Monoclonal antibody mapping of structural and functional plectin epitopes.
To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association.
Foisner
R
R
Institute of Biochemistry, University of Vienna, Austria.
Feldman
B
B
Sander
L
L
Wiche
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D005910:Glioma; D015151:Immunoblotting / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007381:Intermediate Filament Proteins / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008854:Microscopy, Electron; D008970:Molecular Weight; D051190:Plectin; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000276:immunology* / Q000648:ultrastructure; D051381:Rats; D014746:Vimentin / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007381:Intermediate Filament Proteins; C495333:Plec protein, mouse; C495334:Plec protein, rat; D051190:Plectin; D011506:Proteins; D014746:Vimentin