Dataset Information

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein.

ABSTRACT: BACKGROUND: Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. METHODS: 4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. RESULTS: Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. CONCLUSION: The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.

SUBMITTER: Lanzillotti R

PROVIDER: S-EPMC1635724 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein.

Lanzillotti Roberto R Coetzer Theresa L TL

Malaria journal 20061106

<h4>Background</h4>Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise mole ...[more]

PMID: 17087826

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Project description:BackgroundPlasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms and dimorphism have prevented to development of effective vaccines based on this gene. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko Island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175.MethodsThe allelic dimorphism of PfEBA-175 region II of 297 bloods samples from Equatorial Guinea in 2018 and 2019 were investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program.ResultsBoth Bioko Island and Bata district populations, the frequency of the F-fragment was higher than that of the C-fragment of PfEBA-175 gene. The PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and - 0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (FST > 0.15, P < 0.05). A total of 310 global isolates clustered in 92 haplotypes, and only one cluster contained isolates from three continents. The mutations A34T, K109E, D278Y, K301N, L305V and D329N were predicted as probably damaging.ConclusionsThis study demonstrated that the dimorphism of F-fragment PfEBA-175 was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar another region isolates. And the levels of recombination events suggested that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen.

Project description:UnlabelledErythrocyte invasion is an essential step in the pathogenesis of malaria. The erythrocyte binding-like (EBL) family of Plasmodium falciparum proteins recognizes glycophorins (Gp) on erythrocytes and plays a critical role in attachment during invasion. However, the molecular basis for specific receptor recognition by each parasite ligand has remained elusive, as is the case with the ligand/receptor pair P. falciparum EBA-175 (PfEBA-175)/GpA. This is due largely to difficulties in producing properly glycosylated and functional receptors. Here, we developed an expression system to produce recombinant glycosylated and functional GpA, as well as mutations and truncations. We identified the essential binding region and determinants for PfEBA-175 engagement, demonstrated that these determinants are required for the inhibition of parasite growth, and identified the glycans important in mediating the PfEBA-175-GpA interaction. The results suggest that PfEBA-175 engages multiple glycans of GpA encoded by exon 3 and that the presentation of glycans is likely required for high-avidity binding. The absence of exon 3 in GpB and GpE due to a splice site mutation confers specific recognition of GpA by PfEBA-175. We speculate that GpB and GpE may have arisen due to selective pressure to lose the PfEBA-175 binding site in GpA. The expression system described here has wider application for examining other EBL members important in parasite invasion, as well as additional pathogens that recognize glycophorins. The ability to define critical binding determinants in receptor-ligand interactions, as well as a system to genetically manipulate glycosylated receptors, opens new avenues for the design of interventions that disrupt parasite invasion.ImportancePlasmodium falciparum uses distinct ligands that bind host cell receptors for invasion of red blood cells (RBCs) during malaria infection. A key entry pathway involves P. falciparum EBA-175 (PfEBA-175) recognizing glycophorin A (GpA) on RBCs. Despite knowledge of this protein-protein interaction, the complete mechanism for specific receptor engagement is not known. PfEBA-175 recognizes GpA but is unable to engage the related RBC receptor GpB or GpE. Understanding the necessary elements that enable PfEBA-175 to specifically recognize GpA is critical in developing specific and potent inhibitors of PfEBA-175 that disrupt host cell invasion and aid in malaria control. Here, we describe a novel system to produce and manipulate the host receptor GpA. Using this system, we probed the elements in GpA necessary for engagement and thus for host cell invasion. These studies have important implications for understanding how ligands and receptors interact and for the future development of malaria interventions.

Project description:BackgroundDuring erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.MethodsrPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.ResultsrPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.ConclusionStudies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Dataset Information

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein.

Publications

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein.

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