A new monoclonal antibody DAG-6F4 against human alpha-dystroglycan reveals reduced core protein in some, but not all, dystroglycanopathy patients.
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.
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK.
Emma L
EL
Humphrey
Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, UK.
Erica
E
Lacey
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK.
Lam T
LT
Le
Dubowitz Neuromuscular Centre, UCL Institute of Child Health, University College London, London, UK.
Lucy
L
Feng
CNR - Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Catholic University, Rome, Italy.
Francesca
F
Sciandra
Rare Diseases Advisory Group Service for Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental Hospital, Newcastle upon Tyne, UK.
Charlotte R
CR
Morris
Centre for Genetics and Genomics, School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
Jane E
JE
Hewitt
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK.
Ian
I
Holt
CNR - Istituto di Chimica del Riconoscimento Molecolare c/o Istituto di Biochimica e Biochimica Clinica, Catholic University, Rome, Italy.
Andrea
A
Brancaccio
Rare Diseases Advisory Group Service for Neuromuscular Diseases, Muscle Immunoanalysis Unit, Dental Hospital, Newcastle upon Tyne, UK.
Rita
R
Barresi
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Dubowitz Neuromuscular Centre, UCL Institute of Child Health, University College London, London, UK.
Caroline A
CA
Sewry
Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, UK.
Susan C
SC
Brown
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK. Electronic address: glenn.morris@rjah.nhs.uk.
Glenn E
GE
Morris
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002675:Child, Preschool; D049030:Dystroglycans / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000276:immunology; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis / Q000473:pathology*; D012508:Sarcolemma / Q000276:immunology
D000911:Antibodies, Monoclonal; C000600386:DAG-6F4 monoclonal antibody; D008565:Membrane Proteins; D049030:Dystroglycans
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