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Plasmodium falciparum reticulocyte binding-like homologue protein 2 (PfRH2) is a key adhesive molecule involved in erythrocyte invasion.

Sahar T, Reddy KS, Bharadwaj M, Pandey AK, Singh S, Chitnis CE, Gaur D - PLoS ONE (2011)

Bottom Line: One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion.This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway.Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner.

View Article: PubMed Central - PubMed

Affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.

ABSTRACT
Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH2(40)) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH2(40) bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH2(40) blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine.

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Erythrocyte binding activity of native PfRH2a/b and recombinant rPfRH240 proteins.(A) Binding of the native PfRH2a/b protein in 3D7 culture supernatants incubated with untreated (U) erythrocytes, different enzyme-treated erythrocytes (Nm: neuraminidase-treated; T: trypsin-treated; C: chymotrypsin-treated). The processed PfRH2a/b parasite protein fragments (220 kDa and 80 kDa) were detected in the eluate fractions by immunoblotting using the anti-rPfRH240 antibodies. (B) Binding of the recombinant rPfRH240 protein with a similar set of erythrocytes. (C) Binding of native EBA-175 from 3D7 parasite culture supernatant with different enzyme treated erythrocytes. (D) Binding of recombinant EBA-175 region II (rEBA-175 RII) with enzyme treated erythrocytes. (E) Binding of recombinant PfRH4 (rRH430) region (amino acids 328–588) with enzyme treated erythrocytes.
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pone-0017102-g003: Erythrocyte binding activity of native PfRH2a/b and recombinant rPfRH240 proteins.(A) Binding of the native PfRH2a/b protein in 3D7 culture supernatants incubated with untreated (U) erythrocytes, different enzyme-treated erythrocytes (Nm: neuraminidase-treated; T: trypsin-treated; C: chymotrypsin-treated). The processed PfRH2a/b parasite protein fragments (220 kDa and 80 kDa) were detected in the eluate fractions by immunoblotting using the anti-rPfRH240 antibodies. (B) Binding of the recombinant rPfRH240 protein with a similar set of erythrocytes. (C) Binding of native EBA-175 from 3D7 parasite culture supernatant with different enzyme treated erythrocytes. (D) Binding of recombinant EBA-175 region II (rEBA-175 RII) with enzyme treated erythrocytes. (E) Binding of recombinant PfRH4 (rRH430) region (amino acids 328–588) with enzyme treated erythrocytes.

Mentions: The erythrocyte binding characteristics of native PfRH2a/b was studied in standard erythrocyte binding assays using different enzyme treated erythrocytes and culture supernatants of purified P. falciparum schizonts [31], [32]. The processed fragments of PfRH2a/b observed in the parasite detergent based extract were also found in the culture supernatants. The presence of native PfRH2a/b among the proteins eluted from the human erythrocytes incubated with parasite culture supernatants was detected by immunoblotting using anti-PfRH2a/b antibodies (Figures 3A and S6B) and interestingly exhibited differential erythrocyte binding activity.


Plasmodium falciparum reticulocyte binding-like homologue protein 2 (PfRH2) is a key adhesive molecule involved in erythrocyte invasion.

Sahar T, Reddy KS, Bharadwaj M, Pandey AK, Singh S, Chitnis CE, Gaur D - PLoS ONE (2011)

Erythrocyte binding activity of native PfRH2a/b and recombinant rPfRH240 proteins.(A) Binding of the native PfRH2a/b protein in 3D7 culture supernatants incubated with untreated (U) erythrocytes, different enzyme-treated erythrocytes (Nm: neuraminidase-treated; T: trypsin-treated; C: chymotrypsin-treated). The processed PfRH2a/b parasite protein fragments (220 kDa and 80 kDa) were detected in the eluate fractions by immunoblotting using the anti-rPfRH240 antibodies. (B) Binding of the recombinant rPfRH240 protein with a similar set of erythrocytes. (C) Binding of native EBA-175 from 3D7 parasite culture supernatant with different enzyme treated erythrocytes. (D) Binding of recombinant EBA-175 region II (rEBA-175 RII) with enzyme treated erythrocytes. (E) Binding of recombinant PfRH4 (rRH430) region (amino acids 328–588) with enzyme treated erythrocytes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046117&req=5

pone-0017102-g003: Erythrocyte binding activity of native PfRH2a/b and recombinant rPfRH240 proteins.(A) Binding of the native PfRH2a/b protein in 3D7 culture supernatants incubated with untreated (U) erythrocytes, different enzyme-treated erythrocytes (Nm: neuraminidase-treated; T: trypsin-treated; C: chymotrypsin-treated). The processed PfRH2a/b parasite protein fragments (220 kDa and 80 kDa) were detected in the eluate fractions by immunoblotting using the anti-rPfRH240 antibodies. (B) Binding of the recombinant rPfRH240 protein with a similar set of erythrocytes. (C) Binding of native EBA-175 from 3D7 parasite culture supernatant with different enzyme treated erythrocytes. (D) Binding of recombinant EBA-175 region II (rEBA-175 RII) with enzyme treated erythrocytes. (E) Binding of recombinant PfRH4 (rRH430) region (amino acids 328–588) with enzyme treated erythrocytes.
Mentions: The erythrocyte binding characteristics of native PfRH2a/b was studied in standard erythrocyte binding assays using different enzyme treated erythrocytes and culture supernatants of purified P. falciparum schizonts [31], [32]. The processed fragments of PfRH2a/b observed in the parasite detergent based extract were also found in the culture supernatants. The presence of native PfRH2a/b among the proteins eluted from the human erythrocytes incubated with parasite culture supernatants was detected by immunoblotting using anti-PfRH2a/b antibodies (Figures 3A and S6B) and interestingly exhibited differential erythrocyte binding activity.

Bottom Line: One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion.This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway.Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner.

View Article: PubMed Central - PubMed

Affiliation: Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.

ABSTRACT
Erythrocyte invasion by Plasmodium merozoites is a complex, multistep process that is mediated by a number of parasite ligand-erythrocyte receptor interactions. One such family of parasite ligands includes the P. falciparum reticulocyte binding homologue (PfRH) proteins that are homologous with the P. vivax reticulocyte binding proteins and have been shown to play a role in erythrocyte invasion. There are five functional PfRH proteins of which only PfRH2a/2b have not yet been demonstrated to bind erythrocytes. In this study, we demonstrated that native PfRH2a/2b is processed near the N-terminus yielding fragments of 220 kDa and 80 kDa that exhibit differential erythrocyte binding specificities. The erythrocyte binding specificity of the 220 kDa processed fragment of native PfRH2a/2b was sialic acid-independent, trypsin resistant and chymotrypsin sensitive. This specific binding phenotype is consistent with previous studies that disrupted the PfRH2a/2b genes and demonstrated that PfRH2b is involved in a sialic acid independent, trypsin resistant, chymotrypsin sensitive invasion pathway. Interestingly, we found that the smaller 80 kDa PfRH2a/2b fragment is processed from the larger 220 kDa fragment and binds erythrocytes in a sialic acid dependent, trypsin resistant and chymotrypsin sensitive manner. Thus, the two processed fragments of PfRH2a/2b differed with respect to their dependence on sialic acids for erythrocyte binding. Further, we mapped the erythrocyte binding domain of PfRH2a/2b to a conserved 40 kDa N-terminal region (rPfRH2(40)) in the ectodomain that is common to both PfRH2a and PfRH2b. We demonstrated that recombinant rPfRH2(40) bound human erythrocytes with the same specificity as the native 220 kDa processed protein. Moreover, antibodies generated against rPfRH2(40) blocked erythrocyte invasion by P. falciparum through a sialic acid independent pathway. PfRH2a/2b thus plays a key role in erythrocyte invasion and its conserved receptor-binding domain deserves attention as a promising candidate for inclusion in a blood-stage malaria vaccine.

Show MeSH
Related in: MedlinePlus