A recombinant form of the human BP180 ectodomain forms a collagen-like homotrimeric complex

被引:61
作者
Balding, SD
Diaz, LA
Giudice, GJ
机构
[1] MED COLL WISCONSIN, DEPT DERMATOL, MILWAUKEE, WI 53226 USA
[2] MED COLL WISCONSIN, DEPT BIOCHEM, MILWAUKEE, WI 53226 USA
[3] VET AFFAIRS MED CTR, MILWAUKEE, WI 53226 USA
关键词
D O I
10.1021/bi970675n
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (secl80e) in transiently transfected COS-1 cells. Gel filtration and sedimentation analyses demonstrated that secl80e 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 secl80e 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 secl80e complex is 3.25. With a Stokes radius of 13.6 nm, a sedimentation coefficient of 6.5 S, and a frictional ratio of 3.01, the secl80e 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 secl80e 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.
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页码:8821 / 8830
页数:10
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