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

• Published in: Plos One Vol. 9; no. 5
• Main Authors: Fortunato, Marisa J, Nonneman, Dan J, Ball, Charlotte E, Hollinger, Katrin, Patel, Niraj B, Modi, Jill N, Rajasekaran, Vedika, Ross, Jason W, Kennedy, Eileen J, Selsby, Joshua T, Beedle, Aaron M
• Format: Publication
• Language: English
• Published: 2014
• Subjects: animal models; cytoskeletal proteins; epitopes; extracellular matrix; fluorescent antibody technique; glycosylation; laminin; metastasis; mice; molecular weight; monoclonal antibodies; muscles; muscular dystrophy; neoplasms; rabbits; rats; skeletal muscle; swine; Western blotting

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.