Molecular Basis Of The Interaction Between Human Anti-PHD2 Single-chain Variable Fragment And Antigen

Prolyl Hydroxylase 2(PHD2)is a crucial regulator of the stability and activity of hypoxia-inducible factor-1(HIF-1).It dominantly hydroxylates two highly conserved proline residues of HIF-1α to promote its degradation.HIF-1α participates in a variety of physiological processes.The up-regulation of its protein level promotes the angiogenesis and the tissue repair after acute injury.Therefore,PHD2 has become an important target for the repair of liver tissue damage and angiogenesis.Since the PHD2 inhibitors currently used produce a large number of toxic side effects,it is imperative to obtain noval inhibitors that specifically inhibit PHD2 activity.In the previous work of our laboratory,we have obtained a human single-chain variable fragment(sc Fv)that specifically binds to PHD2 and inhibits its function both in vivo and in vitro through screening of phage antibody libraries.Anti-PHD2 intracellular antibodies were prepared with anti-PHD2 sc Fv genes as templates.We found that anti-PHD2 intracellular antibodies can inhibit the activity of PHD2 prolyl hydroxylase,and has pro-angiogenic activity and protective effect on liver injury.However,the molecular basis of the recognition between anti-PHD2 sc Fv and its antigen is still unclear.Epitope is very important for the recognition of antigen and antibody.To analyze the interaction mode of antigen and antibody,it is necessary to clarify the epitope recognized by the antibody.In this study,epitope-prediction tools was used to predict the B cell epitope of PHD2,homology modeling and molecular docking were used to simulate the interaction mode.ELISA combined with mutation assays were conducted to verify the prediction and simulation.The results of the study are as follows:1.SEPPA-3.0 epitope prediction tool was used to predict the epitopes of recombinant human PHD2(rh PHD2),and 25 groups of sites that may participate in the formation of rh PHD2 epitopes were obtained.Based on the results of SEPPA epitope prediction,one rh PHD2 truncation mutant and three rh PHD2 deletion mutants were designed.2.Prokaryotic expression vector of rh PHD2 Δ130-209 mutant was successfully constructed,and soluble recombinant human PHD2 truncated mutant rh PHD2 Δ130-209 protein was expressed and purified.ELISA results showed that anti-PHD2 sc Fv has good binding activity to rh PHD2 Δ130-209.This indicates that the deletion of amino acids 130-209 of recombinant human PHD2 protein does not affect the interaction between antigen and antibody,and the epitopes of interaction between PHD2 and antiPHD2 sc Fv are concentrated in amino acids 1-129.3.rh PHD2 deletion mutants were successfully constructed.Three PHD2 deletion mutants rh PHD2 Δ42-49,rh PHD2 Δ54-58 and rh PHD2 Δ65-74 were solublely expressed and purified.It was found that the mutants with amino acid deletions at amino acids 65-74 lost the ability to bind to anti-PHD2 sc Fv according to ELISA results.This indicates that residues 65-74 of the recombinant human PHD2 protein are the key components of the epitope recognized by anti-PHD2 sc Fv.4.Homology modeling and molecular docking were conducted to simulate the interaction between anti-PHD2 sc Fv and PHD2.Combined with recombinant human PHD2 site-directed mutation verification experiments,it is found that Gly2,Gly67,and Glu73 mutants of recombinant human PHD2 reduce the binding activity to anti-PHD2 sc Fv,and the mutation of Gly at position 67 makes it lose the binding activity with antiPHD2 sc Fv.Therefore,Gly 67 not only participates in the epitope formation of PHD2,but also play a vital role in the interaction between recombinant human PHD2 and antiPHD2 sc Fv.In summary,this study preliminaryly explored the interaction mode of anti-PHD2 sc Fv and PHD2,providing experimental evidence for elucidating the molecular basis of the interaction between anti-PHD2 sc Fv and PHD2,and also laid the foundation for the PHD2-targeted treatment of diseases.