Solution Structural And Functional Relationship Studies Of CyPA-CD147，Cu²⁺-CD147

CD147belongs to the immunoglobulin superfamily. It plays an important role intumor cell invasion and metastasis, rheumatoid disease, viruse infections, lymphocytemigration and activation, Alzheimer’s disease, falciparum infections, and has becomea new drug target molecule for cancer and other major diseases. As a singletransmembrane protein, the multifunction of CD147depends on its extracellular domaininteracting with various extracellular and membrane targets. In order to distinguish thefeatures of these different targets of CD147in pathological processes and design thespecific drugs in different diseases, it is critical to find the target and understand the structural and functional relationship between the target and CD147.It has been well identified that CyPA is the ligand of CD147. The interaction betweenCyPA and CD147plays important roles in rheumatoid arthritis, atherosclerosis, asthmaand other inflammatory diseases. So far, the structural mechanism of CyPA/CD147interaction is still unclear. Also, the receptor or ligand of CD147in the classicCD147-induced MMP secretion, promoting tumor invasion and metastasis function is stillunknown，too. Here, by using nuclear magnetic resonance（NMR）technology combinedwith cellular and molecular biology research tools, we revealed the structural andfunctional relationship of CD147and its interacting molecules in inflammation and cancerprogression. The study is composed of the following two parts.Part1Studies on the relationship between structure and function of CD147andCyPA interaction inducing lymphocytes chemotaxis.Numerous studies show that CyPA/CD147interaction induces lymphocytechemotaxis and contributed to the pathogenesis of inflammation diseases. CyPA is apeptidyl-prolylcis-transisomerase （PPIase） and people believe that CyPA/CD147interaction is a relationship between enzyme and substrate. However, whether CyPAinteracts directly on CD147extracellular domain is disputable. In this study, byGST-Pulldown and NMR titration experiments, we reported that the ectodomain of CD147（CD147ECT） can directly interact with CyPA, and the CD147ECTbinding site on CyPAoverlaped with the PPIase active site. Mutantion of CyPA PPIase activity center （keyresidues R55to Ala） did not affect the binding of CD147ECTwith CyPA, and theCyPAR55Amutant displayed similar chemotactic ability to CyPA. These results indicatedthat the PPIase function of CyPA was not critical for its chemotactic activity mediated byCD147. To further confirm this finding, we mutanted R69, H70and T107to Ala, whichwere not affect the PPIase activity of CyPA. The binding ability of the mutants withCD147ECTwas decreased by70%，46%，55%, respectively, compared to wild type CyPA.The chemotactic ability of the mutants was also decreased by67%，57%，62%,respectively. It was noticed that mutants with reduced binding capacity and decreased chemotactic ability were positively correlated. In summary, our study first reported thatCyPA induced leukocyte chemotaxis through a direct binding with the ectodomain ofCD147and independent of its PPIase activity. Furthermore, due to its non-relationshipwith PPIase activity, but also vital for CyPA chemotactic function, residues R69, H70andT107can be used as specific drug targets for inflammatory diseases.Part2Studies on the relationship between structure and function of CD147andextracellular copper ions promoting tumor invasion and metastasis.Recently numerous studies indicate that both serum and tumor copper levels in cancerpatients are significantly elevated than healthy subjects. Copper chelators can effectivelyinhibit tumor invasion and metastasis. However, the molecular mechanism of copper intumor invasion and metastasis remains unknown. In this study, we found that extracellularCu2+can active FAK, AKT, ERK1/2signaling pathway, up-regulate MMP2and MMP14expression, thus promoting tumor invasion and metastasis on CD147highly expressedtumor cells. These functions by Cu2+were significantly inhibited when CD147wasinterfered or knockout, demonstrating that extracellular Cu2+can promote tumor invasionand metastasis via membrane CD147. By NMR, chemical cross-linking, dynamic lightscattering, surface plasmon resonance（SPR） methods we proved that Cu2+bound onCD147ECTand mediated CD147ECTself-association. To further confirm this result，wemutanted H115，H170，H205to Ala which located on Cu2+/CD147ECTinterface selected byNMR titration experiment. Chemical cross-linking and SPR expriments showed thatCD1473HAmutant almost lost its self-association ability mediated by Cu2+. The capacity ofCu2+promoting tumor invasion and metastasis become invalid in CD1473HAexpressedtumor cells. It further suggested that extracellular Cu2+mediated CD147ECTself-association to promote tumor invasion and metastasis.In summary, we first reported that the PPIase activity-independent interactionbetween CyPA and CD147ECTwas responsible for the leukocyte chemotaxis; the elevatedcopper levels in tumor tissue mediated membrane CD147self-association and promotedtumor invasion and metastasis. The CD147/CyPA, Cu2+/CD147interface determined by NMR provided important theoretical basis for the development of novel anti-tumor,anti-inflammatory drugs.