Leukocyte chemotactic activity of FKBP and inhibition by FK506.

Cyclophilin, the cyclosporin A binding protein and member of the immunophilin family of proteins, demonstrates leukocyte chemotactic activity. In this study we demonstrate that FKBP, the FK506 and rapamycin binding protein, also displays leukocyte chemotactic activity. The chemotactic activity of FKBP is inhibited by FK506, however, FK506 was unable to inhibit cyclophilin-stimulated chemotactic activity. Rapamycin was unable to prevent the chemotactic activity of FKBP, similarly, the CsA analogue Me6Ala-CsA while displaying cyclophilin binding was unable to block cyclophilin-stimulated chemotactic activity. These results suggest that in addition to their intracellular role the immunophilins may also function as chemotactic agents, furthermore this activity is modulated by immunosuppressants.     

FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein, cyclophilin

A novel macrolide antibiotic, FK-506, isolated from Streptomyces tsukubaensis, has been shown to be a potent immunosuppressive agent in vivo and in vitro. FK-506 shares a number of immunosuppressive properties with the cyclic peptide, cyclosporin A (CsA), although 10 to 100 times more potent in this regard. These similarities suggest that both agents may share a similar mechanism(s) of action at the biochemical level. We have identified a cytoplasmic binding protein for FK-506 in the human T cell line, JURKAT, using [3H]FK-506. The FK-506 binding protein has a mr of 10 to 12 kDa (as determined by gel filtration), is heat stable and does not bind CsA. This contrasts with the CsA binding protein, cyclophilin, in that cyclophilin is heat labile and has a mr of 15 to 17 kDa. Our data suggest that FK-506 binds to a low m.w. protein(s) in JURKAT cells, which is distinct from cyclophilin. This protein may mediate the immunosuppressive effects of FK-506 in T cells. In addition, our results suggest that the immunosuppressive activity of FK-506, as with CsA, is mediated by an intracellular mechanism.     

A novel secreted cyclophilin-like protein (SCYLP)

A novel cyclosporin A binding glycoprotein of 21 kDa was isolated from human milk by several steps of cation exchange chromatography. The corresponding gene was cloned from human T cells, expressed in Escherichia coli and the recombinant protein purified. The protein shares 58% amino acid identity with the cytosolic cyclophilin and is initially synthesized with a hydrophobic leader sequence. The cyclophilin-like protein has also peptidyl-prolyl cis/trans-isomerase activity, although less efficient, that is inhibited by cyclosporin A. The existence of a secreted form of cyclophilin-like protein in addition to the previously known cytosolic cyclophilin implies that these proteins act on different in vivo targets.     

Isolation and partial characterization of membrane-associated cyclophilin and a related 22-kDa glycoprotein.

The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5-12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pI approximately 9.3-9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue.     

A Candida albicans homolog of a human cyclophilin gene encodes a peptidyl-prolyl cis-trans isomerase

A Candida albicans cDNA and its genomic counterpart were isolated from lambda phage libraries using a human T-cell cyclophilin (Cyp) cDNA as a hybridization probe. The clones contain a 486-bp open reading frame predicting a 162-amino acid, approx. 18 kDa protein which is similar in size to, and which shares 68 and 81% homology with, human T-cell Cyp and cytosolic Saccharomyces cerevisiae Cyp, respectively. Northern blots show the presence of a single mRNA species of about 800 bp. However, genomic Southern blots suggest the presence of at least one other Cyp-related gene in C. albicans. The cDNA was engineered for expression in Escherichia coli, and the resulting recombinant protein, like mammalian Cyps, exhibited a peptidyl-prolyl cis-trans isomerase (PPIase) activity which was sensitive to inhibition by cyclosporin A in vitro. These results indicate that the gene which we have cloned encodes a C. albicans Cyp. We designate this gene CYP1 (cyclophilin). Interestingly, the predicted C. albicans protein contains only two cysteine residues which do not align with any of the four cysteines conserved among mammalian Cyps. This suggests that the PPIase catalytic mechanism may not involve an enzyme-bound hemithioorthoamide, as previously reported for porcine Cyp.     

Two distinct regions of cyclophilin B are involved in the recognition of a functional receptor and of glycosaminoglycans on T lymphocytes

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and 14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67 residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.     

Expression of a gene for cyclophilin which contains an amino-terminal endoplasmic reticulum-targeting signal

We isolated a novel gene for cyclophilin (CyP) first identified as an intracellular target of the immunosuppressant cyclosporin A and also known to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, named ATCYP5 from Arabidopsis thaliana. ATCYP5 encoded a polypeptide with 201 amino acids with a putative ER-targeting signal sequence at its N-terminal, but without the typical ER-retention signal in its C-terminal. In addition, ATCYP5 protein contained a seven amino-acid long sequence which has been found previously only in cytosolic CyPs from plants. The synthetic mutant green fluorescent protein (sGFP; S65T) was fused to the N-terminal part of ATCYP5, and expressed in tobacco BY-2 cells. The fluorescence derived from the fusion protein was detected mainly around the nucleus, indicating translocation into ER. ATCYP5 was expressed mainly in young stems especially in the apical region and weakly in leaves and roots.     

Cyclophilin of Schistosoma japonicum.

A 623-bp cDNA molecule encoding cyclophilin, a specific cyclosporin A-binding protein, has been isolated from Schistosoma japonicum using a heterologous cDNA probe from Echinococcus granulosus. The nucleotide sequence of this molecule has been determined, and the deduced amino acid sequence has revealed extensive homology with homologues of other species. Southern blot analysis suggests that S. japonicum cyclophilin is the product of a single-copy gene. The cloning of this cDNA will allow an investigation of cyclophilin as a possible target of the antischistosome effects of cyclosporin A.     

Complementary DNA for human T-cell cyclophilin.

Complementary DNA encoding human cyclophilin, a specific cyclosporin A-binding protein, has been isolated from the leukemic T-cell line Jurkat and sequenced. Comparison of the deduced amino acid sequence with the previously determined sequence of bovine thymus cyclophilin reveals only three differences: an additional amino acid at the carboxy terminus end and two internal changes. RNA transfer blot analysis indicates an mRNA size of approximately 1 kb for human T-cell cyclophilin. Phytohaemagglutinin and phorbol myristate acetate induction of T cells treated or not with cyclosporin A affects only marginally the level of cyclophilin mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes strongly suggests the existence of a multigene family for cyclophilin.