Function Analysis Of The Duffy-binding-like Domain α Of Plasmodium Falciparum Erythrocyte Membrane Protein 1

Severe malaria is an important parasitic disease caused by Plasmodium falciparum and epidemic in tropical and subtropical regions. The parasite infected red blood cells adhere to host endothelium (cytoadherence) and to non-infected erythrocytes or infected erythrocytes (Rosetting/autoagglutination) lead to the sequestration of the microvasculature in various tissues and organs. It has been considered as the main reasons of severe malaria .The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates Rosetting,a virulent phenotype associated with the occurrence of severe malaria. Heparan sulfate-like glycosaminoglycans, blood group antigens A and complement receptor 1 (CR1) on human erythrocyte surface have been characterised as Rosetting receptors. An analysis of the binding properties of the different regions of PfEMP1 provides evidence that the DBLαdomain is the predominant ligand involved in heparan sulfate-like GAGs and ABO blood group antigens binding. However, it is not clear that which DBLαdomains are responsible for the specific affinity to heparin sulfate or its analogs It is also unknown whether the recombinant DBLαprotein which has been characterized as the ligand for the binding of PfEMP1 to blood group antigen A could also bind to blood group antigen B. The purpose of this study is screening the strongest affinity with the heparin or heparan sulfate sequence in the structure of PfEMP1-DBLα, and identification of recombinant DBLαprotein whether have specific affinity to blood group antigen B. It is the purpose of this study.Firstly, the sequence of PfEMP1(IT4-var60)DBLαwere optimized according to the characteristics of E. coli codon and synthesized. Secondly, the sequence of PfEMP1 (FCR3S1.2-var1) DBLαoptimized district was cloned. In accordance with the optimized PfEMP1 (FCR3S1.2-var1) DBLαgene designed specific primers and then divided it into three fragments by PCR. The recombinant plasmid were constructed and transformed into E.coli BL21-codonplus, and the cells were culture and induced by IPTG. Five soluble recombinant protein were expressed in E.coli and then purified from bacterial lysates using Gluthathione-Sepharose 4B. Heparin affinity test and GAG inhibition test were used to identify the amino acid sequence or motifs in PfEMP1 (FCR3S1.2-var1) DBLαwhich are responsible for the specific affinity to heparin or heparin sulfate. The recombinant protein was mixed with Heparin-Sepharose and incubated for 5 min at room temperature. GST protein was used as negative control. The Heparin-Sepharose binding protein wad wash with PBST buffer before running for 10% SDS-PAGE. The results showed that five kinds of recombinant proteins can binded to the Heparin-Sepharose,while GST control not. Further study confirmed that GAG concentrations-dependently inhibit the binding of recombinant protein to Heparin-Sepharose. GAG inhibition test results showed that the 285-415 amino acid fragment in PfEMP1(FCR3S1.2-var1)DBLαhave the strongest affinity to Heparin. Base on the molecular basis of heparin-binding with PfEMP1,which is contained a number of similer structure such as XBBXXBX or XBBBXXBX(B represent basic amino acid,such as lysine/ arginine/ histidine, x represent any amino acid)in its DBLαdistrict, the three motifs C R K K K K K L E N L E K Q,S R K G K V R M,D K Q K K Y of PfEMP1(FCR3S1.2-var1)may ply key role in binding of PfEMP1(FCR3S1.2-var1) DBLαto Plasmodium falciparum infected red blood cells .In order to identify if recombinant DBLαprotein have specific affinity with blood group antigen B. The recombinant protein was mixed with A(B)-tri-APE-gel incubated it for 1h at 4℃.GST protein was used as a negative control. The A(B)-tri-APE-gel binding protein was washed in PBST buffer before running for 10% SDS-PAGE. The results showed that the recombination PfEMP1 (IT4-var60) DBLαprotein and recombination PfEMP1 (FCR3S1.2-var1) DBLαprotein can specificly binded blood group antigen A/B, which confirmed the blood group antigen A/B as one of the receptors of Rosetting. The results provided experimental evidence for revealing the molecular pathogenesis of Plasmodium falciparum.