Corsi D, Galluzzi L, Lecomte M C, Magnani M
G. Fornaini Institute of Biological Chemistry, University of Urbino, Via Saffi 2, 61029 Urbino, Italy.
J Biol Chem. 1997 Jan 31;272(5):2977-83. doi: 10.1074/jbc.272.5.2977.
Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane.
此前，我们证明α-血影蛋白是泛素系统的底物，且这种缀合是一个动态过程（科尔西，D.，加卢齐，L.，克里内利，R.，和马尼亚尼，M.（1995年）《生物化学杂志》270，8928 - 8935）。在本研究中，我们绘制了红细胞α-血影蛋白上泛素化位点的图谱。先前进行过体外125I - 泛素缀合的消化α-血影蛋白的肽图谱显示存在四条不同的标记带，分子量分别为40,000、36,000、29,000和25,500。使用针对每个α-血影蛋白结构域的抗体进行的蛋白质印迹实验表明，只有抗αIII结构域的IgG识别29 kDa的125I标记泛素肽，而抗αV结构域的IgG识别分子量为40,000的125I - 泛素标记肽。另外两条分子量为36,000和25,500的标记带被鉴定为四聚体和三聚体多泛素链。通过抗α-血影蛋白结构域免疫亲和色谱进一步证实了αIII和αV结构域的泛素化。将先前与125I - 泛素缀合的内肽酶Lys C消化的血影蛋白与针对α-血影蛋白每个抗胰蛋白酶结构域的抗体一起孵育。通过蛋白A色谱纯化的放射性标记抗原 - 抗体复合物的γ计数显示仅在抗αIII和抗αV复合物中有标记。αIII结构域不与任何已知功能相关，而αV结构域包含α链和β链缔合的成核位点。后一个结构域的泛素化表明泛素在调节红细胞膜的稳定性、可变形性和粘弹性特性中起作用。
