Studies On The Chaperone-like Function And Partial Structure Of Recombinant Human CyPA

The first cis-trans isomerization of peptidyl prolyl bonds was originally found as an 18 kDa protein isolated from porcine kidney in 1984 by Fischer et al, while another 18 kDa protein named cyclophilin A discovered in bovine thymocytes was found to have high affinity for the immunosuppressant drug cyclosporin A(CsA). Then, cyclophilins from calf thymus and human T-cell line were further characterized by sequences. In 1989, Fisher and Takahashi found that PPIase and CyPA were in fact the identical enzyme. Subsequent studies focused on the role of CyPs in cellular biochemistry. Researchers revealed that besides mediating in the CsA-induced immunosuppressive effect, CyPs were involved in various cellular processes including protein-protein interaction, cell division and proliferation, protein folding, chemokine receptor CXCR4-mediated signaling cascades, Influenza A virus replication, HIV infection and viral capsid formation. In 1991, researchers obtained the crystals of recombinant huamn T-cell CyPA, in that year, its three-dimensional structure was resolved out firstly with 2.5A resolution. After that, various three-dimensional structures of hCyPA combined with different substrates or ligands were resolved out by means of X-Ray or NMR. Results of the resolution had showed that hCyPA was composed of 165 amino acid residues in single chain which was a single domian protein. hCypA has an eight-stranded antiparallelβ-barrel structure and two a-helices are arranged to sit on the top and bottom of the barrel.In recent years, with the increasing research in the field of protein folding, the controversial issue that whether the single domain CyPA is just a enzyme with PPIase or a chaperone attracts attention of researchers again. In 1992, Freskgard et al found that the CyPA extracted from a pig showed the ability to suppress the aggregation and improve the folding yield of carbonic anhydrase during its refolding as well as PPIase activity, so he suggested that hCyPA had chaperone function. Soon after, Kern repeated Freskgard's experiments but extended the time of folding reaction from one hour to four hours, and he found that after added CyPA, the folding yield of carbonic anhydrase was almost identical to spontaneous folding, thus, he claimed that CyPA exert effect just as enzyme during carbonic anhydrase refolding. In recent years, Wen-Bin Ou et al got inactive CyPA from pig kidney and found that inactive CyPA could suppress the aggregation and enhance folding yield during refolding of creatine kinase effectively. Anutosh Chakraborty et al found that Leishmania donovani single domain CyPA could resolve the soluble aggregates of adenosine kinase also from Leishmania donovani. But so far, evidences for chaperone function of CyPA is far from enough.We have choosed four sites base on the review with various methods to explore the active sites. By means of site-directed mutagensis, four mutants R55A, F60A, Q63A and H126A were obtained and the corresponding proteins were expressed and purified to electrophoresis homogenous. PPIase activity assay suggested that the residual activity of four mutants were all less than 0.2% compared with WT, that was almost inactive complete. The mutants structures were characterized by UV difference spectra and fluorescence spectroscopy, and the results showed that overall spatial structures of all mutants were more loose compared with WT, Trp local structures of R55A, F60A, and H126A became more condensing, however, Trp local structure of Q63A became more extense than WT. In addition, ANS fluoresence showed that spatial structure of Q63A was more loose than the other three variants. By comparing the ability to suppress aggregation of variants and WT during arginine kinase (arginine kinase, AK, A model protein containing 12 proline residues, the folding process exists prolyl cis-trans isomerism) refolding, we found that rhCyPA's ability to suppress aggregation was independent of its PPIase. More interesting was that ability to suppress aggregation of Q63A was strong than WT, which probably was connected with the fact that adjacent spatial structure of the only one Trp or the overall structure of Q63A became more loose, and all these results suggested that rhCyPA have some chaperone-like characteristics.Using hydrogen peroxide to oxidize, we got the rhCyPA which lost PPIase completely. Then, we investigated the influence on AK folding exerted by different concentrations of native or theinactive rhCyPA, respectively. The results showed that the ability of native rhCyPA to suppress aggregation presented approximately stoichiometric relationship with its concentration, which is different from enzyme and is one of the important features of chaperone. The oxidized rhCyPA accelerated the aggregation of AK instead of suppression, which was probably due to the interaction between the large number of hydrophobic surface induced by oxidization and the hydrophobic surface of AK exposed to the solution during its folding, and the interaction hindered AK from folding in the right direction and promoted the aggregation at last.During the purification, the purified rhCyPA was homogeneous when analyzed with SDS-PAGE, but the non-denaturing gel showed three bands. In addition, the gel filtration presented three protein peaks which had the same behaviour in SDS-PAGE. Further investigation using methods of Western blotting, electrospray mass spectrometry, protein crosslinking, cetyltrimethyl ammonium bromide electrophoresis (CTAB-PAGE), we proved that the rhCyPA existed three forms of monomer, dimer and trimer.