Ryffel B, Woerly G, Murray M, Eugster H P, Car B
Institute of Toxicology, University of Zurich, Switzerland.
Biochem Biophys Res Commun. 1993 Aug 16;194(3):1074-83. doi: 10.1006/bbrc.1993.1931.
The binding properties of several active and inactive cyclosporins to the major intracellular receptor proteins, cyclophilin A and B, as well as the interaction with the phosphatase calcineurin were investigated by ELISA and by means of a photoaffinity labeled probe (PL-CS). Binding to recombinant human cyclophilin A and B was rapid and saturable, and correlated with the in vitro immunosuppressive activity of cyclosporin derivatives. In the presence of cyclophilin A or B and calcium cyclosporin binds specifically to purified bovine calcineurin. PL-CS labeled only the calcineurin A subunit, but not the B subunit or calmodulin. Calcineurin A binding was competed by active (CsA, CsG or CsM), but not inactive (CsH, CsF) derivatives or the structurally unrelated macrolide immunosuppressant FK506. Ternary complexes containing equimolar ratios of cyclophilin A or B, PL-CS and calcineurin were resolved by chemical-crosslinking. The formation of these complexes was apparently specific, calcium-, but not calmodulin-dependent, and only inhibited by active cyclosporins. In vivo labelling of Jurkat T-cells revealed, that cyclophilin A and calcineurin A are the main labeled proteins, which form complexes in the presence of active cyclosporin. Thus, we demonstrate directly, that active cyclosporins have two recognition sites, which allow the in vivo recognition of cyclophilins and calcineurin A.
通过酶联免疫吸附测定（ELISA）以及光亲和标记探针（PL-CS）研究了几种活性和非活性环孢菌素与主要细胞内受体蛋白亲环蛋白A和B的结合特性，以及与磷酸酶钙调神经磷酸酶的相互作用。与重组人亲环蛋白A和B的结合迅速且具有饱和性，并且与环孢菌素衍生物的体外免疫抑制活性相关。在亲环蛋白A或B以及钙离子存在的情况下，环孢菌素特异性结合纯化的牛钙调神经磷酸酶。PL-CS仅标记钙调神经磷酸酶A亚基，而不标记B亚基或钙调蛋白。活性衍生物（环孢素A、环孢素G或环孢素M）可竞争钙调神经磷酸酶A的结合，而非活性衍生物（环孢素H、环孢素F）或结构不相关的大环内酯类免疫抑制剂FK506则不能。通过化学交联解析了含有等摩尔比亲环蛋白A或B、PL-CS和钙调神经磷酸酶的三元复合物。这些复合物的形成显然具有特异性且依赖于钙离子而非钙调蛋白，并且仅被活性环孢菌素抑制。对Jurkat T细胞的体内标记显示，亲环蛋白A和钙调神经磷酸酶A是主要的标记蛋白，它们在活性环孢菌素存在时形成复合物。因此，我们直接证明了活性环孢菌素有两个识别位点，这使得它们能够在体内识别亲环蛋白和钙调神经磷酸酶A。
