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PfRH5: a novel reticulocyte-binding family homolog of plasmodium falciparum that binds to the erythrocyte, and an investigation of its receptor.

Rodriguez M, Lustigman S, Montero E, Oksov Y, Lobo CA - PLoS ONE (2008)

Bottom Line: Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase.We have determined the affinity, copy number and apparent molecular mass of the receptor protein.Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Blood-Borne Parasites, Lindsley Kimball Research Institute, The New York Blood Center, New York, New York, USA.

ABSTRACT
Multiple interactions between parasite ligands and their receptors on the human erythrocyte are a condition of successful Plasmodium falciparum invasion. The identification and characterization of these receptors presents a major challenge in the effort to understand the mechanism of invasion and to develop the means to prevent it. We describe here a novel member of the reticulocyte-binding family homolog (RH) of P. falciparum, PfRH5, and show that it binds to a previously unrecognized receptor on the RBC. PfRH5 is expressed as a 63 kDa protein and localized at the apical end of the invasive merozoite. We have expressed a fragment of PfRH5 which contains the RBC-binding domain and exhibits the same pattern of interactions with the RBC as the parent protein. Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase. We have determined the affinity, copy number and apparent molecular mass of the receptor protein. Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathway.

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PfRH5 binds to a ∼32 kDa protein on human erythrocytes.Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).
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pone-0003300-g006: PfRH5 binds to a ∼32 kDa protein on human erythrocytes.Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).

Mentions: To identify the receptor(s) of PfRH5 on human erythrocytes, we used gel overlay experiments in which erythrocyte ghost proteins, separated on SDS-PAGE, were incubated with rRH5 (Fig. 6, rRH5) or with Dd2 culture supernatant (Fig. 6, Dd2). As a control, GST, the fusion partner pf rRH5.was included in the overlay assay (GST). After exposure of the ghost proteins on the membrane to these three different antigens the blot was treated with anti-RH5 (which contains antibodies to GST too) to detect specific ligand-receptor interactions. A non-specific ∼28 kDa RBC protein (Fig. 6, marked by asterisk) was seen in all the lanes. However, we were able to detect a specific interaction between rRH5 and a component of apparent molecular mass of 32 kDa on the blot of ghost proteins (indicated by arrow, lane rRH5). The same 32 kDa band was seen in the lanes treated with native parasite supernatant (Dd2, indicated by arrow). Thus, the 32 kDa erythrocyte protein interacts with rRH5 as well as the native RH5 from the parasite supernatant. Pre-immune rabbit sera did not react with any of the proteins on the ghost overlay (PI). To determine whether any of the glycophorins migrated at this position on the blot, parallel strips were treated with anti-GPA/B and anti-GPC/D. The 32 kDa band does not migrate near any of the glycophorins (lanes GPA/B and GPC/D), thus, it appears, ruling them out as PfRH5 receptors. Future work will be directed at identifying this putative ∼32 kDa receptor molecule.


PfRH5: a novel reticulocyte-binding family homolog of plasmodium falciparum that binds to the erythrocyte, and an investigation of its receptor.

Rodriguez M, Lustigman S, Montero E, Oksov Y, Lobo CA - PLoS ONE (2008)

PfRH5 binds to a ∼32 kDa protein on human erythrocytes.Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553180&req=5

pone-0003300-g006: PfRH5 binds to a ∼32 kDa protein on human erythrocytes.Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).
Mentions: To identify the receptor(s) of PfRH5 on human erythrocytes, we used gel overlay experiments in which erythrocyte ghost proteins, separated on SDS-PAGE, were incubated with rRH5 (Fig. 6, rRH5) or with Dd2 culture supernatant (Fig. 6, Dd2). As a control, GST, the fusion partner pf rRH5.was included in the overlay assay (GST). After exposure of the ghost proteins on the membrane to these three different antigens the blot was treated with anti-RH5 (which contains antibodies to GST too) to detect specific ligand-receptor interactions. A non-specific ∼28 kDa RBC protein (Fig. 6, marked by asterisk) was seen in all the lanes. However, we were able to detect a specific interaction between rRH5 and a component of apparent molecular mass of 32 kDa on the blot of ghost proteins (indicated by arrow, lane rRH5). The same 32 kDa band was seen in the lanes treated with native parasite supernatant (Dd2, indicated by arrow). Thus, the 32 kDa erythrocyte protein interacts with rRH5 as well as the native RH5 from the parasite supernatant. Pre-immune rabbit sera did not react with any of the proteins on the ghost overlay (PI). To determine whether any of the glycophorins migrated at this position on the blot, parallel strips were treated with anti-GPA/B and anti-GPC/D. The 32 kDa band does not migrate near any of the glycophorins (lanes GPA/B and GPC/D), thus, it appears, ruling them out as PfRH5 receptors. Future work will be directed at identifying this putative ∼32 kDa receptor molecule.

Bottom Line: Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase.We have determined the affinity, copy number and apparent molecular mass of the receptor protein.Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Blood-Borne Parasites, Lindsley Kimball Research Institute, The New York Blood Center, New York, New York, USA.

ABSTRACT
Multiple interactions between parasite ligands and their receptors on the human erythrocyte are a condition of successful Plasmodium falciparum invasion. The identification and characterization of these receptors presents a major challenge in the effort to understand the mechanism of invasion and to develop the means to prevent it. We describe here a novel member of the reticulocyte-binding family homolog (RH) of P. falciparum, PfRH5, and show that it binds to a previously unrecognized receptor on the RBC. PfRH5 is expressed as a 63 kDa protein and localized at the apical end of the invasive merozoite. We have expressed a fragment of PfRH5 which contains the RBC-binding domain and exhibits the same pattern of interactions with the RBC as the parent protein. Attachment is inhibited if the target cells are exposed to high concentrations of trypsin, but not to lower concentrations or to chymotrypsin or neuraminidase. We have determined the affinity, copy number and apparent molecular mass of the receptor protein. Thus, we have shown that PfRH5 is a novel erythrocyte-binding ligand and the identification and partial characterization of the new RBC receptor may indicate the existence of an unrecognized P. falciparum invasion pathway.

Show MeSH