Matthew W. Harding

cyclophilin, A Primary Molecular Target For Cyclosporine

An understanding of the mechanism of action of cyclosporine requires the identification and functional characterization of its molecular target or targets in the cell. Our laboratory has presented evidence that cyclophilin (CYP), a low‐molecular‐weight (Mr 17,737) basic protein, is the primary cytosolic receptor for CsA. The high affinity of CYP for CsA (Kd 30 nM) and specificity for immunosuppressive cyclosporine analogs implicate CYP as a pivotal regulator of T cell and B cell activation. CYP exists in at least two isoforms, is abundant (0.05% to 0.4% of total protein) in the cytosol, and is ubiquitous in cells and tissues of eukaryotic organisms. Its amino acid sequence is highly conserved and there is strong evidence that CYP is a member of a new multigene family. These features suggest that one or more CYP isoforms must play a crucial role in lymphocyte activation and perhaps a multifunctional role in cellular physiology. The ability of CsA to suppress expression of several lymphokine and proto‐oncogene products via a transcriptional control mechanism suggests that CYP may function at some level in a signaling pathway linking membrane receptor stimulation to gene regulatory elements in lymphocytes, and possibly nonlymphoid cell types as well.

The immunochemical distribution of cyclophilin in normal mammalian tissues.

Cyclophilin, a 17 Kd proline cis-trans isomerase, high-affinity (Kd 10(-8) M) target for the immunosuppressive drug cyclosporine has ubiquitous phylogenic distribution, but its tissue localization in mammals has not been detailed. To explore a potential relationship between the multiple systemic effects of CsA and the cellular and tissue distribution of CYP, thirty-three different normal porcine tissues were examined using an immunohistochemical technique. Tissue was obtained from farmbred pigs, immediately fixed in buffered formalin, and prepared as embedded 5-mu sections. Immune-specific staining was accomplished using an ABC immunoperoxidase method and an affinity-purified, monospecific, rabbit anti-CYP IgG. Cut sections served as their own blanks and controls, and all tissues were stained in batch to minimize the effects of variation in technique. Consistent with earlier reports, CYP was present in all tissues studied, however, there was remarkable heterogeneity in CYP distribution. Renal parenchymal cells, cardiac and striated muscle, pulmonary and skin demonstrated cytoplasmic immunospecific CYP--however, the cellular localization varied. Cytoplasmic staining of endothelial, neural, and glandular elements was consistently observed. Contrasting with previous reports, CYP localized to the nucleus as well as the cytoplasm of some lymphoid cells, hepatocytes, and cells of the large intestine. Generally, greater CYP-specific staining was noted in organs amenable to CsA immunosuppression (heart, liver, kidney), compared with organs deemed more immunologically vulnerable when allografted under CsA (pancreas, lung, small bowel). Similarly, CYP-immunospecific staining was abundant in tissues susceptible to CsA toxicities (neural tissue, smooth muscle, kidney, liver). This detailed immunohistological examination affords a correlation between CYP content and sensitivity to CsA. It also raises some new questions about tissues with little extractable CYP but significant histological staining.

Activity of cyclosporin A and a non-immunosuppressive cyclosporin against multidrug resistant leukemic cell lines.

Cyclosporin A (CsA) has been shown to increase the sensitivity of multidrug resistant (MDR) cells to chemotherapeutic agents. Although the concentration of drug required to produce this effect is clinically achievable, the use of this drug would be hampered by significant immunosuppression. We report a comparison of the effects of 11-methyl-leucine cyclosporin (11-met-leu CsA), a non-immunosuppressive homolog to the parent drug, on MDR cell lines. Both cyclosporins sensitized resistant cell lines to doxorubicin, including P388 murine leukemia and GM 3639 human T-cell leukemia. The action of the cyclosporins was more pronounced with resistant cells than with sensitive ones. 11-Met-leu CsA was less potent than, but equally effective as, the parent drug. Both agents increased the intracellular accumulation and retention of doxorubicin in MDR cells. The sensitization caused by the cyclosporins was independent of their effects on cyclophilin, calmodulin, and protein kinase C. Furthermore, there were no differences in the binding of labelled CsA to MDR cells compared to the binding to sensitive cells, suggesting that P-glycoprotein was also not the molecular site of action. These studies demonstrate that a non-immunosuppressive cyclosporin can modulate multidrug resistance and suggest its further evaluation for use in clinical trials.

Structural elements pertinent to the interaction of cyclosporin a with its specific receptor protein, cyclophilin

Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies have revealed 81% of the 156 expected HN-H alpha crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D2O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with beta-sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues.

Autoantibodies against cyclophilin in systemic lupus erythematosus and Lyme disease

Autoantibodies against cyclophilin, a cyclosporin A binding protein, were detected in sera of 29 of 46 (63%) patients with systemic lupus erythematosus and 14 of 40 (35%) Lyme disease patients. The antibodies are directed against the denatured form of both the major and minor isoform of cyclophilin and, can be demonstrated in Western blots. Some first‐degree relatives of lupus patients also express these antibodies. They are specific for cyclophilin and are not the consequence of hypergammaglobulinaetnia. Four monoclonal IgM antibodies from a patient with lepromatous leprosy also bound to cyclophilin. The generation of these antibodies may be of special interest because they are against a protein involved in the control of the immune system not known to be directly associated with DNA or RNA.