Abstract
BackgroundIn several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion. In Plasmodium falciparum and Toxoplasma gondii, the MJ is composed of the Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck Proteins (RONs) complex; specifically, AMA1 interacts with RON2 during host cell invasion.MethodsRecombinant proteins based on Plasmodium vivax RON2 (A2033-P2100) and its synthetic peptide fragments, one cyclic and one linear, based on PvRON2 (D2035-T2074) were generated and used to evaluate the interaction with P. vivax AMA1 (PvAMA1) by the far western blot, surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) methods. The structural studies of peptides were performed by circular dichroism, and the structural analysis of the complex of PvAMA1 with peptides based on PvRON2 (D2035-T2074) was conducted with small-angle X-ray scattering (SAXS).ResultsSurface plasmon resonance (KD = 23.91 ± 2.078 μmol/L) and ITC (K = 3 × 105 mol/L) studies conclusively showed an interaction between the cyclic peptide based on PvRON2 and PvAMA1-His6. In contrast, the linear peptide and recombinant PvRON2 (GST fusion protein) did not show an interaction with PvAMA1. However, the interaction among recombinant proteins PvRON2.2 and PvAMA1-His6 was possible to show by far western blot.ConclusionsThe results show that the PvRON2 structure, particularly the S–S bond between C2051 and C2063, is determinant for the existence of the interaction between PvAMA1 and PvRON2.
Highlights
In several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion
Synthesis, cloning, bacterial expression and purification of PvRON2 recombinant proteins In the amino acid sequence of the P. vivax Rhoptry Neck Protein 2 (RON2) protein (PVX_117880) [23], which was obtained from the base pairs of the gene that encodes this protein, we focused the region between residues 2033 to 2100, which corresponds to the sequence of the fusion proteins GSTRON2.2 and GST-RON2.2 mut (C2051A and C2063A)
This conformational restriction, nonexistent in the linear peptide could help the cyclic peptide to adopt the correct conformation to interact with P. vivax AMA1 (PvAMA1) showed in the work of Vulliez Le Normand et al [15]
Summary
In several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion. In Plasmodium falciparum and Toxoplasma gondii, the MJ is composed of the Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck Proteins (RONs) complex; AMA1 interacts with RON2 during host cell invasion. Plasmodium vivax is globally the most widely distributed and the Aiming to develop vaccines against this species, in recent years, the group studied multiple aspects of naturally acquired immune responses against recombinant proteins representing P. vivax blood-stage antigens. Among the most promising malaria vaccine candidates, it has been found that the recombinant proteins representing Apical Membrane Antigen 1 (AMA1) were highly immunogenic in natural infections [5,6,7,8,9] and after mouse. Despite the use of AMA1 for vaccine development, little is known about the function of this protein or the antibodies induced by natural infection or immunization with recombinant proteins. The functional importance of PvAMA1 in invasion is derived from a study in which antibodies induced by immunization with recombinant PvAMA1 were able to partially inhibit the red blood cell re-invasion ex vivo [9]
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