A recombinant form of the human BP180 ectodomain forms a collagen-like homotrimeric complex.
BP180 is a glycoprotein constituent of the epidermal anchoring complex and a major antigenic target of autoantibodies associated with bullous pemphigoid, a blistering skin disease. The C-terminal extracellular domain of BP180 contains 15 domains composed of Gly-X-Y tandem repeats, which are predicted to form collagen-like triple helices. To facilitate the structural analysis of this protein, the extracellular region of human BP180 was expressed as a secreted protein (sec180e) in transiently transfected COS-1 cells. Gel filtration and sedimentation analyses demonstrated that sec180e exists in two forms: a globular monomeric form and a high-molecular mass multimeric form with an elongated conformation. Pulse-chase and cross-linking experiments established that the sec180e complex is a stable homotrimeric structure which assembles prior to secretion from the cell. On the basis of its calculated molecular mass, the oligomeric state of the sec180e complex is 3.25. With a Stokes radius of 13.6 nm, a sedimentation coefficent of 6.5 S, and a frictional ratio of 3.01, the sec180e protein appears to be highly extended (length to width ratio is between 52 and 60), yet is more flexible than a rigid rod. BP180 isolated from human epidermis was also shown to exist in a high-molecular mass complex which, like sec180e and other collagenous proteins, is SDS-stable but heat-labile. These findings strongly suggest that the BP180 ectodomain exists as an elongate, flexible homotrimer. This trimerization is likely to result from the formation of stable collagen triple-helical and coiled-coil type structures and does not depend upon the presence of the cytoplasmic or transmembrane domains of this protein.
Balding
S D
SD
Department of Dermatology, Medical College of Wisconsin, and Veterans Affairs' Medical Center, Milwaukee, Wisconsin 53226, USA.
Diaz
L A
LA
Giudice
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry*; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002850:Chromatography, Gel; D004817:Epidermis / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D024041:Non-Fibrillar Collagens; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D014162:Transfection; D000097805:Collagen Type XVII
D001324:Autoantigens; D024041:Non-Fibrillar Collagens; D011994:Recombinant Proteins; D000097805:Collagen Type XVII