Kouklis P D, Papamarcaki T, Merdes A, Georgatos S D
Programme of Cell Biology, European Molecular Biology Laboratory, Heidelberg, Germany.
J Cell Biol. 1991 Aug;114(4):773-86. doi: 10.1083/jcb.114.4.773.
To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.
为了确定III型中间丝（IF）蛋白的自我缔合位点，我们采用了“抗独特型抗体”方法。对一种单克隆抗体（抗-Ct）进行了特性分析，该抗体识别波形蛋白、结蛋白和外周蛋白杆状结构域末端附近的相似特征（ε位点或ε表位）。通过用纯化的抗-Ct免疫兔子产生的抗独特型抗体，识别波形蛋白、结蛋白和外周蛋白中共同的一个位点（推测与ε表位“互补”）（β位点或β表位）。通过比较免疫印迹分析可见，β表位存在于以外周蛋白30个COOH末端残基为模型的合成肽（PII）中。与ε位点和β位点之间存在缔合的观点一致，PII在体外与完整的IF蛋白以及含有ε表位的片段结合，但不与不与抗-Ct反应的IF蛋白结合。体内进行的显微注射实验和体外进行的丝状体重建分析进一步证明，这种位点特异性缔合的“解偶联”（通过与PII或抗-Ct竞争）会干扰正常的IF结构，导致形成直径远大于正常IF的丝状体和丝束。这些粗纤维与之前在体外组装缺失27个COOH末端残基的结蛋白衍生物时观察到的纤维非常相似（考夫曼，E.，K. 韦伯，和N. 盖斯勒。1985年。《分子生物学杂志》185:733 - 742）。作为一种分子解释，我们在此提出III型IF蛋白的ε位点和β位点是“互补的”，并在丝状体组装过程中缔合。由于这种缔合，我们进一步推测形成了一种表面暴露的“环”或“发夹”结构，该结构可能在空间上阻止不适当的丝状体 - 丝状体聚集并调节丝状体厚度。
