Two Distinct Regions of Cyclophilin B Are Involved in the Recognition of a Functional Receptor and of Glycosaminoglycans on T Lymphocytes
1999; Elsevier BV; Volume: 274; Issue: 16 Linguagem: Inglês
10.1074/jbc.274.16.10990
ISSN1083-351X
AutoresMathieu Carpentier, Fabrice Allain, Bernard Haendler, A Denys, Christophe Mariller, Monique Benaı̈ssa, Geneviève Spik,
Tópico(s)Peptidase Inhibition and Analysis
ResumoCyclophilin 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 and14Tyr-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 Phe67residues, 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. 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 and14Tyr-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 Phe67residues, 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. Cyclophilins are highly conserved proteins first identified as the main binding proteins for cyclosporin A (CsA), 1The abbreviations used are: CsA, cyclosporin A; CyPA, cyclophilin A; CyPB, cyclophilin B; CyPC, cyclophilin C; GAG, glycosaminoglycans; HBP, heparin-binding protein; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PPIase, peptidyl-prolylcis/trans isomerase.1The abbreviations used are: CsA, cyclosporin A; CyPA, cyclophilin A; CyPB, cyclophilin B; CyPC, cyclophilin C; GAG, glycosaminoglycans; HBP, heparin-binding protein; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PPIase, peptidyl-prolylcis/trans isomerase. an immunosuppressive drug widely used in the prevention of graft rejection (1Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar, 2Harding M.W. Handschumacher R.E. Speicher D.W. J. Biol. Chem. 1986; 261: 8547-8555Abstract Full Text PDF PubMed Google Scholar). They were later identified as peptidyl-prolyl cis/trans isomerases (PPIase) (3Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1201) Google Scholar, 4Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (936) Google Scholar). Such an activity consists of the acceleration of thecis/trans isomerization of Xaa-Pro peptide bonds and has been proposed to be involved in protein folding (5Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar). The enzymatic activity of cyclophilins is strongly inhibited by CsA because of the binding of the drug over the catalytic site of these proteins. Different members of the cyclophilin family have been described. They all contain a conserved core domain, carrying both the CsA binding and isomerase sites, flanked by distinct N and C termini accounting for their specificities (6Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar). The prototype of this family is the abundant cytosolic 18-kDa form now named cyclophilin A (CyPA) (1Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar). Cyclophilin B (CyPB) (7Spik G. Haendler B. Delmas O. Mariller C. Chamoux M. Maes P. Tartar A. Montreuil J. Stedman K. Kocher H. Keller R. Hiestand P.C. Movva N.R. J. Biol. Chem. 1991; 266: 10735-10738Abstract Full Text PDF PubMed Google Scholar) and cyclophilin C (CyPC) (8Schneider H. Charara N. Schmitz R. Wehrli S. Mikol V. Zurini M.G.M. Quesniaux V.F.J. Movva N.R. Biochemistry. 1994; 33: 8218-8224Crossref PubMed Scopus (68) Google Scholar) are closely related, but their mRNA encodes a signal peptide that directs them to the secretory pathway. A mitochondrial form called cyclophilin D (9Bergsma D.J. Eder C. Gross M. Kersten H. Sylvester D. Appelbaum E. Cusimano D. Livi G.P. McLaughlin M.M. Kasyan K. Porter T.G. Silverman C. Dunnington D. Hand A. Prichett W.P. Bossard M.J. Brandt M. Levy M.A. J. Biol. Chem. 1991; 266: 23204-23214Abstract Full Text PDF PubMed Google Scholar) and a second larger cytosolic form named cyclophilin 40 (10Kieffer L.J. Thalhammer T. Handschumacher R.E. J. Biol. Chem. 1992; 267: 5503-5507Abstract Full Text PDF PubMed Google Scholar) have also been described. Alignment of amino acid sequences reveals 65% identity between CyPA and CyPB (6Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar, 7Spik G. Haendler B. Delmas O. Mariller C. Chamoux M. Maes P. Tartar A. Montreuil J. Stedman K. Kocher H. Keller R. Hiestand P.C. Movva N.R. J. Biol. Chem. 1991; 266: 10735-10738Abstract Full Text PDF PubMed Google Scholar) and more than 70% between CyPB and CyPC (8Schneider H. Charara N. Schmitz R. Wehrli S. Mikol V. Zurini M.G.M. Quesniaux V.F.J. Movva N.R. Biochemistry. 1994; 33: 8218-8224Crossref PubMed Scopus (68) Google Scholar). In the central core of the three forms the conservation in amino acid sequence is over 80%, implying that the regions bearing the CsA binding and isomerase activity are very similar in the different cyclophilins. The three-dimensional structures of CyPA (11Ke H. Zydowsky L.D. Liu J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9483-9487Crossref PubMed Scopus (167) Google Scholar), CyPB (12Mikol V. Kallen J. Walkinshaw M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5183-5186Crossref PubMed Scopus (98) Google Scholar), and CyPC (13Ke H. Zhao Y. Luo F. Weissman I. Friedman J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11850-11854Crossref PubMed Scopus (62) Google Scholar) have been solved and as expected the central core is similarly shaped. The structure includes eight antiparallel β strands forming a right-handed β-barrel overlaid by connecting loops and α helices (11Ke H. Zydowsky L.D. Liu J. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9483-9487Crossref PubMed Scopus (167) Google Scholar). Both active sites are closely localized in a large hydrophobic pocket formed by four β strands and their connecting loops, whereas the N and C termini are located on opposite sides of the molecule.In the case of CyPB, the C-terminal VEKPFAIAKE sequence has been described as a signal for retention in intracellular vesicles (14Arber S. Krause K.H. Caroni P. J. Cell Biol. 1992; 116: 113-125Crossref PubMed Scopus (98) Google Scholar). The protein however was reported to follow the secretory pathway (15Price E.R. Jin D. Lin D. Pati S. Walsh C.T. McKeon F.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3931-3935Crossref PubMed Scopus (105) Google Scholar). Our finding that CyPB is present in human milk and blood plasma provided the first evidence that it is effectively secreted into biological fluids (7Spik G. Haendler B. Delmas O. Mariller C. Chamoux M. Maes P. Tartar A. Montreuil J. Stedman K. Kocher H. Keller R. Hiestand P.C. Movva N.R. J. Biol. Chem. 1991; 266: 10735-10738Abstract Full Text PDF PubMed Google Scholar, 15Price E.R. Jin D. Lin D. Pati S. Walsh C.T. McKeon F.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3931-3935Crossref PubMed Scopus (105) Google Scholar, 16Allain F. Boutillon C. Mariller C. Spik G. J. Immunol. Methods. 1995; 178: 113-120Crossref PubMed Scopus (53) Google Scholar). Mariller et al. (17Mariller C. Allain F. Kouach M. Spik G. Biochim. Biophys. Acta. 1996; 1293: 31-38Crossref PubMed Scopus (17) Google Scholar) furthermore demonstrated that CyPB isolated from milk is truncated because of the absence of the C-terminal AIAKE sequence. The presence of CyPB in plasma spurred us on to find out whether specific binding sites for this protein existed on blood cells. Indeed we were able to detect surface binding sites for CyPB on human peripheral blood T lymphocytes (18Allain F. Denys A. Spik G. J. Biol. Chem. 1994; 269: 16537-16540Abstract Full Text PDF PubMed Google Scholar). The binding parameters were estimated to be 10–20 nmfor the dissociation constant (K d) and 30,000–120,000 for the number of binding sites/cell. We also found that the surface-bound ligand was specifically internalized into T cells and that CsA-complexed CyPB retained its cellular binding properties while promoting increased uptake of the drug and enhanced immunosuppressive activity (19Allain F. Denys A. Spik G. Biochem. J. 1996; 317: 565-570Crossref PubMed Scopus (21) Google Scholar, 20Denys A. Allain F. Foxwell B. Spik G. Immunology. 1997; 91: 609-617Crossref PubMed Scopus (19) Google Scholar, 21Denys A. Allain F. Masy E. Dessaint J.P. Spik G. Transplantation. 1998; 65: 1076-1084Crossref PubMed Scopus (17) Google Scholar). More recently, we were able to distinguish two classes of CyPB binding sites on the surface of peripheral blood T lymphocytes (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). Although both types of sites exhibit similar binding affinities, they are easily discriminated by their sensitivity to 0.6 m NaCl. The type I class of binding sites is insensitive to ionic strength and corresponds to functional lymphocyte receptors for CyPB, because endocytosis of the ligand follows the binding (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). Interaction with type I sites is reduced in the presence of CsA, suggesting that the conserved CsA binding region of CyPB is involved. CyPC also interacts with the lymphocyte receptors, however with a 5-fold lower affinity, whereas CyPA does not, indicating that fine differences in the three-dimensional structure and/or a few specific amino acids may be responsible for variations in binding affinity. The type II class of binding sites represents at least 70% of the total lymphocyte binding capacity and corresponds to sulfated glycosaminoglycans (GAG) (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). Interestingly, the interaction of CyPB with GAG is strongly inhibited by a peptide corresponding to the 24 N-terminal amino acid residues of CyPB, but remains unchanged in the presence of CsA (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). On the other hand, the binding to lymphocyte type II sites is found exclusively for CyPB, because neither CyPA nor CyPC is able to reduce the ligand binding (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). These results suggest that two distinct regions of CyPB may be involved in the interactions with lymphocyte GAG and specific membrane receptors.In this study, which aims to understand the functional implications of CyPB association with the two classes of binding sites on T cells, we have tried to identify the GAG binding region and the functional receptor binding region in the CyPB molecule. Using either fragments of CyPB obtained by proteolysis or mutant forms modified by genetic engineering, we localized two amino acid clusters necessary for binding to sulfated GAG. In addition, we provide evidence that the region interacting with the type I binding sites is close to the CsA binding site of CyPB and that the amino acids of the enzymatic site of the protein are not required for the interactions with the receptor.DISCUSSIONBefore mapping the CyPB regions involved in functional receptor and GAG binding, we checked that the two types of binding sites described by Denys et al. (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar) on the membrane of peripheral blood T lymphocytes were also expressed on the membrane of Jurkat cells. Indeed we found that 75% of the total Jurkat cell binding capacity was due to interactions with type II sites and that the remainder corresponded to type I functional receptors previously shown to be involved in ligand endocytosis. Binding to type I receptors was also found for CyPC and was strongly reduced in the presence of CsA, suggesting that part of the conserved CsA binding domains of CyPB and CyPC corresponds to the binding region. The type II binding sites represent about 75% of the total binding capacity and probably involve interactions with sulfated GAG present on cell surface. This binding is highly specific for CyPB and can be effectively reduced in the presence of a synthetic peptide corresponding to the N-terminal part of the protein (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). The role of this N-terminal extension of CyPB in the cellular binding has first been emphasized by Mariller et al. (24Mariller C. Haendler B. Allain F. Denys A. Spik G. Biochem. J. 1996; 317: 571-576Crossref PubMed Scopus (7) Google Scholar) who have also suggested that another region of CyPB may be involved in the interactions.The results presented here identify two regions of CyPB involved in the interactions with both types of CyPB binding sites present on the T cell membrane. We clearly demonstrate that CyPB possesses two receptor binding regions: one, located in the specific N-terminal extension of the protein, required for the interactions with sulfated GAG and the second one, corresponding to a part of the central conserved core of CyPB, involved in the recognition of a specific functional receptor. Both binding regions are spatially located on opposite sides of the CyPB molecule (12Mikol V. Kallen J. Walkinshaw M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5183-5186Crossref PubMed Scopus (98) Google Scholar), raising the possibility that interactions with both types of binding sites could occur simultaneously.The use of CyPB mutants corresponding to the protein modified within its N-terminal extension provided evidence for the role of the14Tyr-Phe-Asp16 cluster in the interactions with heparin and the type II binding sites constituted by sulfated GAG. Proteolytic cleavage confirmed the requirement of the13VYFDLR18 peptide of CyPB in the interactions with these binding sites, indicating that this region covers at least in part the actual binding region present in the N-terminal end of the protein. Most probably the 13VYFDLR18 peptide and more specifically the 14Tyr-Phe-Asp16sequence are necessary but not sufficient for interactions with the type II binding sites. Conceivably the14Tyr-Phe-Asp16 sequence is required to stabilize the primary interactions of a highly exposed basic sequence with the type II sites, explaining why a large excess of free13VYFDLR18 peptide inhibits the binding of CyPB. In this respect, we found that another region which contains the3Lys-Lys-Lys5 basic cluster was also required for tight interactions, in line with the involvement of two regions from the N-terminal part of CyPB in the binding to GAG chains. CyPBKKK- and CyPBΔYFD were eluted from a heparin-Sepharose column, respectively, with 0.1 and 0.3 mNaCl whereas CyPA, which lacks a KKK cluster, and CyPC, which lacks a YFD cluster, were eluted from 0.2 and 0.25 m NaCl (28Price E.R. Zydowsky L.D. Jin M. Baker C.H. McKeon F.D. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1903-1907Crossref PubMed Scopus (275) Google Scholar). The three-dimensional model of the N-terminal extension of CyPB showed that both the 3Lys-Lys-Lys5 and14Tyr-Phe-Asp16 clusters can be spatially juxtaposed and may act synergistically to form a cradle-like binding site for the sulfated polysaccharide chain. Unfortunately, the published crystallographic data on CyPB (12Mikol V. Kallen J. Walkinshaw M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5183-5186Crossref PubMed Scopus (98) Google Scholar) do not give information on the three-dimensional conformation of the1Asp-Glu-Lys-Lys-Lys5 extremity, which would help confirm this model. This is because of the absence in the crystal structure of the five N-terminal amino acid residues, which were probably cleaved off by proteases during the isolation procedure. Using the molecular modeling, we showed that the3Lys-Lys-Lys5 and the14Tyr-Phe-Asp16 clusters form a structural arrangement that could permit the interactions with the GAG. In addition, a YFDLR peptide was described in type IV collagen as a heparin binding domain (32Koliakos G.G. Kouzi-Koliakos K. Furcht L.T. Reger L.A. Tsilibary E.C. J. Biol. Chem. 1989; 264: 2313-2323Abstract Full Text PDF PubMed Google Scholar, 33Wilke M.S. Furcht L.T. J. Invest. Dermatol. 1990; 95: 264-270Abstract Full Text PDF PubMed Google Scholar). Indeed, this YFDLR peptide is located in a discontinuity of the triple helix of collagen and was reported to be directly involved in promoting keratinocyte adhesion to heparin sulfate proteoglycans. Taken together, these data clearly document the role of such regions in the interactions with GAG and strongly support our hypothesis that the binding region of CyPB involved in the interactions with type II sites and heparin is probably restricted to the 3Lys-Lys-Lys5 and14Tyr-Phe-Asp16 clusters.A role of sulfated GAG was suggested to be related to localization or local presentation of HBP. Because the biological functions of secreted cyclophilins are as yet largely unclear, it is difficult to speculate on the implications of the interaction with GAG. Some cyclophilins were reported to exhibit chemotactic activity for eosinophils, neutrophils, and monocytes (34Xu Q. Leiva M.C. Fischkoff S.A. Handschumacher R.E. Lyttle C.R. J. Biol. Chem. 1992; 267: 11968-11971Abstract Full Text PDF PubMed Google Scholar, 35Sherry B. Zybarth G. Alfano M. Dubrovsky L. Mitchell R. Rich D. Ulrich P. Bucala R. Cerami A. Bukrinsky M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1758-1763Crossref PubMed Scopus (92) Google Scholar). Also, CyPB levels measured in blood plasma from CsA-treated graft recipients (21Denys A. Allain F. Masy E. Dessaint J.P. Spik G. Transplantation. 1998; 65: 1076-1084Crossref PubMed Scopus (17) Google Scholar) and from patients suffering from HIV infection (36Endrich M.M. Gehring H. Eur. J. Cell. Biochem. 1998; 252: 441-446Crossref Scopus (39) Google Scholar) or sepsis (37Tegeder I. Schumacher A. John S. Geiger H. Geisslinger G. Bang H. Brune K. J. Clin. Immunol. 1997; 17: 380-386Crossref PubMed Scopus (92) Google Scholar) are increased for reasons not yet understood. Taken together, these observations suggest however that secreted cyclophilins might act as pro-inflammatory mediators. Another family of small chemoattractant proteins are the chemokines that are implicated in the attraction and activation of a variety of leukocytes (38Schall T.J. Cytokine. 1991; 3: 165-183Crossref PubMed Scopus (635) Google Scholar). Like CyPB, they bind to functional receptors on target cells and interact with sulfated GAG. The importance of the interactions with sulfated GAG present on cell membrane has also been shown for many growth factors and cytokines. Indeed these primary interactions are a prerequisite for the binding to specific receptors and the enhancement of cellular responses. For instance, soluble heparin was reported to support binding of interleukin-8 to its neutrophil receptor and to increase the intracellular free calcium concentration (39Rot A. Eur. J. Immunol. 1993; 23: 303-306Crossref PubMed Scopus (192) Google Scholar). On the other hand, it has been suggested that interactions with GAG, either at the surface of endothelial cells or in the extracellular matrix, could be responsible for the establishment of an immobilized chemokine gradient and the presentation of these molecules to leukocytes (40Tanaka Y. Adams D.H. Shaw S. Immunol. Today. 1993; 14: 111-115Abstract Full Text PDF PubMed Scopus (382) Google Scholar,41Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2073) Google Scholar). In support of this idea, it was demonstrated that MIP-1β and RANTES (regulated on activation normal T cell expressed) interact with solid phase GAG or activated endothelium and that these immobilized chemokines are then capable of stimulating leukocyte adhesion (42Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (843) Google Scholar, 43Gilat D. Hershkoviz R. Mekori A. Vlodavsky I. Lider O. J. Immunol. 1994; 153: 4899-4906PubMed Google Scholar). It is therefore conceivable that the type II sites contribute to the binding of CyPB to its type I lymphocyte receptor and regulate its activity. This possibility may only be further clarified by elucidating the biological role of CyPB and identifying the functional receptor.Finally, we examined in detail the implication of the CsA binding and PPIase domains of CyPB in type I receptor binding, because the interaction between CyPB and the drug leads to impaired receptor binding. Upon analysis of the homologies between the sequences of CyPA and CyPB, three amino acids residues, namely Arg62, Phe67, Trp128, were identified in CyPB as being possibly required for either PPIase, CsA binding, or both activities. We found that substitution of the Trp128 residue in CyPB prevented the interaction with CsA and markedly reduced the affinity of the protein for the type I sites. These results agree with our previous finding that addition of CsA inhibits the interactions of CyPB with the type I sites present on blood T lymphocytes (22Denys A. Allain F. Carpentier M. Spik G. Biochem. J. 1998; 336: 689-697Crossref PubMed Scopus (31) Google Scholar). Surprisingly, PPIase activity was not directly related to the cellular binding properties of CyPB. The substitution of Arg62 and Phe67effectively resulted in the loss of enzymatic activity but did not affect the interactions with the type I sites. Taken together, our results indicate that the second binding region of CyPB is probably conformational and emphasize the crucial role of the Trp128residue in the interactions with either CsA or type I sites. The presence of a Trp residue in the CsA binding domain is a common feature of all cyclophilins and may explain in part why CyPC exhibits binding activity for the type I sites. The binding affinity of CyPC was however found to be 6-fold lower than that of CyPB, whereas CyPA was unable to interact with any of the binding sites present on T cells. Such a discrimination of the cyclophilins for the recognition of a receptor has been already reported for CyPC with which a 77-kDa protein, termed CyCAP for CyPC-associated protein, was found to specifically interact (44Friedman J. Weissmann I. Cell. 1991; 66: 799-806Abstract Full Text PDF PubMed Scopus (358) Google Scholar, 45Friedman J. Trahey M. Weissman I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6815-6819Crossref PubMed Scopus (91) Google Scholar). CsA inhibits this interaction although neither CyPA nor CyPB is able to bind to this membrane protein. The areas of CyPC that interact with CyCAP are thought to be localized in a loop close to the catalytic site but without any sequence homology with the other cyclophilins, explaining the specificity of recognition. Inhibition of CyCAP binding by CsA is probably because of steric hindrance (13Ke H. Zhao Y. Luo F. Weissman I. Friedman J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11850-11854Crossref PubMed Scopus (62) Google Scholar). Such fine divergences in the spatial conformation of the central domain of the different cyclophilins might therefore well explain the variations seen in the binding affinities of CyPB and CyPC for their specific receptors. In addition, Sherry et al. (35Sherry B. Zybarth G. Alfano M. Dubrovsky L. Mitchell R. Rich D. Ulrich P. Bucala R. Cerami A. Bukrinsky M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1758-1763Crossref PubMed Scopus (92) Google Scholar) have very recently characterized a signaling receptor for CyPA on T lymphocytes and suggested that here also the CsA binding domain is involved in the interaction. Possibly, the type I binding sites for CyPB, CyCAP and the lymphocyte CyPA receptor might all be members of a family of related cyclophilin-binding proteins. The possibility that the different cyclophilins interact to some extent with the various receptor family members cannot be ruled out presently. Resolution of this question will require purification and structure elucidation of the different cyclophilin-binding proteins expressed on the membrane of T cells.Our studies clearly localize two distinct binding regions in the CyPB molecule that are separately involved in the recognition of GAG and of a functional receptor on T cells. This should allow the design of new experiments to test the importance of the cellular binding of CyPB and to provide new insights into the biological activity of this protein. Cyclophilins are highly conserved proteins first identified as the main binding proteins for cyclosporin A (CsA), 1The abbreviations used are: CsA, cyclosporin A; CyPA, cyclophilin A; CyPB, cyclophilin B; CyPC, cyclophilin C; GAG, glycosaminoglycans; HBP, heparin-binding protein; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PPIase, peptidyl-prolylcis/trans isomerase.1The abbreviations used are: CsA, cyclosporin A; CyPA, cyclophilin A; CyPB, cyclophilin B; CyPC, cyclophilin C; GAG, glycosaminoglycans; HBP, heparin-binding protein; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PPIase, peptidyl-prolylcis/trans isomerase. an immunosuppressive drug widely used in the prevention of graft rejection (1Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar, 2Harding M.W. Handschumacher R.E. Speicher D.W. J. Biol. Chem. 1986; 261: 8547-8555Abstract Full Text PDF PubMed Google Scholar). They were later identified as peptidyl-prolyl cis/trans isomerases (PPIase) (3Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1201) Google Scholar, 4Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (936) Google Scholar). Such an activity consists of the acceleration of thecis/trans isomerization of Xaa-Pro peptide bonds and has been proposed to be involved in protein folding (5Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar). The enzymatic activity of cyclophilins is strongly inhibited by CsA because of the binding of the drug over the catalytic site of these proteins. Different members of the cyclophilin family have been described. They all contain a conserved core domain, carrying both the CsA binding and isomerase sites, flanked by distinct N and C termini accounting for their specificities (6Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3575) Google Scholar). The prototype of this family is the abundant cytosolic 18-kDa form now named cyclophilin A (CyPA) (1Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1442) Google Scholar). Cyclophilin B (CyPB) (7Spik G. Haendler B. Delmas O. Mariller C. Chamoux M. Maes P. Tartar A. Montreuil J. Stedman K. Kocher H. Keller R. Hiestand P.
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