Epitopes in the interacting regions of beta-dystroglycan (PPxY motif) and dystrophin (WW domain).
The dystroglycan gene produces two products from a single mRNA, the extracellular alpha-dystroglycan and the transmembrane beta-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via beta-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in beta-dystroglycan. A panel of four monoclonal antibodies (MANDAG1-4) was produced using the last 16 amino acids of beta-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four beta-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first beta-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin-dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.
Pereboev
A V
AV
MRIC Biochemistry Group, North East Wales Institute, Mold Road, LL11 2AW, Wrexham, UK.
Ahmed
N
N
thi Man
N
N
Morris
G E
GE
D020816:Amino Acid Motifs / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000276:immunology* / Q000378:metabolism; D049030:Dystroglycans; D016189:Dystrophin / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; D016189:Dystrophin; D000939:Epitopes; D008562:Membrane Glycoproteins; D049030:Dystroglycans