Limits...
The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking.

Gilberger TW, Thompson JK, Reed MB, Good RT, Cowman AF - J. Cell Biol. (2003)

Bottom Line: The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process.In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain.These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia.

ABSTRACT
The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

Show MeSH

Related in: MedlinePlus

Immunoblot analysis of transgenic parasites expressing different EBA-175 mutant proteins. Proteins from synchronized parasite cultures were separated by SDS-PAGE on a 6% gel under reducing conditions. Proteins were detected with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and anti-HSP70. Approximately equal signal was obtained with the anti-HSP70 antibodies, suggesting that each lane was loaded equally. In 3D7-derived parasite pellets, an additional cross-reactive band of ∼100 kD is detected by anti-EBA-175 antibodies. (B) Bar graph of the relative expression ratio of EBA-175 in late schizont stages of selected parasites. This ratio was calculated by dividing the average of three EBA175 assays with the average of the actin and histone 2B signals. The SD represents the sum of the EBA-175 and housekeeping SDs. The averages are calculated and the relative expression ratio is obtained by dividing the EBA-175 value with a mean of each control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172798&req=5

fig2: Immunoblot analysis of transgenic parasites expressing different EBA-175 mutant proteins. Proteins from synchronized parasite cultures were separated by SDS-PAGE on a 6% gel under reducing conditions. Proteins were detected with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and anti-HSP70. Approximately equal signal was obtained with the anti-HSP70 antibodies, suggesting that each lane was loaded equally. In 3D7-derived parasite pellets, an additional cross-reactive band of ∼100 kD is detected by anti-EBA-175 antibodies. (B) Bar graph of the relative expression ratio of EBA-175 in late schizont stages of selected parasites. This ratio was calculated by dividing the average of three EBA175 assays with the average of the actin and histone 2B signals. The SD represents the sum of the EBA-175 and housekeeping SDs. The averages are calculated and the relative expression ratio is obtained by dividing the EBA-175 value with a mean of each control.

Mentions: To confirm that the EBA-175 protein was expressed, we analyzed parasites in schizont stages of parental lines W2mef and 3D7, as well as the transfectants W2mef3′R, W2mefY1419, W2mefY1464, W2mefYY, W2mefEDD, W2mefΔ15, W2mefYΔ15, W2mefTRAP, W2mefΔtail, W2mefΔ230, 3D7Δtail, and 3D73′R parasites by Western blots with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and also anti-HSP70 antibodies as a loading control (Fig. 2 A). Anti-EBA175 antibodies detect the ectodomain of the protein, and all of the parasite lines show expression of the appropriately sized protein. Additionally, in 3D7 and in 3D7-derived transgenic parasites, a strong (∼100 kD) cross-reactive protein is detected. The W2mefΔtail, 3D7Δtail, and W2mefΔ230 parasites express a protein smaller than that observed for W2mef consistent with expression of the truncated protein. Antibodies specific to the cytoplasmic tail of EBA-175 detect the protein in W2mef, and all of the transfectants except for W2mefTRAP, W2mefΔtail, W2mefΔ230, and 3D7Δtail. This shows that these transfected parasite lines express EBA-175 that lacks the cytoplasmic domain. The anti-EBA175-CT antibodies detect EBA-175 in W2mefYΔ15 and W2mefΔ15 despite the deletion of 15 amino acids. This is not surprising, as both of these truncated proteins retain 49 amino acids of the 64-amino acid cytoplasmic tail to which the antibody was raised.


The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking.

Gilberger TW, Thompson JK, Reed MB, Good RT, Cowman AF - J. Cell Biol. (2003)

Immunoblot analysis of transgenic parasites expressing different EBA-175 mutant proteins. Proteins from synchronized parasite cultures were separated by SDS-PAGE on a 6% gel under reducing conditions. Proteins were detected with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and anti-HSP70. Approximately equal signal was obtained with the anti-HSP70 antibodies, suggesting that each lane was loaded equally. In 3D7-derived parasite pellets, an additional cross-reactive band of ∼100 kD is detected by anti-EBA-175 antibodies. (B) Bar graph of the relative expression ratio of EBA-175 in late schizont stages of selected parasites. This ratio was calculated by dividing the average of three EBA175 assays with the average of the actin and histone 2B signals. The SD represents the sum of the EBA-175 and housekeeping SDs. The averages are calculated and the relative expression ratio is obtained by dividing the EBA-175 value with a mean of each control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172798&req=5

fig2: Immunoblot analysis of transgenic parasites expressing different EBA-175 mutant proteins. Proteins from synchronized parasite cultures were separated by SDS-PAGE on a 6% gel under reducing conditions. Proteins were detected with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and anti-HSP70. Approximately equal signal was obtained with the anti-HSP70 antibodies, suggesting that each lane was loaded equally. In 3D7-derived parasite pellets, an additional cross-reactive band of ∼100 kD is detected by anti-EBA-175 antibodies. (B) Bar graph of the relative expression ratio of EBA-175 in late schizont stages of selected parasites. This ratio was calculated by dividing the average of three EBA175 assays with the average of the actin and histone 2B signals. The SD represents the sum of the EBA-175 and housekeeping SDs. The averages are calculated and the relative expression ratio is obtained by dividing the EBA-175 value with a mean of each control.
Mentions: To confirm that the EBA-175 protein was expressed, we analyzed parasites in schizont stages of parental lines W2mef and 3D7, as well as the transfectants W2mef3′R, W2mefY1419, W2mefY1464, W2mefYY, W2mefEDD, W2mefΔ15, W2mefYΔ15, W2mefTRAP, W2mefΔtail, W2mefΔ230, 3D7Δtail, and 3D73′R parasites by Western blots with anti-EBA175, anti-EBA175-CT, anti-TRAP-CT, and also anti-HSP70 antibodies as a loading control (Fig. 2 A). Anti-EBA175 antibodies detect the ectodomain of the protein, and all of the parasite lines show expression of the appropriately sized protein. Additionally, in 3D7 and in 3D7-derived transgenic parasites, a strong (∼100 kD) cross-reactive protein is detected. The W2mefΔtail, 3D7Δtail, and W2mefΔ230 parasites express a protein smaller than that observed for W2mef consistent with expression of the truncated protein. Antibodies specific to the cytoplasmic tail of EBA-175 detect the protein in W2mef, and all of the transfectants except for W2mefTRAP, W2mefΔtail, W2mefΔ230, and 3D7Δtail. This shows that these transfected parasite lines express EBA-175 that lacks the cytoplasmic domain. The anti-EBA175-CT antibodies detect EBA-175 in W2mefYΔ15 and W2mefΔ15 despite the deletion of 15 amino acids. This is not surprising, as both of these truncated proteins retain 49 amino acids of the 64-amino acid cytoplasmic tail to which the antibody was raised.

Bottom Line: The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process.In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain.These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Australia.

ABSTRACT
The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

Show MeSH
Related in: MedlinePlus