2 mM) affect both tryptophan fluorescence and circular dichroism of β-DG, and significantly perturb the interaction with the α-DG subunit as shown by solid-phase binding assays and fluorescence titrations in solution. This result confirms, as recently proposed for natively unfolded proteins, that β-DG(654-750) exists in a native state, which is crucial to fulfill its biological function. Two-dimensional NMR analysis shows that SDS does not induce any evident conformational rearrangement within the ectodomain of β-DG. Its first 70 amino acids, which show a lower degree of mobility, interact with the detergent, but this does not change the amount of secondary structure, whereas the highly flexible and mobile C-terminal region of β-DG(654-750) remains largely unaffected, even at a very high SDS concentration (up to 50 mM). Our data indicate that SDS can be used as a useful tool for investigating natively unfolded proteins, and confirm that the β-DG ectodomain is an interesting model system. [ABSTRACT FROM AUTHOR]

Dystroglycan (DG) is a member of the glycoprotein complex associated to dystrophin and composed by two subunits, the β-DG, a transmembrane protein, and the α-DG, an extensively glycosylated extracellular protein. The β-DG ectodomain degradation by the matrix metallo-proteinases (i.e., MMP-2 and MMP-9) in both, pathological and physiological conditions, has been characterized in detail in previous publications. Since the amounts of α-DG and β-DG at the cell surface decrease when gelatinases are up-regulated, we investigated the degradation of α-DG subunit by MMP-2. Present data show, for the first time, that the proteolysis of α-DG indeed occurs on a native glycosylated molecule enriched from rabbit skeletal muscle. In order to characterize the α-DG portion, which is more prone to cleavage by MMP-2, we performed different degradations on tailored recombinant domains of α-DG spanning the whole subunit. The overall bulk of results casts light on a relevant susceptibility of the α-DG to MMP-2 degradation with particular reference to its C-terminal domain, thus opening a new scenario on the role of gelatinases (in particular of MMP-2) in the degradation of this glycoprotein complex, taking place in the course of pathological processes. [ABSTRACT FROM AUTHOR]

Abstract: Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG–ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated. [Copyright &y& Elsevier]

Abstract: Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely α- and β-DG. In skeletal muscle, DG is the central component of the dystrophin–glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of α-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine α-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30%) sequence homology with the N-terminal region of α-DG, protein fold recognition methods found a significant resemblance to the α-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues ∼500–600 of α-DG were predicted to adopt an immunoglobulin-like (Ig-like) β-sandwich fold. Such modeled domain includes the β-DG binding epitope of α-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550–565) assumes a β-strand conformation. The remaining segment of the α-DG C-terminal region (residues 601–653) is organized in a coil–helix–coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the α/β DG interface. Preliminary characterization of a novel recombinant peptide (505–600) encompassing this second Ig-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysis. [Copyright &y& Elsevier]

Abstract: Mutations of the GNE gene are responsible for autosomal recessive hereditary inclusion-body myopathy (HIBM). In this study we searched for the presence of any significant abnormality of α-dystroglycan (α-DG), a highly glycosylated component of the dystrophin-glycoprotein complex, in 5 HIBM patients which were previously clinically and genetically characterized. Immunocytochemical and immunoblot analysis showed that α-DG extracted from muscle biopsies was normally expressed and displayed its typical molecular mass. Immunoblot analysis on the wheat germ lectin-enriched glycoprotein fraction of muscles and primary myotubes showed a reduced amount of α-DG in 4 out of 5 HIBM patients, compared to normal and other diseased muscles. However, such altered lectin-binding behaviour, possibly reflecting a partial hyposialylation of α-DG, did not affect the laminin binding properties of α-DG. Therefore, the subtle changes within the α-DG glycosylation pattern, detected in HIBM muscles, likely do not play a key pathogenic role in this disorder. [Copyright &y& Elsevier]