The Interaction Between The Extracellular Domain Of PLA2R And PAHs In Vitro

Idiopathic membranous nephropathy（IMN）is an autoimmune disease.The main molecular marker for diagnosis of IMN is serum autoantibodies（anti-PLA2R）against M-type phospholipase A2 receptor（PLA2R）,which Exists in more than 80% of IMN patients,and does not exist in healthy people.Anti-PLA2R combines with PLA2R on the surface of glomerular podocytes to form an in situ immune complexes,activates the complement lectin pathway,produces C5b-9 membrane attack complex,attacks glomerular podocytes,destroys podocyte structure,and damages the kidney.The ball filters through the barrier,eventually leading to IMN.Studies have found that the incidence of IMN is significantly related to the concentration of PM2.5 in the atmosphere.Researchers have been committed to clarifying the role of the body’s immune system in the pathogenesis of IMN,and have not established the relationship between environmental pollution and the pathogenesis of IMN.Polycyclic Aromatic Hydrocarbons（PAHs）,as an important organic component of PM2.5,have toxic effects on the kidneys.PAHs are easy to accumulate in the kidney,especially in the basement membrane of podocytes.Therefore,PLA2R is a potential toxic target molecule of PAHs.To explore the interaction between PAHs and PLA2R,this thesis constructed a prokaryotic expression system of human-derived PLA2R extracellular domain CTLD1,screened and characterized PAHs interacting with CTLD1 protein,and successfully crystallized CTLD1.The work of this thesis provides an important scientific basis for revealing and cognizing the pathogenesis of INM induced by PM2.5,especially PAHs.In the thesis,the human PLA2R（Ncbi:NM₀07366.5）gene was codon-optimized and linked to the pET40 b vector;the recombinant plasmid with the target gene was transformed into the expression host Shuffle T7 and induced at low temperature to obtain the soluble protein DsbC-8His-CTLD1;DsbC-8His-CTLD1 fusion protein with a purity of 95% was obtained by nickel column affinity chromatography and gel filtration chromatography;the DsbC fusion tag was removed with human enterokinase hEK-6His and further purified to obtain CTLD1 with a purity of more than 95%;The actual molecular weight of CTLD1 measured by MALDI-TOF MS is 17199 Da,which is consistent with the theoretical value.At the same time,the pET28aCTLD1-6His prokaryotic expression vector was constructed and subjected to the same induction conditions to obtain soluble CTLD1-6His;after chromatographic purification and mass spectrometry characterization,CTLD1-6His with a purity of more than 95% was obtained.Using circular dichroism（CD）analysis of CTLD1 and CTLD1-6His,the CD spectra of the two are consistent,indicating that CTLD1 can still be soluble in E.coli in its natural conformation without the presence of DsbC（disulfide-promoting isomerase）expression.The temperature-variable circular dichroism analysis of CTLD1 was performed,and the Tm was determined to be 52℃.In total,six kinds of PAHs that interact with CTLDlwere screened using the isothermal titration calorimetry experiment CTLD1:among them,benzo[a]anthracene,benzo[a]pyrene,benzo[b]fluoranthene and pyrene can combine with CTLD1,the binding ability from strong to weak is benzo[a]anthracene> benzo[a]pyrene>benzo[b]fluoranthene> pyrene.Further using CD analysis to co-incubate CTLD1 and PAHs solution found that the combination of benzo[a]anthracene,benzo[a]pyrene,pyrene and CTLD1 affected the secondary structure of CTLD1.Finally,in order to analyze the X-ray crystal structure of CLTD1,the CLTD1 crystal culture conditions were screened and determined as follows:CLTD1 protein concentration 11 mg/mL,25℃ 0.075 M Na₂HPO₄,0.075 M K₂HPO₄,0.075 M MES pH=6.5.In summary,the thesis has realized the high-efficiency expression of some extracellular domains of PLA2R under the prokaryotic system.The PAHs interacting with CTLD1-6His protein were screened and characterized by isothermal titration calorimetry technology,and CTLD1-6His protein crystals were obtained at the same time.