A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients

  • Neuromuscul Disord. 2015 Jan;25(1):32-42. doi: 10.1016/j.nmd.2014.09.005.
Emma L Humphrey  1 ,  Erica Lacey  2 ,  Lam T Le  1 ,  Lucy Feng  3 ,  Francesca Sciandra  4 ,  Charlotte R Morris  5 ,  Jane E Hewitt  6 ,  Ian Holt  7 ,  Andrea Brancaccio  4 ,  Rita Barresi  5 ,  Caroline A Sewry  8 ,  Susan C Brown  2 ,  Glenn E Morris  9
Affiliations
  • 1. Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK.
  • 2. Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, UK.
  • 3. Dubowitz Neuromuscular Centre, UCL Institute of Child Health, University College London, London, UK.
  • 4. CNR - Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Catholic University, Rome, Italy.
  • 5. Rare Diseases Advisory Group Service for Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental Hospital, Newcastle upon Tyne, UK.
  • 6. Centre for Genetics and Genomics, School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
  • 7. Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK.
  • 8. Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Dubowitz Neuromuscular Centre, UCL Institute of Child Health, University College London, London, UK.
  • 9. Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK. Electronic address: [email protected].
PMID: 25387694 DOI: 10.1016/j.nmd.2014.09.005
Abstract

We generated a novel monoclonal antibody, DAG-6F4, against alpha-dystroglycan 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 alpha-dystroglycan. On Western blots of Recombinant alpha-dystroglycan, 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 alpha-dystroglycan). 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 alpha-dystroglycan 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.

Keywords
Alpha-dystroglycan; Beta-dystroglycan; Congenital muscular dystrophy; DAG-6F4; Dystroglycanopathy; Dystrophin; Epitope mapping; Fukutin-related protein; Limb-girdle muscular dystrophy; Monoclonal antibody; Phage-displayed peptides; glycosylation.
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