Structural and Functional Characterization of the Interaction between Cyclophilin B and a Heparin-derived Oligosaccharide

Abstract

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp 179 -Pro 180 bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.First characterized as the molecular targets of the immunosuppressive drug cyclosporin A (CsA), 3 cyclophilins (Cyps) constitute one class of the prolyl cis/trans isomerases that catalyze the cis/trans interconversion of the peptide bond preceding a proline (1, 2). Members of this class such as the predominantly cytoplasmic CypA, the secreted CypB, and the mitochondrial CypD are small ubiquitous proteins sharing a high sequence homology (65% identity between human CypA and CypB), that translates into a closely related three-dimensional fold. Indeed, the NMR and crystal structures of CypA free and in complex with CsA (3-6), as well as the crystal structure of CypB in complex with a cyclosporine analogue (7) all show the same core structure composed of eight antiparallel ␤-strands forming a ␤-barrel surrounded by ␣-helices and loops. Whereas the nearly identical active site and CsA binding pocket further underscore their close relationship, both proteins do differ in their N and C termini, CypB containing two peptides of some 10 residues long that are lacking in CypA.CypA and CypB act in the progression of inflammatory diseases such as rheumatoid arthritis and psoriasis, but are equally involved in the first steps of certain viral infections (8 -10). Their inflammatory activity is conditioned by their interaction with heparan sulfate proteoglycans (HSPGs) and the membrane receptor CD147, two binding partners at the cell surface of T cell lymphocytes, granulocytes and macrophages (11-14). Significantly, both molecular partners have equally been described as co-receptors for the HIV-1 virus (10,12,15).Both intact prolyl cis/trans activity of the cyclophilins and the presence of the Pro 180 residue of CD147, located on one of the two ex...

A novel functional site of extracellular matrix metalloproteinase inducer (EMMPRIN) that limits the migration of human uterine cervical carcinoma cells

“…Thus, the loop I domain of EMMPRIN is unlikely to contribute to the regulation of cell migration in SKG-II cells. On the other hand, the loop II domain of EMMPRIN has been reported to form a complex with caveolin-1 and cyclophilin (14,16). In the present study, we found that the synthetic EM9 peptide of EMMPRIN, 170 HIENLNMEADPGQYR…”

“…On the other hand, the loop II domain of EMMPRIN has been reported to form a complex with caveolin-1, which may be associated with the regulation of MMP production and the localization of EMMPRIN in lipid rafts (13)(14)(15). In addition, cyclophilin B-mediated isomerization of proline in the loop II domain of EMMPRIN has been reported to participate in the chemotactic activity of T lymphocytes (16 EMMPRIN may help with the understanding of its pathophysiological roles and the development of anti-metastatic strategies, they remain to be elucidated. Tumor cell migration is likely to be a crucial event for the progression of tumor invasion and metastasis (17,18).…”

“…A recent study by Hanoulle et al (16) reported that the cis/trans isomerization of Pro 180 in CD147 by cyclophilin B is required for the CD147-associated chemotactic activity in T lymphocytes. In addition to our confirmation that Pro 180 is contained in the EM9 sequence, we have demonstrated that cyclophilin B is constitutively expressed but does not interact with EMMPRIN in SKG-II cells (data not shown).…”

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

“…Assignments of NS5A-D2 backbone resonances were achieved using two-dimensional 1 Assignments of the CypB spectrum were taken from our previous study (43 15 N, 13 C-labeled protein component, whereas the HNCO spectrum with modified phases to select for the non-13 C-labeled 15 N nuclei (which we will further call the HN(noCO) spectrum (47) Cyclophilin PPIase Activity toward NS5A-D2-PPIase activity of CypA and CypB on NS5A-D2 were assessed using EXSY spectra, whereby the exchange was monitored on the proton resonance (in homonuclear 1 H, 1 H spectra (48) (49)). The ratio between the cis and trans populations for a given residue (p c and p t , respectively) was measured on the basis of a 1 H, 15 N HSQC spectrum in the absence of any cyclophilin assuming that an exchange peak for this residue was observed.…”

“…Expression and Purification of Nonlabeled and 15 N, 13 C-Labeled Cyclophilin B-Production and purification of recombinant human cyclophilin B in E. coli were done as described previously (43). Briefly, the pET15b-CypB plasmid was introduced into E. coli BL21(DE3) strain, recombinant bacteria were grown in LB medium ( NMR Data Collection and Assignments-Spectra were acquired on either a Bruker Avance 600 MHz equipped with a cryogenic triple resonance probe head or a Bruker Avance 800 MHz with a standard triple resonance probe (Bruker, Karlsruhe, Germany).…”