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Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Chen Q, Barragan A, Fernandez V, Sundström A, Schlichtherle M, Sahlén A, Carlson J, Datta S, Wahlgren M - J. Exp. Med. (1998)

Bottom Line: Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs.A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix.The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, the Swedish Institute for Infectious Disease Control, S-171 77 Stockholm, Sweden.

ABSTRACT
Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

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Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with  GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and  FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7  × 10−3 IU.)
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Figure 5: Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7 × 10−3 IU.)

Mentions: To establish the role of heparan sulfate also in rosetting, we studied the disruptive activity of different GAGs on FCR3S1.2 rosettes. FCR3S1.2 rosettes were sensitive to both heparin and to heparan sulfate, but neither chondroitin sulfate A, keratan sulfate, nor hyaluronic acid had any effect on the rosettes (Fig. 5 A). Chondroitin sulfate B had a slight effect only at high concentrations (Fig. 5 A). These findings were confirmed by enzyme treatment of the uninfected erythrocyte; heparinase, but not chondroitinase or neuraminidase, treatment blocked the rosetting (Fig. 5 B). Addition of heparan sulfate to the heparinase–cell mixture completely blocked the heparinase activity suggesting that the heparinase activity seen was indeed due to the cleavage of a heparan sulfate–like GAG and not due to a contamination causing proteolytic cleavage of erythrocyte surface–located polypeptides.


Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum.

Chen Q, Barragan A, Fernandez V, Sundström A, Schlichtherle M, Sahlén A, Carlson J, Datta S, Wahlgren M - J. Exp. Med. (1998)

Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with  GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and  FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7  × 10−3 IU.)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199182&req=5

Figure 5: Effect of GAGs on P. falciparum rosetting. A shows disruption of rosettes exerted by different GAGs. FCR3S1.2 cultures were incubated with GAGs for 1 h at 37°C and compared to control culture. Results are the means and standard error of three separate experiments. B shows the effect of enzyme treatment of uninfected, C-FDA–labeled erythrocytes in a competitive assay of rosette reformation in the presence of normal erythrocytes and FCR3S1.2-infected pRBCs. Results are the means and standard error of three separate experiments, or two experiments for neuraminidase. Neuraminidase and chondroitinase ABC concentrations are in IU, whereas the heparinase III concentration is in Sigma units. (One Sigma unit corresponds to ∼1.7 × 10−3 IU.)
Mentions: To establish the role of heparan sulfate also in rosetting, we studied the disruptive activity of different GAGs on FCR3S1.2 rosettes. FCR3S1.2 rosettes were sensitive to both heparin and to heparan sulfate, but neither chondroitin sulfate A, keratan sulfate, nor hyaluronic acid had any effect on the rosettes (Fig. 5 A). Chondroitin sulfate B had a slight effect only at high concentrations (Fig. 5 A). These findings were confirmed by enzyme treatment of the uninfected erythrocyte; heparinase, but not chondroitinase or neuraminidase, treatment blocked the rosetting (Fig. 5 B). Addition of heparan sulfate to the heparinase–cell mixture completely blocked the heparinase activity suggesting that the heparinase activity seen was indeed due to the cleavage of a heparan sulfate–like GAG and not due to a contamination causing proteolytic cleavage of erythrocyte surface–located polypeptides.

Bottom Line: Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs.A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix.The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Tumor Biology Center, Karolinska Institutet, the Swedish Institute for Infectious Disease Control, S-171 77 Stockholm, Sweden.

ABSTRACT
Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.

Show MeSH
Related in: MedlinePlus