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.
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.
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 Rosettagami 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.
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