Abstract
The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells. BPAG1 structural organization is similar to the intermediate filament-associated proteins desmoplakin I (DPI) and plectin. All three proteins have predicted dumbbell-like structure with central alpha-helical coiled-coil rod and regions of N- and C-terminal homology. To characterize the size of the N-terminal globular domain in BPAG1, two polypeptides spanning possible boundaries with the coiled-coil rod domain of BPAG1 were expressed in Escherichia coli. BP-1 (Mr = 111,000), containing amino acids 663-1581 of BPAG1 (Sawamura, D., Li, K., Chu, M.-L., and Uitto, J. (1991) J. Biol. Chem. 266, 17784-17790), and BP-1A, with a 186 amino acid N-terminal deletion, were purified. BP-1 and BP-1A behave as highly asymmetric dimers in aqueous solution according to velocity sedimentation and gel filtration. Both have globular heads with rod-like tails of roughly equal length, 55-60 nm, upon rotary shadowing. BP-1A content of alpha-helix, determined by circular dichroism, is approximately 90%, consistent with alpha-helical coiled-coil formation in the rod-like tails. The estimated rod length, 383 +/- 57 amino acids (0.15 nm/amino acid), implies that globular folding in the BPAG1 N-terminal extends to the end of N-terminal homology with DPI and plectin. These findings support the existence of a common domain structure in the N-terminal regions of the BPAG1/DPI/plectin family.
Highlights
The bullous pemphigoid antigen BPAG1 is required for keratin filament linkage to the hemidesmosome, an adhesion complex in epithelial basal cells
BPAG1 is homologous to two proteins which associate with intermediate filaments (IF): desmoplakin I (DPI), which is part of the desmosome, a cell-cell junction; and plectin, a ubiquitous cytoskeletal protein that binds IF subunits in vitro [15,16,17,18,19,20]
Protein Purification—Two polypeptides encompassing the predicted structural transitions from the N-terminal homology domain to the central ␣-helical coiled-coil rod portion of BPAG1 were expressed in E. coli
Summary
Materials—cDNA clones II-1 and III-1, representing a portion of the BPAG1 sequence, were obtained from J. For extraction of BP-1, 0.2 M NaCl, 1% deoxycholic acid, 1% Nonidet P-40, 20 mM Tris, pH 7.6, 2 mM EDTA [30] was added to the lysate which was centrifuged at 5,000 ϫ g for 10 min at 4 °C. The protein was further purified on a 375-ml Sephacryl HR 500 gel filtration column (2.5 ϫ 70 cm) (Pharmacia Biotech Inc.) in buffer containing 20 mM Tris-HCl, pH 7.4, 0.5 mM DTT, 0.2 mM EDTA, and 0.1 M NaCl at a flow rate of about 6 ml/h. Determination of Protein Concentration and Analysis of Circular Dichroism—Samples for CD were dialyzed three times in 20 mM sodium phosphate, pH 7.4, and 0.2 mM DTT, and centrifuged at 200,000 ϫ g for 30 min at 4 °C.
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