An epitope structure for the C-terminal domain of dystrophin and utrophin.
The muscular dystrophy protein, dystrophin, and the closely related protein, utrophin, are large cytoskeletal proteins which link actin microfilaments to the plasma membrane. A panel of 38 monoclonal antibodies (mAbs) has been produced against the C-terminal domains of dystrophin and utrophin. This domain interacts with both dystrobrevins, via their "leucine zipper" coiled-coil helices, and syntrophins, adaptor proteins which also interact with nitric oxide synthetase and transmembrane sodium channels. The amino acid sequences recognized by the mAbs have now been identified using a variety of epitope mapping techniques, including fragmentation by transposon mutagenesis, synthetic peptides, phage-displayed peptide libraries, and mutant dystrophins expressed in transgenic mice. In addition to defining antibody recognition sites, mapping was sufficiently precise to provide structural information, since individual amino acids accessible on the surface of the native protein were identified in many cases. In two regions of the domain, short linear epitopes were found in proline-rich sequences which may form surface loops, turns, or linkers, but these were separated by a third region which contained mainly conformational epitopes. The results are consistent with a loose and flexible structure for much of the C-terminal domain, especially around the highly conserved second leucine zipper or coiled-coil helix (CC-H2), but there is evidence for denaturation-resistant tertiary structure in the syntrophin-binding region and the first coiled-coil helix (CC-H1).
MRIC Biochemistry Group, North East Wales Institute, Wrexham, UK.
G E
GE
Morris
S G
SG
Sedgwick
J M
JM
Ellis
A
A
Pereboev
J S
JS
Chamberlain
Nguyen thi Man
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004251:DNA Transposable Elements / Q000235:genetics; D016189:Dystrophin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D018101:Mice, Inbred mdx; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D049411:Utrophin
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; D004251:DNA Transposable Elements; D016189:Dystrophin; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; C483600:Utrn protein, mouse; D049411:Utrophin