Baculovirus-expressed Plasmodium reichenowi EBA-140 merozoite ligand is host specific.

Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained. Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway. A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction. In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart, P. falciparum.

Studies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines.

The baculovirus-expressed binding region of Plasmodium falciparum EBA-140 ligand and its glycophorin C binding specificity.

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36-63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.

Dynamic interplay of ParA with the polarity protein, Scy, coordinates the growth with chromosome segregation in Streptomyces coelicolor.

Prior to bacterial cell division, the ATP-dependent polymerization of the cytoskeletal protein, ParA, positions the newly replicated origin-proximal region of the chromosome by interacting with ParB complexes assembled on parS sites located close to the origin. During the formation of unigenomic spores from multi-genomic aerial hyphae compartments of Streptomyces coelicolor, ParA is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregation of tens of chromosomes into prespore compartments. Here, we show that in addition to being a segregation protein, ParA also interacts with the polarity protein, Scy, which is a component of the tip-organizing centre that controls tip growth. Scy recruits ParA to the hyphal tips and regulates ParA polymerization. These results are supported by the phenotype of a strain with a mutant form of ParA that uncouples ParA polymerization from Scy. We suggest that the ParA-Scy interaction coordinates the transition from hyphal elongation to sporulation.

[Human erythrocyte glycophorin C as the receptor for EBA-140 Plasmodium falciparum merozoite ligand]. / Glikoforyna C erytrocytów ludzkich jako receptor dla liganda EBA-140 merozoitów Plasmodium falciparum.

Erythrocyte invasion by the blood-stage Plasmodium falciparum parasites is a multistep process involving specific interactions between parasites and red blood cells. Several proteins are involved in this process, including EBL ligands. The structure of the EBA-140 ligand, a member of the EBL protein family, provides a full description of its molecular interactions with the erythrocyte receptor. The crystal structure of the EBA-140 Region II in a complex with sialolactose revealed that the binding region is monomeric. Two glycan binding pockets, one in each F1 or F2 domain, were identified. Stark differences in the receptor binding for the F1 and F2 domains suggests that each domain performs a distinct function. Although both domains are required for effective glycan binding, it seems that the interaction may be mediated solely by the F1 domain. The structure of the binding region and the interaction with glycan are unique to the EBA-140 ligand and not shared by other EBL ligands. The EBA-140 ligand binds specifically to human erythrocytes through the membrane sialoglycoprotein glycophorin C. The receptor site for the EBA-140 ligand was suggested to be a cluster of N-and O-linked sialylated glycans on the GPC molecule, whose conformation is dependent on the polypeptide chain region composed of amino acid residues 36-63. Precise definition of the binding site for the EBA-140 ligand on glycophorin C may be important with respect to human erythrocyte invasion inhibition strategies based on a receptor.

Bacterially expressed truncated F2 domain of Plasmodium falciparum EBA-140 antigen can bind to human erythrocytes.

The recently identified erythrocyte binding antigen-140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 antigen. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte interaction during invasion. It was shown that the F2 domain of EBA-175 antigen seems to be more important for erythrocyte binding. In order to study activity and immunogenicity of EBA-140 antigen F2 domain, it is necessary to obtain recombinant protein of high purity and in a sufficient amount, which used to pose a challenge due to the high content of disulphide bridges. Here, we present a new method for expression and purification of Plasmodium falciparum EBA-140 antigen F2 domain in E. coli Rosetta-gami strain in fusion with the maltose binding protein (MBP). The truncated F2 domain formed by spontaneous proteolytic degradation of the fusion protein was purified by affinity chromatography on Ni-NTA resin followed by size exclusion chromatography. Molecular mass of this protein was confirmed by mass spectrometry. Its N-terminal amino acid sequencing revealed a proteolytic cleavage site within the F2 domain. The proper folding of the recombinant, truncated F2 domain of EBA-140 antigen was confirmed by circular dichroism analysis. The truncated F2 domain can specifically bind to human erythrocytes but its binding is not as efficient as that of full Region II. This confirms that both the F1 and F2 domains of EBA-140 antigen are required for effective erythrocyte binding.

[Proteins involved in invasion of human red blood cells by malaria parasites]. / Bialka biorace udzial w procesie inwazji erytrocytów ludzkich przez zarodzce wywolujace malarie.

Malaria is a disease caused by parasites of Plasmodium species. It is responsible for around 1-2 million deaths annually, mainly children under the age of 5. It occurs mainly in tropical and subtropical areas. Malaria is caused by five Plasmodium species: P. falciparum, P. malariae, P. vivax, P. knowlesi and P. ovale. Mosquitoes spread the disease by biting humans. The malaria parasite has two stages of development: the human stage and the mosquito stage. The first stage occurs in the human body and is divided into two phases: the liver phase and the blood phase. The invasion of erythrocytes by Plasmodium merozoites is a multistep process of specific protein interactions between the parasite and red blood cell. The first step is the reversible merozoite attachment to the erythrocyte followed by its apical reorientation, then formation of an irreversible "tight" junction and finally entry into the red cell in a parasitophorous vacuole. The blood phase is supported by a number of proteins produced by the parasite. The merozoite surface GPI-anchored proteins (MSP-1, 2, 4, 5, 8 and 10) assist in the process of recognition of susceptible erythrocytes, apical membrane antigen (AMA-1) may be directly responsible for apical reorientation of the merozoite and apical proteins which function in tight junction formation. These ligands are members of two families: Duffy binding-like (DBL) and reticulocyte binding-like (RBL) proteins. In Plasmodium falciparum the DBL family includes: EBA-175, EBA-140 (BAEBL), EBA-181 (JESEBL), EBA-165 (PEBL) and EBL-1 ligands. To date, no effective antimalarial vaccine has been developed, but there are several studies for this purpose. Therefore, it is crucial to understand the molecular basis of host cells invasion by parasites. Major efforts are focused on developing a multiantigenic and multiepitope vaccine preventing all steps of Plasmodium invasion.