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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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Characterization of binding kinetics of rRH5 and RBC.A Competition of binding to RBCs of rhodamine-labeled against unlabeled rRH5. Binding is inhibited by unlabeled rRH5 in a dose-dependent manner. The inhibition curve derived from scans of two independent assays shows that rRH5 competes against the binding of labeled rRH5 at an IC50 of ca. 5×10−5 M. B Binding profile of rRH5 to resealed RBC ghosts. The curve is the calculated least-squares best fit for a population of identical independent binding sites, giving Kd = 1.25 (±3)×10−5 M, with a saturation level (number of sites) at 0.42 µM.
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pone-0003300-g005: Characterization of binding kinetics of rRH5 and RBC.A Competition of binding to RBCs of rhodamine-labeled against unlabeled rRH5. Binding is inhibited by unlabeled rRH5 in a dose-dependent manner. The inhibition curve derived from scans of two independent assays shows that rRH5 competes against the binding of labeled rRH5 at an IC50 of ca. 5×10−5 M. B Binding profile of rRH5 to resealed RBC ghosts. The curve is the calculated least-squares best fit for a population of identical independent binding sites, giving Kd = 1.25 (±3)×10−5 M, with a saturation level (number of sites) at 0.42 µM.

Mentions: A competitive binding assay was set up by mixing labeled and unlabeled rRH5 using increasing amounts of the unlabeled ligand, to give total concentrations of 0.1 to 400 µM with 25 µl of packed ghosts in a total volume of 100 µl. As Fig. 5A shows, the unlabeled protein competes against the labeled protein, with a concentration for 50% inhibition of ca. 5×10−5 M, implying a slightly smaller affinity (ca threefold increase in Kd). Thus specificity of binding is retained after labeling, and the affinity is almost unperturbed. Similar experiments with rhodamine-GST did not reveal any binding to the ghosts (data not shown).


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)

Characterization of binding kinetics of rRH5 and RBC.A Competition of binding to RBCs of rhodamine-labeled against unlabeled rRH5. Binding is inhibited by unlabeled rRH5 in a dose-dependent manner. The inhibition curve derived from scans of two independent assays shows that rRH5 competes against the binding of labeled rRH5 at an IC50 of ca. 5×10−5 M. B Binding profile of rRH5 to resealed RBC ghosts. The curve is the calculated least-squares best fit for a population of identical independent binding sites, giving Kd = 1.25 (±3)×10−5 M, with a saturation level (number of sites) at 0.42 µM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003300-g005: Characterization of binding kinetics of rRH5 and RBC.A Competition of binding to RBCs of rhodamine-labeled against unlabeled rRH5. Binding is inhibited by unlabeled rRH5 in a dose-dependent manner. The inhibition curve derived from scans of two independent assays shows that rRH5 competes against the binding of labeled rRH5 at an IC50 of ca. 5×10−5 M. B Binding profile of rRH5 to resealed RBC ghosts. The curve is the calculated least-squares best fit for a population of identical independent binding sites, giving Kd = 1.25 (±3)×10−5 M, with a saturation level (number of sites) at 0.42 µM.
Mentions: A competitive binding assay was set up by mixing labeled and unlabeled rRH5 using increasing amounts of the unlabeled ligand, to give total concentrations of 0.1 to 400 µM with 25 µl of packed ghosts in a total volume of 100 µl. As Fig. 5A shows, the unlabeled protein competes against the labeled protein, with a concentration for 50% inhibition of ca. 5×10−5 M, implying a slightly smaller affinity (ca threefold increase in Kd). Thus specificity of binding is retained after labeling, and the affinity is almost unperturbed. Similar experiments with rhodamine-GST did not reveal any binding to the ghosts (data not shown).

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