Cysteine-rich Interdomain Region Domains Research Articles

Expression of Single-Domain Soluble and Disulfide-Folded PfEMP1 Antigens in the Escherichia coli SHuffle Expression System.

The genome of Plasmodium falciparum has an A/T content of around 81%. This, together with a high cysteine content and the high molecular weight of several proteins, make the expression of recombinant parasite proteins in heterologous systems challenging. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of proteins composed of several Duffy-binding like (DBL) and cysteine-rich inter-domain region (CIDR) domains involved in cytoadhesion to human host receptors and development of severe malaria. Expression of correctly folded single- and multiple-domain PfEMP1 fragment regions containing cysteines forming disulfide bonds, remains particularly difficult. Nevertheless, expression of single DBL and CIDR domains has been successful and this protocol describes the expression and purification of single-domain soluble PfEMP1 fragments using the Escherichia coli SHuffle expression system.

Genetic diversity and distribution patterns of PfEMP1 in Plasmodium falciparum isolates along the Thai-Myanmar border.

Duffy binding-like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encode malaria virulence proteins. The variants of these genes have been reported to be associated with severe/complicated malaria. The present study investigated the prevalence and distribution patterns of DBLα0.6/9, DBLα1.1, DBLα1 not var3 genes, DBLα2/α1.1/2/4/7, DBLβ12 & DBLβ3/5, DBLε8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between PfEMP1 variants and parasite density was also investigated. Two hundred and thirteen finger-prick dried blood spot (DBS) or whole blood samples were collected in 2007 and 2015, from patients with acute uncomplicated P. falciparum in Tak, Kanchanaburi, and Ranong provinces. Analysis of the variant genes was performed using polymerase chain reaction (PCR). The DBLs variant which was found at the highest and lowest frequencies in the three provinces were DBLα1 not var3 (72.77%), and DBLε8 (17.37%). The two CIDR domain variants were found at relatively lower frequencies compared with DBL domain variants (9.9% and 30.1%). P. falciparum isolates carrying the four PfEMP1 variants, i.e., DBLα0.6/9, DBLα1.1, DBLα2/α.1.1/2/4/7, and DBLε8 were found to be significantly associated with low parasitemia. Both DBLα0.6/9 and DBLα2/α1.1/2/4/7 variant genes which were present at high frequencies in this border area could be potential candidate markers for predicting P. falciparum hyperparasitemia and in this border area. Furthermore, the information could be exploited as candidate proteins for the development of an effective malaria vaccine in specific malaria-endemic areas.

Dynamics of PfEMP1 Antibody Profile From Birth to 12 Months of Age in Beninese Infants.

Plasmodium falciparum-infected erythrocytes bind to specific endothelial cell receptors via members of the PfEMP1 family exported onto the erythrocyte surface. These interactions are mediated by different types of cysteine-rich interdomain region (CIDR) domains found in the N-terminal region of all PfEMP1. CIDRα1 domains bind endothelial protein C receptor (EPCR), CIDRα2-6 domains bind CD36, whereas the receptor specificity of CIDRβ/γ/δ domains is unknown. In this study, we investigated the level of immunoglobulin (Ig)G targeting the different types of PfEMP1 CIDR during the first year of life. We used plasma collected longitudinally from children of pregnant women who had been followed closely through pregnancy. Antibodies to CIDRα1 domains were more frequent in cord blood compared with antibodies to CIDRα2-6 domains. Higher IgG levels to EPCR-binding CIDRα1 variants positively correlated with the timing of first infections. Antibodies to all PfEMP1 types declined at similar rates to the point of disappearance over the first 6 months of life. At 12 months, children had acquired antibody to all types of CIDR domains, mostly in children with documented P falciparum infections. These observations agree with the notion that the timing and phenotype of first P falciparum infections in life are influenced by the immune status of the mother.

Acquisition of Antibodies Against Endothelial Protein C Receptor–Binding Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 in Children with Severe Malaria

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration in postcapillary venules in P. falciparum malaria. PfEMP1 types can be classified based on their cysteine-rich interdomain region (CIDR) domains. Antibodies to different PfEMP1 types develop gradually after repeated infections as children age, and antibodies to specific CIDR types may confer protection. Levels of immunoglobulin G to 35 recombinant CIDR domains were measured by means of Luminex assay in acute-stage (baseline) and convalescent-stage plasma samples from Papua New Guinean children with severe or uncomplicated malaria and in healthy age-matched community controls. At baseline, antibody levels were similar across the 3 groups. After infection, children with severe malaria had higher antibody levels than those with uncomplicated malaria against the endothelial protein C receptor (EPCR) binding CIDRα1 domains, and this difference was largely confined to older children. Antibodies to EPCR-binding domains increased from presentation to follow-up in severe malaria, but not in uncomplicated malaria. The acquisition of antibodies against EPCR-binding CIDRα1 domains of PfEMP1 after a severe malaria episode suggest that EPCR-binding PfEMP1 may have a role in the pathogenesis of severe malaria in Papua New Guinea.

Comprehensive analysis of antibody responses to Plasmodium falciparum erythrocyte membrane protein 1 domains

Acquired antibodies directed towards antigens expressed on the surface of merozoites and infected erythrocytes play an important role in protective immunity to Plasmodium falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major parasite component of the infected erythrocyte surface, has been implicated in malaria pathology, parasite sequestration and host immune evasion. However, the extent to which unique PfEMP1 domains interact with host immune response remains largely unknown. In this study, we sought to comprehensively understand the naturally acquired antibody responses targeting different Duffy binding-like (DBL), and Cysteine-rich interdomain region (CIDR) domains in a Ugandan cohort. Consequently, we created a protein library consisting of full-length DBL (n = 163) and CIDR (n = 108) domains derived from 62-var genes based on 3D7 genome. The proteins were expressed by a wheat germ cell-free system; a system that yields plasmodial proteins that are comparatively soluble, intact, biologically active and immunoreactive to human sera. Our findings suggest that all PfEMP1 DBL and CIDR domains, regardless of PfEMP1 group, are targets of naturally acquired immunity. The breadth of the immune response expands with children’s age. We concurrently identified 10 DBL and 8 CIDR domains whose antibody responses were associated with reduced risk to symptomatic malaria in the Ugandan children cohort. This study highlights that only a restricted set of specific domains are essential for eliciting naturally acquired protective immunity in malaria. In light of current data, tandem domains in PfEMP1s PF3D7_0700100 and PF3D7_0425800 (DC4) are recommended for extensive evaluation in larger population cohorts to further assess their potential as alternative targets for malaria vaccine development.

Plasmodium falciparum var genes expressed in children with severe malaria encode CIDRα1 domains.

Most severe Plasmodium falciparum infections are experienced by young children. Severe symptoms are precipitated by vascular sequestration of parasites expressing a particular subset of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion molecules. Parasites binding human endothelial protein C receptor (EPCR) through the CIDRα1 domain of certain PfEMP1 were recently associated with severe malaria in children. However, it has remained unclear to which extend the EPCR‐binding CIDRα1 domains epitomize PfEMP1 expressed in severe malaria. Here, we characterized the near full‐length transcripts dominating the var transcriptome in children with severe malaria and found that the only common feature of the encoded PfEMP1 was CIDRα1 domains. Such genes were highly and dominantly expressed in both children with severe malarial anaemia and cerebral malaria. These observations support the hypothesis that the CIDRα1‐EPCR interaction is key to the pathogenesis of severe malaria and strengthen the rationale for pursuing a vaccine or adjunctive treatment aiming at inhibiting or reducing the damaging effects of this interaction.

IgG Antibodies to Endothelial Protein C Receptor-Binding Cysteine-Rich Interdomain Region Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 Are Acquired Early in Life in Individuals Exposed to Malaria

Severe malaria syndromes are precipitated by Plasmodium falciparum parasites binding to endothelial receptors on the vascular lining. This binding is mediated by members of the highly variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. We have previously identified a subset of PfEMP1 proteins associated with severe malaria and found that the receptor for these PfEMP1 variants is endothelial protein C receptor (EPCR). The binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDR) of the subtypes α1.1 and α1.4 to α1.8. In this study, we investigated the acquisition of anti-CIDR antibodies using plasma samples collected in four study villages with different malaria transmission intensities in northeastern Tanzania during a period with a decline in malaria transmission. We show that individuals exposed to high levels of malaria transmission acquire antibodies to EPCR-binding CIDR domains early in life and that these antibodies are acquired more rapidly than antibodies to other CIDR domains. The rate by which antibodies to EPCR-binding CIDR domains are acquired in populations in areas where malaria is endemic is determined by the malaria transmission intensity, and on a population level, the antibodies are rapidly lost if transmission is interrupted. This indicates that sustained exposure is required to maintain the production of the antibodies.

Structural and Immunological Correlations between the Variable Blocks of the VAR2CSA Domain DBL6ε from Two Plasmodium falciparum Parasite Lines

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a family of adhesins of the falciparum species of the malaria parasite, is exposed on the surface of the infected erythrocyte. In general, only one PfEMP1 variant is expressed at a time but switching between variants occurs, changing both host-cell receptor specificity and serotype. The PfEMP1 variant VAR2CSA causes sequestration of infected erythrocytes in the intervillous spaces of the placenta via the glycosaminoglycan chondroitin sulfate A. This leads to pregnancy-associated malaria, which has severe consequences for the fetus and mother. The extracellular region of VAR2CSA comprises six DBL (Duffy-binding-like) domains and a single CIDR (cysteine-rich inter-domain region) domain. The C-terminal domain DBL6ε, the most polymorphic domain of VAR2CSA, has seven regions of high variability termed variable blocks (VBs). Here we have determined the crystal structure of DBL6ε from the FCR3 parasite line and have compared it with the previously determined structure of that from the 3D7 line. We found significant differences particularly in the N-terminal region, which contains the first VB (VB1). Although DBL6ε is the most variable VAR2CSA domain, DBL6ε-FCR3 and DBL6ε-3D7 react with IgG purified from immune sera of pregnant women. Furthermore, IgG purified on one domain cross-reacts with the other, confirming the presence of cross-reactive epitopes. We also examined reactivity of immune sera to the four least variable VB (VB1, VB2, VB4 and VB5) using peptides with the consensus sequence closest, in turn, to the FCR3 or 3D7 domain. These results provide new molecular insights into immune escape by parasites expressing the VAR2CSA variant.

The Cysteine-Rich Interdomain Region from the Highly Variable Plasmodium falciparum Erythrocyte Membrane Protein-1 Exhibits a Conserved Structure

Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1α domains bind CD36. Here we describe the CD36-binding portion of a CIDR1α domain, MC179, as a bundle of three α-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1α three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1α and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1α domains are approximately 100 residues larger and that CIDR1α domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the molecular basis of cytoadherence.

Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BackgroundCellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally.MethodsA computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli.ResultsSolubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1α (46.8%) > CIDR2β (42.9%) > DBL2-4γ (31.7%) > DBL2β + C2 (30.6%) > DBL1α (24.9%) > DBL2-7ε (23.1%) > DBL2-5δ (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions.ConclusionThe acidic terminal segment (ATS) and CIDR α/β domain types are suitable for recombinant expression in E. coli while all DBL subtypes (α, β, γ, δ, ε) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.

Classification of adhesive domains in the Plasmodium falciparum Erythrocyte Membrane Protein 1 family

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (α, β, and γ) and DBL domains as five sequence classes (α, β, γ, δ, and ε). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations — such as DBLαCIDRα, DBLδCIDRβ, and DBLβC2 — are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.

Plasmodium falciparum: Cloned and Expressed CIDR Domains of PfEMP1 Bind to Chondroitin Sulfate A

Degen, R., Weiss, N., and Beck, H.-P. 2000. Plasmodium falciparum: Cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A. Experimental Parasitology95, 113–121. Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.