Location: Reproduction ResearchTitle: Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue Author
|Nonneman, Danny - Dan|
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/21/2014
Publication Date: 5/13/2014
Publication URL: http://handle.nal.usda.gov/10113/59658
Citation: Fortunato, M.J., Ball, C.E., Hollinger, K., Patel, N.B., Modi, J.N., Rajasekaran, V., Nonneman, D.J., Ross, J.W., Kennedy, E.J., Selsby, J.T., Beedle, A.M. 2014. Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue. PLoS One. 9(5):e97567. Interpretive Summary: Alpha-dystroglycan is a dystrophin-associated glycoprotein first isolated from skeletal muscle membranes. It is encoded by the gene dystroglycan 1 (DAG1) gene and is cleaved to form two separate proteins, alpha-dystroglycan (alphaDG) and beta-dystroglycan (ßDG), which remain in complex to provide a structural connection between the muscle fiber and the basement membrane by binding to other extracellular proteins. Dystroglycan is expressed in a wide variety of tissues and plays a role in muscle integrity, cell signaling, and development. Alpha-dystroglycan undergoes extensive glycosylation (addition of carbohydrate chains), which is required for functional binding to extracellular matrix proteins. Disruption of dystroglycan is associated with muscle, brain, and other tissue abnormalities and loss of alphaDG and ßDG protein in muscle occurs in disorders, such as dystrophin-deficient Duchenne and Becker muscular dystrophies. Altered glycosylation of alphaDG due to mutations in the DAG1 gene or in alphaDG processing genes leads to mild to severe muscular dystrophies known as secondary dystroglycanopathies. Alpha-dystroglycan glycosylation defects have been observed in cancer tissues and are associated with enhanced metastatic potential. Currently, the standard antibody reagents used for detection of alphaDG require the glycosylation modification on alphaDG. The detection of unglycosylated alphaDG and the study of alphaDG in disease have been restricted by limited antibody reagents against the alphaDG protein. Therefore, we sought to develop a reagent with the ability to detect alphaDG regardless of glycosylation state. We generated three rabbit monoclonal antibodies against the C-terminal of alphaDG and demonstrated their ability to specifically detect alphaDG in mouse, rat, and pig tissue as well as in two models of muscular dystrophy. In addition, we demonstrated that these antibodies are not dependent on glycosylation and are valuable tools for investigating alphaDG in disease states.
Technical Abstract: 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 address this reagent bottleneck, we generated rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus. 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. We further tested the antibodies on two disease models. 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, antibody detection of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antisera are sustainable reagents for alpha-dystroglycan core protein detection for the advancement of dystroglycan-related studies.