Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase.

Abstract

Peptidyl-prolyl cis-trans-isomerase accelerates otherwise slow, rate-limiting isomerization steps during folding of proteins in vitro, but is not yet securely identified with any specific physiologic role. Peptidyl-prolyl cis-trans-isomerase and the cyclosporin A (CsA)-binding protein cyclophilin are identical, and peptidyl-prolyl cis-trans-isomerase activity is inhibited by the immunosuppressive drug CsA in vitro. To establish a possible physiologic role of peptidyl-prolyl cis-trans-isomerase, we have studied the folding of procollagen I in suspended chick embryo tendon fibroblasts. Folding of procollagen I is slowed by CsA: the time needed for 50% of the molecules to reach a completely helical confirmation is 8.5 min in the absence and 13.5 min in the presence of 5 microM CsA; and the calculated products, k x K, of the rate constant (k) and the equilibrium constant (K) of peptidyl-prolyl cis-trans isomerization are 2.10 and 1.30 s-1, respectively. In contrast, folding of purified collagen III in vitro is unaffected by CsA. In cultured human fibroblasts, CsA caused posttranslational overmodification (hydroxylation of lysine 32.1 versus 22.1%) and increased intracellular degradation (18.7 versus 12.5%), and hence decreased production (10.2 versus 13.2% of total protein synthesis) of collagens I and III, indicating that procollagen folding is slowed by CsA also in human fibroblasts. We conclude that peptidyl-prolyl cis-transisomerase (and hence cyclophilin) accelerates protein folding in living cells. Furthermore, the CsA-induced changes in collagen metabolism are reminiscent of those observed in several variants of osteogenesis imperfecta caused by structural abnormalities in the pro-collagen chains which impair helix formation.