Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.
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.
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, United States of America.
Marisa J
MJ
Fortunato
Center for Undergraduate Research, University of Georgia, Athens, Georgia, United States of America.
Charlotte E
CE
Ball
Department of Animal Science, Iowa State University, Ames, Iowa, United States of America.
Katrin
K
Hollinger
Center for Undergraduate Research, University of Georgia, Athens, Georgia, United States of America.
Niraj B
NB
Patel
Center for Undergraduate Research, University of Georgia, Athens, Georgia, United States of America.
Jill N
JN
Modi
Center for Undergraduate Research, University of Georgia, Athens, Georgia, United States of America.
Vedika
V
Rajasekaran
United States Department of Agriculture Agricultural Research Service, United States Meat Animal Research Center, Clay Center, Nebraska, United States of America.
Dan J
DJ
Nonneman
Department of Animal Science, Iowa State University, Ames, Iowa, United States of America.
Jason W
JW
Ross
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, United States of America.
Eileen J
EJ
Kennedy
Department of Animal Science, Iowa State University, Ames, Iowa, United States of America.
Joshua T
JT
Selsby
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, United States of America.
Aaron M
AM
Beedle
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D049030:Dystroglycans / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006825:Hybridomas; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D013552:Swine
D000911:Antibodies, Monoclonal; C483081:DAG1 protein, human; D017931:DNA Primers; D049030:Dystroglycans