Properties of the desmin tail domain: studies using synthetic peptides and antipeptide antibodies.

Abstract

Intermediate filament (IF) proteins have a common structural motif consisting of an alpha-helical rod domain flanked by non-alpha-helical amino-terminal head and carboxy-terminal tail domains. Coiled-coil interaction between neighboring rod domains is though to generate the backbone of the 10-nm filament. There must also be other interactions between subunits to bring them into alignment and to effect elongation of the filament, but these are poorly understood. To examine the involvement of the tail domain in filament structure and stabilization, we have studied the interaction between a synthetic peptide corresponding to residues 442-450 of avian desmin, and authentic desmin protein. The potential importance of this region lies in its hydrophilic nature and its high degree of homology among the Type III IF proteins and cytokeratins 8 and 18. The peptide, D442-450, binds to a 27-residue region between lys-436 and leu-463, the carboxy terminus. The presence of the peptide during assembly causes the filaments to appear much more loosely packed than normal desmin IF. We have also generated polyclonal antibodies against this peptide and attempted to localize this portion of the tailpiece along desmin IFs by immunological procedures. By immunoblotting, we found that anti-D442-450 antibodies recognize desmin and only those proteolytic fragments that contain the tailpiece. In contrast, the antibodies do not label any structure in adult gizzard smooth muscle and skeletal muscle myofibrils in immunofluorescence experiments during which conventional antidesmin antibodies do. At the ultrastructural level, anti-D442-450 antibodies label free desmin tetramers but not desmin IFs. These results show that, as part of an assembled IF, the epitope of anti-D442-450 is inaccessible to the antibodies, and suggest that either the tailpiece of an IF protein may not be entirely peripheral to the filament backbone, or the interaction between end domains during assembly masks this particular region of the IF molecule.