Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue

• Published in: PloS one 2014-05, Vol.9 (5), p.e97567-e97567
• Main Authors: Fortunato, Marisa J, Ball, Charlotte E, Hollinger, Katrin, Patel, Niraj B, Modi, Jill N, Rajasekaran, Vedika, Nonneman, Dan J, Ross, Jason W, Kennedy, Eileen J, Selsby, Joshua T, Beedle, Aaron M
• Format: Article
• Language: English
• Subjects: Animal sciences; Animals; Antibodies, Monoclonal - biosynthesis; Becker's muscular dystrophy; Biology and Life Sciences; Blotting, Western; Brain diseases; Brain research; C-Terminus; Cancer; Cloning, Molecular; Congenital diseases; Core protein; Disease; Disease control; DNA Primers - genetics; Dystroglycan; Dystroglycans - chemistry; Dystroglycans - genetics; Dystroglycans - immunology; Dystrophin; Dystrophy; Electrophoresis, Polyacrylamide Gel; Epitopes; Extracellular matrix; FCMD protein; Fluorescent Antibody Technique; Genes; Glycan; Glycoproteins; Glycosylation; Hybridomas; Image Processing, Computer-Assisted; Immunofluorescence; Immunoglobulins; Laboratory animals; Laminin; Low molecular weights; Mannose; Medicine and Health Sciences; Metastases; Mice; Molecular weight; Monoclonal antibodies; Muscle, Skeletal - metabolism; Muscles; Muscular dystrophy; Muscular Dystrophy, Duchenne - metabolism; Musculoskeletal system; Mutation; Pharmaceuticals; Proteins; Rabbits; Rats; Rats, Sprague-Dawley; Reagents; Relative abundance; Research and Analysis Methods; Rodents; Sequence Analysis, DNA; Skeletal muscle; Swine; Zoology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0097567

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.