Study On The Epitope Of CD147 Recognized By Monoclonal Antibody 6H8 And Anti-malaria Mechanism

Malaria is one of the most important infectious diseases that threaten global public health.In 2016,a total of 216 million malaria cases and 445,000 deaths occurred globally,and more than 90% death cases were children from tropical and subtropical regions.Malaria is caused by Plasmodium and transmitted by mosquito bite.Among the four types of malaria parasites that infect human,Plasmodium falciparum causes the most serious clinical symptoms.It is difficult to develop effective vaccine against malaria,due to the complex life cycle of Plasmodium falciparum,invasion-associated protein polymorphism and immune evasion.What's worse,with the spread of drug-resistant Plasmodium strains and insecticide-resistant mosquitoes,it becomes more challenging to control and prevent malaria.CD147 is a transmembrane glycoprotein which is widely expressed on the surface of red blood cells.Studies have demonstrated that CD147 plays a key role in the process of Plasmodium falciparum invasion into erythrocyte.The knockout of CD147 or antibodies targeting it could effectively inhibit the invasion process.6H8 is a mouse-derived monoclonal antibody that targets CD147 and its humanized form is named HP6H8.Experiments in vitro and in humanized mouse model showed that HP6H8 completely abolished the parasite invasion and showed both therapeutic and preventative effects.This CD147-oriented antimalarial strategy could avoid drug resistance of Plasmodium falciparum and possible side effects caused by chemical drugs,thus has good clinical prospect.My project intends to determine the three-dimensional structure of the CD147-6H8 complex using X-ray crystallography,so as to elucidate the functional mechanism of antimalarial effect of m Ab 6H8,and to facilitate the optimization of antibody or development of antimalarial peptidomimetics based on the structure.The study includes three parts:Part ?,Preparation of CD147-6H8 complex.Firstly,the codon-optimized CD147 extracellular portion and two Ig domains were expressed by prokaryotic expression system respectively,and then purified by Ni affinity chromatography,anion exchange chromatography and gel exclusion chromatography;After papain digestion of m Ab6H8,the Fab fragment was purified by cation exchange chromatography and gel exclusion chromatography;3D modeling of 6H8 Fv segment followed by epitope prediction combined with His pull-down experiment confirmed that 6H8 binds the domain1 of CD147;Finally,antigen-antibody complex with more than 95% purity and high homogeneity can be obtained by gel exclusion chromatography.Part ?,Crystallization and structure determination of antigen-antibody complex and free Fab of 6H8.Firstly,we screened and optimized the crystallization conditions of CD147-6H8 Fab,CD147D1-6H8 Fab,and free Fab fragment respectively.After the phase was solved by molecular replacement,we successfully determined the crystal structures of 6H8 Fab at 1.45? and 6H8 Fab in complex with CD147 D1 at 2.6?.The temperature factor and stereochemistry related parameters of both structural models were within reasonable range and could be used for fine structural analysis.Part ?,Crystal structure analysis and preliminary study on anti-malarial mechanism of 6H8.Structural analysis of the complex revealed that the epitope of 6H8 was mapped to 60Val-Val-Leu-Lys-Glu-Asp-Ala-Leu-Pro-Gly-Glu-Phe-Lys75 on CD147,which interacted with the binding pocket formed by CDRs by salt bridge,hydrogen bonds and non-hydrogen bond interactions;Affinity determination after mutation of CD147 and further analysis validated that the salt bridge formed by negatively charged AspH102 in 6H8 CDR-H3 and positively charged Lys63 on CD147 was critical for the high binding affinity;Furthermore,by comparing the structures of free Fab and Fab in complex with CD147D1,we found that the side chain of key amino acid residues involved in interaction showed obvious displacement,indicating the induced fit effect upon complex formation.Structural alignment and SPR affinity determination showed that 6H8 did not affect the interaction between CD147 and its key ligand Pf Rh5.Furthermore,our study showed that the synthetic epitope peptide of CD147 can inhibit the invasion of erythrocytes by Plasmodium falciparum in vitro,indicating that the invasion-inhibit effect of 6H8 was neither due to blocking the interaction between CD147 and Pf Rh5,nor to simple steric hindrance,but likely due to blocking the interaction of CD147 with other invasion-related proteins from Plasmodium falciparum.