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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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Invasion inhibitory activity of anti-rPfRH240 antibodies.Rabbit purified IgG against rPfRH240 was tested for its invasion inhibitory activity against the 3D7 and Dd2 parasite clones. Rabbit anti-PfRH240 IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% and with neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition was 50–53%. The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 with untreated erythrocytes. The invasion inhibitory activity with neuraminidase treated erythrocytes was not tested for Dd2 as the Dd2 parasite clone is sialic acid dependent and fails to invade neuraminidase treated erythrocytes. For studying the reversal of antibody inhibition, rPfRH240, was added at a final concentration of 30 µg/ml.
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pone-0017102-g004: Invasion inhibitory activity of anti-rPfRH240 antibodies.Rabbit purified IgG against rPfRH240 was tested for its invasion inhibitory activity against the 3D7 and Dd2 parasite clones. Rabbit anti-PfRH240 IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% and with neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition was 50–53%. The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 with untreated erythrocytes. The invasion inhibitory activity with neuraminidase treated erythrocytes was not tested for Dd2 as the Dd2 parasite clone is sialic acid dependent and fails to invade neuraminidase treated erythrocytes. For studying the reversal of antibody inhibition, rPfRH240, was added at a final concentration of 30 µg/ml.

Mentions: Since PfRH2a/b is involved in erythrocyte invasion with an erythrocyte binding role and the rPfRH240 region binds with the same specificity as the native protein, we tested if antibodies against rPfRH240 could block erythrocyte invasion. We measured the invasion inhibitory activity of purified rabbit IgG raised against rPfRH240 in standard GIA (growth inhibition assays) as described earlier [23], [24]. Anti-rPfRH240 rabbit IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% (Figure 4). Since, PfRH2a/b mediates invasion through the sialic acid independent, trypsin resistant pathway, we tested the GIA activity of the PfRH2a/b antibodies for invasion into neuraminidase and trypsin treated erythrocytes. The 3D7 parasite line has a sialic acid independent, trypsin resistant invasion phenotype. With neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition (Figure 4) was higher (50–53%) than that observed for invasion into normal erythrocytes indicating that under conditions where the 3D7 parasite is restricted to invade through only sialic acid independent or trypsin resistant pathways, the invasion inhibition was more efficient. Further, the invasion inhibition of the purified IgG could be neutralized by the addition of recombinant rPfRH240 protein (30 µg/ml), which clearly demonstrated that the invasion inhibition was specifically due to the anti- rPfRH240 antibodies. To further confirm the GIA specificity of our antibodies, we tested the GIA activity of the anti-PfRH240 antibodies with the P. falciparum clone Dd2 that invades only through sialic acid dependent pathways (Figure 4). The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 (Figure 4), which is consistent with the fact that PfRH2 is not involved in sialic acid dependent invasion pathways and further confirms the specific invasion inhibitory effect of anti-PfRH2a/b antibodies.


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)

Invasion inhibitory activity of anti-rPfRH240 antibodies.Rabbit purified IgG against rPfRH240 was tested for its invasion inhibitory activity against the 3D7 and Dd2 parasite clones. Rabbit anti-PfRH240 IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% and with neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition was 50–53%. The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 with untreated erythrocytes. The invasion inhibitory activity with neuraminidase treated erythrocytes was not tested for Dd2 as the Dd2 parasite clone is sialic acid dependent and fails to invade neuraminidase treated erythrocytes. For studying the reversal of antibody inhibition, rPfRH240, was added at a final concentration of 30 µg/ml.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017102-g004: Invasion inhibitory activity of anti-rPfRH240 antibodies.Rabbit purified IgG against rPfRH240 was tested for its invasion inhibitory activity against the 3D7 and Dd2 parasite clones. Rabbit anti-PfRH240 IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% and with neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition was 50–53%. The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 with untreated erythrocytes. The invasion inhibitory activity with neuraminidase treated erythrocytes was not tested for Dd2 as the Dd2 parasite clone is sialic acid dependent and fails to invade neuraminidase treated erythrocytes. For studying the reversal of antibody inhibition, rPfRH240, was added at a final concentration of 30 µg/ml.
Mentions: Since PfRH2a/b is involved in erythrocyte invasion with an erythrocyte binding role and the rPfRH240 region binds with the same specificity as the native protein, we tested if antibodies against rPfRH240 could block erythrocyte invasion. We measured the invasion inhibitory activity of purified rabbit IgG raised against rPfRH240 in standard GIA (growth inhibition assays) as described earlier [23], [24]. Anti-rPfRH240 rabbit IgG blocked invasion of normal untreated erythrocytes by the 3D7 parasite line by 25% (Figure 4). Since, PfRH2a/b mediates invasion through the sialic acid independent, trypsin resistant pathway, we tested the GIA activity of the PfRH2a/b antibodies for invasion into neuraminidase and trypsin treated erythrocytes. The 3D7 parasite line has a sialic acid independent, trypsin resistant invasion phenotype. With neuraminidase treated erythrocytes and trypsin treated erythrocytes, the percent inhibition (Figure 4) was higher (50–53%) than that observed for invasion into normal erythrocytes indicating that under conditions where the 3D7 parasite is restricted to invade through only sialic acid independent or trypsin resistant pathways, the invasion inhibition was more efficient. Further, the invasion inhibition of the purified IgG could be neutralized by the addition of recombinant rPfRH240 protein (30 µg/ml), which clearly demonstrated that the invasion inhibition was specifically due to the anti- rPfRH240 antibodies. To further confirm the GIA specificity of our antibodies, we tested the GIA activity of the anti-PfRH240 antibodies with the P. falciparum clone Dd2 that invades only through sialic acid dependent pathways (Figure 4). The anti-rPfRH240 antibodies did not exhibit any invasion inhibition effect on Dd2 (Figure 4), which is consistent with the fact that PfRH2 is not involved in sialic acid dependent invasion pathways and further confirms the specific invasion inhibitory effect of anti-PfRH2a/b antibodies.

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