Regulation of CD147 cell surface expression: involvement of the proline residue in the CD147 transmembrane domain

CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and serves as a signaling receptor for extracellular cyclophilins. Here we demonstrate that the cell surface expression of CD147 is regulated by cyclophilins via the transmembrane domain of CD147. Solution binding experiments demonstrated that the transmembrane domain was both necessary and sufficient for CD147 binding to cyclophilin A (CypA). Treatment with cyclosporin A significantly reduced surface expression of CD147 and of CD8-CD147 fusion protein carrying the extracellular domain of CD8 fused to the transmembrane and cytoplasmic domains of CD147, but did not affect expression of CD8. Peptide binding studies demonstrated specific interaction between CypA and the proline-containing peptide from the CD147 transmembrane domain. Mutation of this proline residue reduced binding of CD147-derived peptides to CypA and also diminished transport of CD147 to the plasma membrane without reducing the total level of CD147 expression. These results suggest involvement of a cyclophilin-related protein in CD147 cell surface expression and provide molecular details for regulation of CD147 trafficking by cyclophilins.     

A novel cyclophilin from parasitic and free-living nematodes with a unique substrate- and drug-binding domain

A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus (OvCYP-16). This 25-kDa cyclophilin shares 43-46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans (CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu(124) (C. elegans) or Asp(123) (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A. CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism.     

Streptomycetes possess peptidyl-prolyl cis-trans isomerases that strongly resemble cyclophilins from eukaryotic organisms

A functionally active 17.5 kDa peptidyl-prolyl cis-trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram-positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram-negative enterobacteria revealed much less homology (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.     

Structural and evolutionary relationships among the immunophilins: two ubiquitous families of peptidyl-prolyl cis-trans isomerases.

The immunophilins, protein receptors for the immunosuppressing drugs cyclosporin A and FK506 and related proteins from plants, fungi, and bacteria, have been analyzed structurally and evolutionarily. The cyclosporin A binding proteins (cyclophilins) represent one ubiquitous family of homologous proteins, and the FK506- and rapamycin-binding proteins (FKBPs) constitute a second, unrelated family. Multiple sequence alignments of members of each of these two protein families define the highly conserved residues that are likely to play important structural and functional roles, and mutations in representative members of these two families that abolish or alter function have been evaluated. FKBPs have undergone greater evolutionary divergence than the cyclophilins. Evolutionary trees were constructed using two distinct programs, and these trees establish the structural relationships that allow division of each of these families into subgroups. The results lead to the suggestion that several genes encoding isozymic forms of the FKBPs and possibly also of the cyclophilins existed in prokaryotes before the emergence of eukaryotes on earth and that representatives of these genes were transmitted to both kingdoms to give rise to current subfamilies of these proteins. By contrast, compartmentalization of both classes of immunophilins appears to have arisen independently in prokaryotes and eukaryotes, late in evolutionary history.     

A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A

Peptidyl prolyl cis/trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca(2+)-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.     

Catalysis of protein folding by cyclophilins from different species.

Cyclophilins are a class of ubiquitous proteins with yet unknown function. They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A. The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides. This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase. This enzyme accelerates slow, proline-limited steps in the refolding of several proteins. Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding. This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins. It could be related with de novo protein folding or be involved in regulatory processes. Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.     

The neuronal choline transporter CHT1 is regulated by immunosuppressor-sensitive pathways

The immunosuppressor cyclosporin A inhibits the peptidyl-prolyl-cis/trans-isomerase activity of cyclophilins and the resulting complex inhibits the phosphatase activity of calcineurin. Both enzymes were detected in peripheral nerve endings isolated from the electric organ of Torpedo and shown to be affected by 10 micro m cyclosporin A. Among the cholinergic properties studied, choline uptake was specifically inhibited by cyclosporin A to a maximum of 40%. Cyclosporin A decreased the rate of choline transport but not the binding of the non-transportable choline analogue hemicholinium-3, indicating that the number of membrane transporters was not affected. Through the use of two other immunosuppressors, FK506, which also inhibits calcineurin, and rapamycin, which does not, two different mechanisms of choline uptake inhibition were uncovered. FK506 inhibited the rate of choline transport, whereas rapamycin diminished the affinity for choline. The Torpedo homologue of the high affinity choline transporter CHT1 was cloned and its activity was reconstituted in Xenopus oocytes. Choline uptake by oocytes expressing tCHT1 was inhibited by all three immunosuppressors and also by microinjection of the specific calcineurin autoinhibitory domain A457-481, indicating that the phosphatase calcineurin regulates CHT1 activity and could be the common target of cyclosporin and FK506. Rapamycin, which changed the affinity of the transporter, may have acted through an immunophilin on the isomerization of critical prolines that are found in the tCHT1 sequence.     

Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA.

We report the cloning and characterization of a new binding protein for the immunosuppressive drug cyclosporin A (CsA). This new cyclophilin, cyclophilin C (cyp C), shows extensive homology with all previously identified cyclophilins. Cyp C mRNA is expressed in a restricted subset of tissues relative to cyclophilins A and B, but is present in those tissues reported to be most affected by CsA therapy. A cyp C fusion protein has peptidyl-prolyl isomerase activity, and CsA inhibits this activity. Using the cyp C fusion protein as an affinity ligand to probe cellular extracts, we find that the cyp C fusion protein binds specifically to a 77 kd protein in the absence of CsA, while in the presence of CsA it instead binds specifically to a 55 kd protein. We propose that the p77 is involved in cyp C native function and that the p55 is involved in signal transduction events blocked by treatment with immunosuppressive levels of CsA.     

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.     

Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4(+) T cells by homologous recombination. HIV-1 replication in PPIA(-/-) cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag's interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA(+/+) and PPIA(-/-) cells. Stable re-expression of CypA in PPIA(-/-) cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA(-/-) cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions.     

Mutation of FKBP associated protein 48 (FAP48) at proline 219 disrupts the interaction with FKBP12 and FKBP52

Immunophilins are known as intracellular receptors for the immunosuppressant drugs, cyclosporin A, FK506, and rapamycin. They can be divided into two groups, cyclophilins that bind cyclosporin A and FK506 binding proteins (FKBPs) that bind FK506 and rapamycin. Many efforts were made to elucidate the physiological role of the immunophilins. Many of them are involved in intracellular signalling as they bind to calcium channels or to steroid receptor complexes. A yeast two-hybrid screen was used to identify further target proteins that interact with known proteins. Recently, a 48-kDa FKBP associated protein (FAP48) was isolated that binds to FKBP12 and FKBP52. Binding of FAP48 to FKBPs is inhibited by the macrolide FK506 indicating that the binding sites on the immunophilins coincide with the binding site for FK506. A peptidyl-prolyl motif on FAP48 should be responsible for the binding of the protein to FKBPs. We sequentially point mutated proline sites on FAP48 and checked the mutant proteins for interaction with FKBP12 and FKBP52. Mutation of proline 219 to alanine leads to a loss of interaction indicating that a cysteinyl prolyl site might be responsible for the binding of FAP48 to FKBPs. Thus we identified proline 219 being essential for the interaction.     

CD147 is a signaling receptor for cyclophilin B.

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins.     

Crystal structure of the cyclophilin-like domain from the parasitic nematode Brugia malayi.

Cyclophilins are a family of proteins that exhibit peptidyl-prolyl cis-trans isomerase activity and bind the immunosuppressive agent cyclosporin A (CsA). Brugia malayi is a filarial nematode parasite of humans, for which a cyclophilin-like domain was identified at the N-terminal of a protein containing 843 amino acid residues. There are two differences in sequence in the highly conserved CsA binding site: A histidine and a lysine replace a tryptophan and an alanine, respectively. The crystal structure of this domain has been determined by the molecular replacement method and refined to an R-factor of 16.9% at 2.15 A resolution. The overall structure is similar to other cyclophilins; however, major differences occur in two loops. Comparison of the CsA binding site of this domain with members of the cyclophilin family shows significant structural differences, which can account for the reduced sensitivity of the Brugia malayi protein to inhibition by CsA.     

Cyclophilin sensitivity to sanglifehrin A can be correlated to the same specific tryptophan residue as cyclosporin A

Sanglifehrin A (SFA) is a recently discovered immunosuppressant drug that shares its intracellular target with the major immunosuppressant drug cyclosporin A (CsA). Both bind to and inhibit the cyclophilins, a diverse family of proteins found throughout nature that share a conserved catalytic domain. Although they share this common protein target, the mechanism of action of the cyclophilin-SFA complex has been reported as distinct from that of the well-studied cyclophilin-CsA complex. The X-ray structure of a macrolide analogue of SFA's cyclic region complexed with cyclophilin A has recently been resolved, but this left the placement of the linear region of SFA unresolved. Using five cyclophilins from the fission yeast Schizosaccharomyces pombe, and a mutant of one of these proteins, SpCyp3-F128W, we have shown that the sensitivity of cyclophilins to SFA can be correlated to the same specific tryptophan residue that has previously been identified to correlate to CsA sensitivity, and that the tail of SFA may be responsible for mediating this sensitivity.     

Cyclosporin-binding proteins of Plasmodium falciparum

A number of cyclosporins, including certain non-immunosuppressive ones, are potent inhibitors of the intraerythrocytic growth of the human malarial parasite Plasmodium falciparum. The major cyclosporin-binding proteins of P. falciparum were investigated by affinity chromatography on cyclosporin-Affigel followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting, and peptide mass fingerprinting. The two bands obtained on gels were shown to correspond to cyclophilins, PfCyP-19A (formerly PfCyP-19) and PfCyP-19B, whose genes had been characterised previously. PfCyP-19B was an abundant protein of intraerythrocytic P. falciparum (up to 0.5% of parasite protein) that was present in the highest amounts in schizont-stage parasites. Unexpectedly, given its apparent signal sequence, it was located primarily in the cytosol of the parasite. The peptidyl-prolyl cis-trans isomerase activity of recombinant PfCyP-19B had the same profile of susceptibility to cyclosporin derivatives as the bulk isomerase activity of crude P. falciparum extracts. The binding of cyclosporins to cyclophilins may be relevant to the mechanism of action of the drug in the parasite.     

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.     

Host-cell cyclophilin is important for the intracellular replication of Leishmania major

The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host–parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.     

Genetic dissection of cyclophilin function. Saturation mutagenesis of the Drosophila cyclophilin homolog ninaA.

Cyclophilins, the intracellular receptors for the widely used immunosuppressant cyclosporin A have been found to be peptidyl-prolyl cis/trans isomerases and have been implicated in intracellular protein folding and trafficking. The Drosophila ninaA gene encodes a photoreceptor-specific cyclophilin homolog involved in rhodopsin biogenesis. ninaA mutants have a 90% reduction in the levels of Rh1 rhodopsin. To gain insight into the role of cyclophilins in vivo, we carried out a genetic screen designed to identify functionally important regions in the ninaA protein. Over 700,000 mutagenized flies were screened for a visible ninaA phenotype and 70 independent mutations in ninaA were isolated and characterized. These mutations provide a detailed dissection of the structure/function relationships in cyclophilin. We also show that mammalian cyclophilins engineered to contain missense mutations found in two temperature-sensitive ninaA alleles display temperature-sensitive prolyl cis/trans isomerase activity.