Cyclophilin: A Specific Cytosolic Binding Protein for Cyclosporin A

TLDR

Cyclophilin is a cytosolic protein that concentrates cyclosporin A in lymphoid cells and was purified from bovine thymocytes. The NH₂‑terminal sequence of the major cyclophilin species was partially characterized. Two cyclophilin species (~15 kDa, pI 9.6) bind cyclosporin A with a dissociation constant of ~2 × 10⁻⁷ M, exhibit sulfhydryl‑dependent, heat‑ and pH‑sensitive binding, and correlate with cyclosporin activity, indicating an intracellular immunosuppressive mechanism.

Abstract

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 × 10 -7 moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH 2 -terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56°C and at p H 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.