Sasaki T, Kostka G, Göhring W, Wiedemann H, Mann K, Chu M L, Timpl R
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
J Mol Biol. 1995 Jan 20;245(3):241-50. doi: 10.1006/jmbi.1994.0020.
Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.
从小鼠纤维连接蛋白-1的两个C末端变体C和D是从稳定转染的人肾细胞克隆的培养基中纯化得到的。旋转投影后，它们呈现出长度约为33nm的哑铃状结构。胃蛋白酶消化显示，二硫键连接的结构域1（类过敏毒素）和结构域II（多个表皮生长因子样基序）具有稳定性，但变体中不同的结构域III则不然。免疫化学分析显示变体之间有密切的相似性，表明结构域III表位的抗原性不强。固相分析中的结合分析表明，变体C与基底膜蛋白巢蛋白的结合比变体D强100倍。两种相互作用都对EDTA敏感。表面等离子体共振分析证实了这种差异，显示变体C的KD = 60 nM，变体D的KD > 1 μM。对于与纤连蛋白、层粘连蛋白-1和IV型胶原的钙依赖性结合，观察到较低的结合活性和两种变体之间较小的差异。在高浓度的纤维连接蛋白-1下观察到自聚集形成巢状寡聚体，这对EDTA不敏感。
