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

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

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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