Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils
Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils
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DOI：
10.1074/jbc.m210611200
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发表时间：
2002-12-27
影响因子：
4.8
通讯作者：
Reinhardt, DP
中科院分区：
文献类型：
--
作者：
Lin, GQ;Tiedemann, K;Reinhardt, DP
原纤维蛋白-1和纤维蛋白-2构成细胞外丝的骨架，称为微纤维。原纤蛋白组装涉及复杂的多步骤机制，以导致微纤维中周期性的头-尾排列。受损的装配可能在遗传性疾病的分子发病机制中起作用，这些遗传性疾病是由马凡氏综合征（Marfan syndrome）和先天性挛缩性蛛网膜下腔出血（acrylatarachinalactyly）引起的。目前，基本的分子间相互作用涉及的组装是模糊的。在这里，我们已经产生了重组全长人β-内酰胺酶-1，和两个重叠的重组多肽跨越整个人β-内酰胺酶-2在哺乳动物表达系统。通过凝胶电泳、旋转阴影后的电子显微镜和与抗体的反应性表征这些重组多肽的正确折叠。同型和异型相互作用谱的分析表明，N-至C-末端结合的Escherichin-1，和Escherichin-1与Escherichin-2。的相互作用是高亲和力的解离常数在低纳摩尔范围内。然而，N端和C端的蛋白多肽不相互作用。这些结果表明，原纤维蛋白可以以N-至C-末端的方式直接相互作用以形成同型的β-淀粉样蛋白-1或异型的β-淀粉样蛋白-1/β-淀粉样蛋白-2微纤维。这一结论进一步加强了双免疫荧光标记的微纤维。此外，结合表位以及整个的cavin分子显示出非常稳定的性质。
Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the Nand C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/ fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.