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Alpha Dystroglycan

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Jane E. Hewitt – One of the best experts on this subject based on the ideXlab platform.

  • A new monoclonal antibody against human AlphaDystroglycan has potential diagnostic applications
    Neuromuscular Disorders, 2014
    Co-Authors: Emma L. Humphrey, Francesca Sciandra, Andrea Brancaccio, E. Lacey, L.t. Le, Christopher Morris, Jane E. Hewitt, Ian Holt, Rita Barresi, Caroline Sewry

    Abstract:

    We have generated novel monoclonal antibodies (mAbs) against AlphaDystroglycan to immunolabel the sarcolemma in human muscle biopsies. For one of these, DAG-6F4, a seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of AlphaDystroglycan. On western blots of recombinant AlphaDystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with a disruption in the Dystroglycan complex. In a range of Dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was generally less reduced than staining by IIH6 (an antibody commonly used to identify glycosylated AlphaDystroglycan), though the extent of the differences between the two antibodies varied between different patients, some biopsies showing reductions in core protein as well as its glycosylation. A second mAb, DAG-3H9, gave similar results, but recognised a different epitope, GDRAP, closer to the C-terminus. There are currently few antibodies available against core AlphaDystroglycan, so DAG-6F4 represents a useful addition to the antibody repertoire for evaluating the Dystroglycan complex in neuromuscular disorders.

  • A new monoclonal antibody DAG-6F4 against human AlphaDystroglycan reveals reduced core protein in some, but not all, Dystroglycanopathy patients.
    Neuromuscular disorders : NMD, 2014
    Co-Authors: Emma L. Humphrey, Francesca Sciandra, Andrea Brancaccio, Lucy Feng, E. Lacey, Jane E. Hewitt, Ian Holt, Charlotte R. Morris, Rita Barresi

    Abstract:

    We generated a novel monoclonal antibody, DAG-6F4, against AlphaDystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of AlphaDystroglycan. On Western blots of recombinant AlphaDystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the Dystroglycan complex. In a range of Dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated AlphaDystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect AlphaDystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the Dystroglycan complex in neuromuscular disorders.

  • P.1.17 Immunostaining of the sarcolemma with a new monoclonal antibody against AlphaDystroglycan core and its relevance to diagnosis
    Neuromuscular Disorders, 2013
    Co-Authors: E. Wilson, Kazuhiro Kobayashi, Tatsushi Toda, Francesca Sciandra, Andrea Brancaccio, Caroline Sewry, E. Lacey, Jane E. Hewitt, Ian Holt, Susan C. Brown

    Abstract:

    AlphaDystroglycan ( α DG) is an extracellular, glycosylated protein attached to the outer plasma membrane by its interaction with the transmembrane protein, beta-Dystroglycan. Genetic mutations that alter the glycosylation state of α DG cause a number of inherited disorders, including Fukuyama MD and certain congenital and limb-girdle MDs. Many of the pathogenic mutations are in proteins that carry out glycosylation of α DG and not in the DG gene itself. These enzymes are usually located in the Golgi where proteins are modified before export to the extracellular space. Amino-acid mutations in α DG itself can also cause congenital MD when sugar attachment to the α DG backbone is affected. Antibodies against Dystroglycans immunostain the sarcolemma in a similar manner to dystrophin antibodies. It would be useful to the muscle pathologist to have monoclonal antibodies (mAbs) that recognize specific glycosylated forms of α DG associated with different mutations or disease states, since such mAbs would be useful for diagnosis using muscle biopsy sections. There are problems of reproducibility with existing mAbs against α DG, but it is still unclear whether those problems are attributable to the antibodies themselves or to the nature of the glycosylated epitopes on α DG. To determine whether it is possible to obtain improved mAbs for diagnostic use, we have begun a collaborative programme, supported by LGMD2I and CureCMD, to produce new mAbs against glycosylated and non-glycosylated forms of α DG. As a first step, we now describe a new mAb raised against bacterial recombinant α DG core sequence, which gives good staining of the sarcolemma in human muscle biopsy sections. We have mapped the epitope recognized by this mAb to short sequence that appears to be accessible to antibody in BOTH glycosylated and non-glycosylated forms of α DG.

Andrea Brancaccio – One of the best experts on this subject based on the ideXlab platform.

  • Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.
    Frontiers in molecular biosciences, 2019
    Co-Authors: Christopher E. Dempsey, Maria Giulia Bigotti, Josephine C. Adams, Andrea Brancaccio

    Abstract:

    Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular Alpha-DG associated noncovalently with a transmembrane beta-DG whose cytodomain interacts with dystrophin and its isoforms. AlphaDystroglycan (Alpha-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbours at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the Alpha-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural and functional information on the LG domains reported to bind AlphaDystroglycan. In addition, we have incorporated bioinformatics and modelling to explore whether specific motifs responsible for AlphaDystroglycan recognition can be identified within isolated LG domains. In particular, we analysed the LG domains of slits and agrin as well as those of paradigmatic Alpha-DG non-binders such as laminin-Alpha3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of Alpha-DG, although it appears that this alone is insufficient to mediate significant Alpha-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with Alpha-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

  • A new monoclonal antibody against human AlphaDystroglycan has potential diagnostic applications
    Neuromuscular Disorders, 2014
    Co-Authors: Emma L. Humphrey, Francesca Sciandra, Andrea Brancaccio, E. Lacey, L.t. Le, Christopher Morris, Jane E. Hewitt, Ian Holt, Rita Barresi, Caroline Sewry

    Abstract:

    We have generated novel monoclonal antibodies (mAbs) against AlphaDystroglycan to immunolabel the sarcolemma in human muscle biopsies. For one of these, DAG-6F4, a seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of AlphaDystroglycan. On western blots of recombinant AlphaDystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with a disruption in the Dystroglycan complex. In a range of Dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was generally less reduced than staining by IIH6 (an antibody commonly used to identify glycosylated AlphaDystroglycan), though the extent of the differences between the two antibodies varied between different patients, some biopsies showing reductions in core protein as well as its glycosylation. A second mAb, DAG-3H9, gave similar results, but recognised a different epitope, GDRAP, closer to the C-terminus. There are currently few antibodies available against core AlphaDystroglycan, so DAG-6F4 represents a useful addition to the antibody repertoire for evaluating the Dystroglycan complex in neuromuscular disorders.

  • A new monoclonal antibody DAG-6F4 against human AlphaDystroglycan reveals reduced core protein in some, but not all, Dystroglycanopathy patients.
    Neuromuscular disorders : NMD, 2014
    Co-Authors: Emma L. Humphrey, Francesca Sciandra, Andrea Brancaccio, Lucy Feng, E. Lacey, Jane E. Hewitt, Ian Holt, Charlotte R. Morris, Rita Barresi

    Abstract:

    We generated a novel monoclonal antibody, DAG-6F4, against AlphaDystroglycan which immunolabels the sarcolemma in human muscle biopsies. Its seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of AlphaDystroglycan. On Western blots of recombinant AlphaDystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with disruption of the Dystroglycan complex. In a range of Dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was often less reduced than staining by IIH6 (antibody against the glycan epitope added by LARGE and commonly used to identify glycosylated AlphaDystroglycan). Whereas IIH6 was reduced in all patients, DAG-6F4 was hardly changed in a LARGE patient, less reduced than IIH6 in limb-girdle muscular dystrophy type 2I, but as reduced as IIH6 in some congenital muscular dystrophy patients. Although absence of the LARGE-dependent laminin-binding site appears not to affect AlphaDystroglycan stability at the sarcolemma, the results suggest that further reduction in aDG glycosylation may reduce its stability. These studies suggest that DAG-6F4 may be a useful addition to the antibody repertoire for evaluating the Dystroglycan complex in neuromuscular disorders.

Markus A. Rüegg – One of the best experts on this subject based on the ideXlab platform.

  • G.P.212
    Neuromuscular Disorders, 2014
    Co-Authors: Sarina Meinen, Geraldine Maier, M. Chauhan, Shuo Lin, Karen K. Mckee, Stephanie C. Crosson, Peter D. Yurchenco, Markus A. Rüegg

    Abstract:

    The basement membrane surrounding skeletal muscle fibers (myomatrix) and its binding to the sarcolemma is important for the structural stability of muscle. The myomatrix consists of laminin-211 (Lm-211), collagen IV and several associated components including nidogen and perlecan. Assembly of the myomatrix is initiated by Lm-211 that binds to its cell surface receptors (AlphaDystroglycan and integrins) and then creates a primary scaffold by self-assembly. Mutations in the α2 chain of Lm-211 result in a severe form of congenital muscular dystrophy, called MDC1A. In MDC1A patients and the dyW/dyW mouse model expression of Lm-411 is increased as an attempt to compensate for the loss of Lm-211. However, Lm-411 cannot self-assemble and binds only weakly to the sarcolemma. Previous proof-of-principle studies in dyW/dyW mice have shown that transgenic expression of mini-agrin, which binds to Lm-411 and to AlphaDystroglycan, strongly ameliorates the dystrophic phenotype. We here present data that introduction of a transgene that allows self-polymerization of Lm-411 has some, although rather weak therapeutic activity in dyW/dyW mice. However, when combined with mini-agrin, this construct shows a strong synergistic effect. The improvements are seen on all levels, such as behavior, weight gain and the histology of skeletal muscles. We are currently evaluating its effect on muscle force and survival. Moreover, we analyze the signaling pathways involved in the amelioration. We also plan to test a new construct that combines the laminin polymerization and the AlphaDystroglycan binding regions. In summary, our data provide formal proof that MDC1A is caused by defects in myomatrix assembly and its connection to the sarcolemma. Our data will have significant impact in future attempts to develop new treatment strategies for MDC1A.

  • Activation of muscle-specific receptor tyrosine kinase and binding to Dystroglycan are regulated by alternative mRNA splicing of agrin
    The Journal of biological chemistry, 2006
    Co-Authors: Patrick Scotton, Francesca Sciandra, Andrea Brancaccio, Thomas Meier, Dorothee Bleckmann, Michael Stebler, Jörg Stetefeld, Markus A. Rüegg

    Abstract:

    Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to AlphaDystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate-isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to AlphaDystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.

  • Dystroglycan is a dual receptor for agrin and laminin 2 in schwann cell membrane
    Journal of Biological Chemistry, 1996
    Co-Authors: Hiroki Yamada, Teruo Shimizu, Louise V. B. Anderson, Hisae Hori, Markus A. Rüegg, Alain J Denzer, Takeshi Tanaka, Sachiko Fujita, Hiroko Fukutaohi, Kiichiro Matsumura

    Abstract:

    We have shown previously that AlphaDystroglycan with a molecular mass of 120 kDa is a Schwann cell receptor of laminin-2, the endoneurial isoform of laminin comprised of the Alpha2, beta1, and gamma1 chains. In this paper, we show that Schwann cell AlphaDystroglycan is also a receptor of agrin, an acetylcholine receptor-aggregating molecule having partial homology to laminin Alpha chains in the C terminus. Immunochemical analysis demonstrates that the peripheral nerve isoform of agrin is a 400-kDa component of the endoneurial basal lamina and is co-localized with AlphaDystroglycan surrounding the outermost layer of myelin sheath of peripheral nerve fibers. Blot overlay analysis demonstrates that both endogenous peripheral nerve agrin and laminin-2 bind to Schwann cell AlphaDystroglycan. Recombinant C-terminal fragment of the peripheral nerve isoform of agrin also binds to Schwann cell AlphaDystroglycan, confirming that the binding site for Schwann cell AlphaDystroglycan resides in the C terminus of agrin molecule. Furthermore, the binding of recombinant agrin C-terminal fragment to Schwann cell AlphaDystroglycan competes with that of laminin-2. All together, these results indicate that AlphaDystroglycan is a dual receptor for agrin and laminin-2 in the Schwann cell membrane.