Desmoplakin I and desmoplakin II. Purification and characterization.

Desmoplakins I and II (DP1 and DP2), major cytoskeletal structural proteins concentrated in desmosomes, have been purified in milligram quantities from keratomed pig tongue epithelium. DP1 and DP2 extracted from purified desmosomes in 4 M urea were chromatographed on DEAE-cellulose and remained soluble after removal of urea during subsequent chromatography. The two proteins differed by only about 15% in molecular weight (Mr = 285,000 for DP1 and 225,000 for DP2 on sodium dodecyl sulfate-polyacrylamide gels) were found to have similar Svedberg constants, 6.7 S (DP1) and 6.4 S (DP2); nevertheless, separation was readily achieved by gel filtration, since DP1 has a Stokes radius (Rs) of 164 nm, but DP2 has a Rs = 90 nm. Calculated molecular mass was 462,000 daltons for DP1 and 242,000 daltons for DP2, suggesting that DP1 may be a dimer in solution and DP2 a monomer. Cross-linking by disuccinimidyl suberate of 125I-labeled DP1 or DP2 at nanomolar concentrations confirmed that DP1 is a dimer by doubling of its apparent Mr on sodium dodecyl sulfate gels and indicated that DP2, which failed to become cross-linked, is a monomer. DP1 in the presence of 8 M urea could not be cross-linked, indicating that urea dissociated the dimers. Calculated frictional ratios (f/f0 = 3 for DP1 and 2 for DP2) indicate that both proteins are highly asymmetric. Rotary shadowing of DP1 demonstrated flexible dumbbell-like extended shapes with a maximal length of about 180 nm with a central rod and coiled or folded end domains. DP2 showed variable extended shapes of maximal length of 78-93 nm. The increased length and Rs of desmoplakin I is probably accounted for by formation of tail-to-tail dimers. Two-dimensional peptide maps and amino acid analysis showed very similar profiles for the two proteins. Purified keratin filaments failed to bind DP1 or DP2, and prekeratins polymerized in vitro and sedimented failed to remove desmoplakins, suggesting that desmoplakins do not bind keratins directly. These studies provide a basis for functional and detailed structural studies with purified native desmosomal proteins.