Limits...
The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

Arévalo-Pinzón G, Bermúdez M, Curtidor H, Patarroyo MA - Malar. J. (2015)

Bottom Line: Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion.Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein.

View Article: PubMed Central - PubMed

Affiliation: Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50 # 26-20, Bogotá, Colombia. gabarpi@gmail.com.

ABSTRACT

Background: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes.

Methods: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.

Results: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes.

Conclusions: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

Show MeSH

Related in: MedlinePlus

rPvRON5 binding to human reticulocytes. (A) Immunoprecipitation and WB assays with reticulocytes (lane 1) and packed RBC (lane 2) with rPvRON5. 15 μg rPvRON5 was incubated with human reticulocytes or packed RBC passed through CF11 columns, followed by separation by dibutyl phthalate gradient. Cell-associated protein was eluted with 1 M NaCl in PBS and the eluted fraction was immunoprecipitated with anti-polyhistidine antibodies. The precipitate was run on an SDS-PAGE gel and revealed with anti-polyhistidine antibodies. (B) Indirect immunofluorescence binding studies. (i) reticulocyte labelling with antibodies against the transferrin receptor (CD71) which is expressed at high levels during erythroid development phases and is absent in mature cells (RBC). (ii) rPvRON5 recognition by polyclonal antibodies directed against the recombinant bound to reticulocytes and RBC. (iii) The cells were seen in white light and those which were positive for CD71 (red). (iv) The cells were observed in white light simultaneously with rPvRON5 binding (green) to cell membrane. (v) Merging between red fluorescence (CD71) and green (rPvRON5). Greater rPvRON5 preference for reticulocytes than mature RBCs was observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4359499&req=5

Fig4: rPvRON5 binding to human reticulocytes. (A) Immunoprecipitation and WB assays with reticulocytes (lane 1) and packed RBC (lane 2) with rPvRON5. 15 μg rPvRON5 was incubated with human reticulocytes or packed RBC passed through CF11 columns, followed by separation by dibutyl phthalate gradient. Cell-associated protein was eluted with 1 M NaCl in PBS and the eluted fraction was immunoprecipitated with anti-polyhistidine antibodies. The precipitate was run on an SDS-PAGE gel and revealed with anti-polyhistidine antibodies. (B) Indirect immunofluorescence binding studies. (i) reticulocyte labelling with antibodies against the transferrin receptor (CD71) which is expressed at high levels during erythroid development phases and is absent in mature cells (RBC). (ii) rPvRON5 recognition by polyclonal antibodies directed against the recombinant bound to reticulocytes and RBC. (iii) The cells were seen in white light and those which were positive for CD71 (red). (iv) The cells were observed in white light simultaneously with rPvRON5 binding (green) to cell membrane. (v) Merging between red fluorescence (CD71) and green (rPvRON5). Greater rPvRON5 preference for reticulocytes than mature RBCs was observed.

Mentions: Figure 4A shows that rPvRON5 bound to umbilical cord RBC and reticulocyte-enriched samples. IFA involving reticulocytes labelled with anti-CD71 antibody and polyclonal serum (rabbit 3) against rPvRON5 having high reactivity for the recombinant protein as shown by WB (Figure 3, lane R), were used for determining recombinant protein preference and/or tropism for human reticulocytes or mature RBC. The results showed that rPvRON5 bound to both mature RBC and reticulocytes (Figure 4B). The differences between cell-associated fluorescence measured by Image J 1.48 in RBCs compared to reticulocytes showed that rPvRON5 had greater ability to bind human reticulocytes (p < 0.01) than RBCs (as expected), as reticulocytes are target cells for P. vivax. This pattern was similar to that reported for PvMSP1 which contains reticulocyte binding HABPs, binding to erythrocytes to a lesser degree [58,61]. The foregoing could depend on the few RON5 receptors on RBC membrane compared to reticulocytes as part of RBC maturation and differentiation [62]. No fluorescence was observed in the negative controls (Additional file 2). All the above data suggested that PvRON5 appears to be an adhesin, which might participate in invasion by binding to reticulocyte surface.Figure 4


The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

Arévalo-Pinzón G, Bermúdez M, Curtidor H, Patarroyo MA - Malar. J. (2015)

rPvRON5 binding to human reticulocytes. (A) Immunoprecipitation and WB assays with reticulocytes (lane 1) and packed RBC (lane 2) with rPvRON5. 15 μg rPvRON5 was incubated with human reticulocytes or packed RBC passed through CF11 columns, followed by separation by dibutyl phthalate gradient. Cell-associated protein was eluted with 1 M NaCl in PBS and the eluted fraction was immunoprecipitated with anti-polyhistidine antibodies. The precipitate was run on an SDS-PAGE gel and revealed with anti-polyhistidine antibodies. (B) Indirect immunofluorescence binding studies. (i) reticulocyte labelling with antibodies against the transferrin receptor (CD71) which is expressed at high levels during erythroid development phases and is absent in mature cells (RBC). (ii) rPvRON5 recognition by polyclonal antibodies directed against the recombinant bound to reticulocytes and RBC. (iii) The cells were seen in white light and those which were positive for CD71 (red). (iv) The cells were observed in white light simultaneously with rPvRON5 binding (green) to cell membrane. (v) Merging between red fluorescence (CD71) and green (rPvRON5). Greater rPvRON5 preference for reticulocytes than mature RBCs was observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359499&req=5

Fig4: rPvRON5 binding to human reticulocytes. (A) Immunoprecipitation and WB assays with reticulocytes (lane 1) and packed RBC (lane 2) with rPvRON5. 15 μg rPvRON5 was incubated with human reticulocytes or packed RBC passed through CF11 columns, followed by separation by dibutyl phthalate gradient. Cell-associated protein was eluted with 1 M NaCl in PBS and the eluted fraction was immunoprecipitated with anti-polyhistidine antibodies. The precipitate was run on an SDS-PAGE gel and revealed with anti-polyhistidine antibodies. (B) Indirect immunofluorescence binding studies. (i) reticulocyte labelling with antibodies against the transferrin receptor (CD71) which is expressed at high levels during erythroid development phases and is absent in mature cells (RBC). (ii) rPvRON5 recognition by polyclonal antibodies directed against the recombinant bound to reticulocytes and RBC. (iii) The cells were seen in white light and those which were positive for CD71 (red). (iv) The cells were observed in white light simultaneously with rPvRON5 binding (green) to cell membrane. (v) Merging between red fluorescence (CD71) and green (rPvRON5). Greater rPvRON5 preference for reticulocytes than mature RBCs was observed.
Mentions: Figure 4A shows that rPvRON5 bound to umbilical cord RBC and reticulocyte-enriched samples. IFA involving reticulocytes labelled with anti-CD71 antibody and polyclonal serum (rabbit 3) against rPvRON5 having high reactivity for the recombinant protein as shown by WB (Figure 3, lane R), were used for determining recombinant protein preference and/or tropism for human reticulocytes or mature RBC. The results showed that rPvRON5 bound to both mature RBC and reticulocytes (Figure 4B). The differences between cell-associated fluorescence measured by Image J 1.48 in RBCs compared to reticulocytes showed that rPvRON5 had greater ability to bind human reticulocytes (p < 0.01) than RBCs (as expected), as reticulocytes are target cells for P. vivax. This pattern was similar to that reported for PvMSP1 which contains reticulocyte binding HABPs, binding to erythrocytes to a lesser degree [58,61]. The foregoing could depend on the few RON5 receptors on RBC membrane compared to reticulocytes as part of RBC maturation and differentiation [62]. No fluorescence was observed in the negative controls (Additional file 2). All the above data suggested that PvRON5 appears to be an adhesin, which might participate in invasion by binding to reticulocyte surface.Figure 4

Bottom Line: Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion.Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein.

View Article: PubMed Central - PubMed

Affiliation: Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50 # 26-20, Bogotá, Colombia. gabarpi@gmail.com.

ABSTRACT

Background: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes.

Methods: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.

Results: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes.

Conclusions: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

Show MeSH
Related in: MedlinePlus