Structure analysis of 230 kilodaltons bullous pemphigoid antigen

Abstract

The 230 kD bullous pemphigoid antigen (BP230 or BPAG1) is a major protein of the cytoplasmic dense plaque of the epidermal hemidesmosome (HD) which may link keratin filaments to transmembrane proteins. The BP230 sequence is highly homologous to desmoplakin I (DPI) and plectin, both keratin-associated proteins. All are predicted to have globular N- and C-terminal domains flanking a central a-helical coiled coil rod, consistent with dumbbell like rotary shadowing images of DPI and plectin. Using genetic cloning, two polypeptides spanning the putative N-terminal globular and coiled coil rod domains of BP230 have been expressed in E. coli. BP-1 (MW=111kD), spanning amino acids 663-1581 of BP230 (Sawamura et al., 1991) is partially purified following urea extraction from insoluble bacterial inclusion bodies and renatured into a soluble form by dialysis. It has an S[20,w] value of 4 .8 on a sucrose density gradient, can be crosslinked to a dimer, and behaves on gel filtration as a highly asymmetric dimer or higher order multimer. BP-1A, 186 amino acids shorter than BP-1 at its N-terminal, is soluble when expressed in E. coli and has been purified [greater than or equal to] 95% . Like BP-1, it is highly asymmetric and has a high content of a-helix by circular dichroism, as expected for significant coiled coil tertiary structure. Rotary shadowing of BP-1 and BP-1 A gives a high percentage of images with globular head and rod-like tails. The tails have roughly equal lengths: 60 [plus or minus] 9 nm (N = 185) for BP-1 and 55 [plus or minus] 8 nm (N = 159) for BP-1 A, implying that the head domains must be formed at the N-termini of BP-1 and BP-1 A. The estimated rod length, 383 [plus or minus] 57 amino acids (.15 nm/amino acid), supports the largest of three recent predictions for N-terminal domain size (boundary with rod at residue 1145 rather than at 708 or 875; Green et al., 1992) and provides a basis for modeling structure and function in the BP230/DPI/plectin family.