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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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Rosetting FCR3S1.2-PfEMP1 binds to heparan sulfate. All the gels are 10% SDS-PAGE stained with Coomassie. A shows the expressed  GST, DBL-1–GST, or ATS–GST after purification on glutathione–Sepharose and SDS-PAGE. B shows the binding capacity of different fusion proteins  to heparin–Sepharose after SDS-PAGE. C shows the inhibition produced by different GAGs (20 μl, 5 mg/ml) on the binding of DBL-1–GST to heparin–Sepharose followed by SDS-PAGE. D and E show the binding of DBL-1–GST (D) and ATS–GST (E) to monolayers of normal RBCs as visualized  by an mAb to GST labeled with biotin and FITC-avidin.
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Figure 3: Rosetting FCR3S1.2-PfEMP1 binds to heparan sulfate. All the gels are 10% SDS-PAGE stained with Coomassie. A shows the expressed GST, DBL-1–GST, or ATS–GST after purification on glutathione–Sepharose and SDS-PAGE. B shows the binding capacity of different fusion proteins to heparin–Sepharose after SDS-PAGE. C shows the inhibition produced by different GAGs (20 μl, 5 mg/ml) on the binding of DBL-1–GST to heparin–Sepharose followed by SDS-PAGE. D and E show the binding of DBL-1–GST (D) and ATS–GST (E) to monolayers of normal RBCs as visualized by an mAb to GST labeled with biotin and FITC-avidin.

Mentions: To confirm the above findings experimentally, we subsequently expressed two domains of the FCR3S1.2-var1 transcript: one that had eight GAG-binding motifs (DBL-1, 1,008 bp corresponding to 336 aa) and one that was the highly charged, ATS that lacks GAG-binding motifs (1,353 bp corresponding to 451 aa). The purified DBL-1–GST efficiently bound to heparin-coupled Sepharose already after a few minutes at room temperature, whereas both the ATS–GST fusion protein and a second DBL-1–GST construct covering a distinct var sequence (var 2; reference 6) that lacks GAG-binding motifs failed to bind to the heparin matrix (Fig. 3 B and not shown). The adhesion could be competed out in a dose dependent manner with heparin or heparan sulfate but not with chondroitin sulfate, another negatively charged erythrocyte surface expressed GAG (Fig. 3 C). Taken together, these indicate that the binding of DBL-1 to heparan sulfate is dependent on structure and not merely ionic interactions between the two molecules. Therefore, heparan sulfate or a heparan sulfate–like GAG seems to be the binding target for this PfEMP1.


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)

Rosetting FCR3S1.2-PfEMP1 binds to heparan sulfate. All the gels are 10% SDS-PAGE stained with Coomassie. A shows the expressed  GST, DBL-1–GST, or ATS–GST after purification on glutathione–Sepharose and SDS-PAGE. B shows the binding capacity of different fusion proteins  to heparin–Sepharose after SDS-PAGE. C shows the inhibition produced by different GAGs (20 μl, 5 mg/ml) on the binding of DBL-1–GST to heparin–Sepharose followed by SDS-PAGE. D and E show the binding of DBL-1–GST (D) and ATS–GST (E) to monolayers of normal RBCs as visualized  by an mAb to GST labeled with biotin and FITC-avidin.
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Related In: Results  -  Collection

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

Figure 3: Rosetting FCR3S1.2-PfEMP1 binds to heparan sulfate. All the gels are 10% SDS-PAGE stained with Coomassie. A shows the expressed GST, DBL-1–GST, or ATS–GST after purification on glutathione–Sepharose and SDS-PAGE. B shows the binding capacity of different fusion proteins to heparin–Sepharose after SDS-PAGE. C shows the inhibition produced by different GAGs (20 μl, 5 mg/ml) on the binding of DBL-1–GST to heparin–Sepharose followed by SDS-PAGE. D and E show the binding of DBL-1–GST (D) and ATS–GST (E) to monolayers of normal RBCs as visualized by an mAb to GST labeled with biotin and FITC-avidin.
Mentions: To confirm the above findings experimentally, we subsequently expressed two domains of the FCR3S1.2-var1 transcript: one that had eight GAG-binding motifs (DBL-1, 1,008 bp corresponding to 336 aa) and one that was the highly charged, ATS that lacks GAG-binding motifs (1,353 bp corresponding to 451 aa). The purified DBL-1–GST efficiently bound to heparin-coupled Sepharose already after a few minutes at room temperature, whereas both the ATS–GST fusion protein and a second DBL-1–GST construct covering a distinct var sequence (var 2; reference 6) that lacks GAG-binding motifs failed to bind to the heparin matrix (Fig. 3 B and not shown). The adhesion could be competed out in a dose dependent manner with heparin or heparan sulfate but not with chondroitin sulfate, another negatively charged erythrocyte surface expressed GAG (Fig. 3 C). Taken together, these indicate that the binding of DBL-1 to heparan sulfate is dependent on structure and not merely ionic interactions between the two molecules. Therefore, heparan sulfate or a heparan sulfate–like GAG seems to be the binding target for this PfEMP1.

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