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Analyses of interactions between heparin and the apical surface proteins of Plasmodium falciparum.

Kobayashi K, Takano R, Takemae H, Sugi T, Ishiwa A, Gong H, Recuenco FC, Iwanaga T, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Bottom Line: Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion.Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles.These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, the University of Tokyo [2] Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, the University of Tokyo [3] Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, the University of Tokyo.

ABSTRACT
Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

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Related in: MedlinePlus

Affinity chromatography of schizont proteins on a heparin column.(A) The elution profile of heparin-binding proteins of the P. falciparum HB3 clone. A schizont lysate was diluted with the binding buffer and separated by affinity chromatography on a heparin column. The proteins were washed and eluted from the column with a stepwise gradient of NaCl (0.2–1.5 M; thin line). Flow-through fractions of the lysate (FT), the wash buffer (wash), and the elution buffer (eluate) were collected (1.0 mL each) and subjected to protein quantification (thick line). (B) The eluate fractions containing proteins (E2–12) were analyzed by use of SDS-PAGE and silver staining. The molecular masses (kDa) are indicated on the right. (C) Eight fractions (FT and E3–9) were analyzed by immunoblotting. The arrowheads indicate specific bands. The molecular masses (kDa) are indicated on the left.
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f5: Affinity chromatography of schizont proteins on a heparin column.(A) The elution profile of heparin-binding proteins of the P. falciparum HB3 clone. A schizont lysate was diluted with the binding buffer and separated by affinity chromatography on a heparin column. The proteins were washed and eluted from the column with a stepwise gradient of NaCl (0.2–1.5 M; thin line). Flow-through fractions of the lysate (FT), the wash buffer (wash), and the elution buffer (eluate) were collected (1.0 mL each) and subjected to protein quantification (thick line). (B) The eluate fractions containing proteins (E2–12) were analyzed by use of SDS-PAGE and silver staining. The molecular masses (kDa) are indicated on the right. (C) Eight fractions (FT and E3–9) were analyzed by immunoblotting. The arrowheads indicate specific bands. The molecular masses (kDa) are indicated on the left.

Mentions: To isolate proteins that interact with heparin, we used affinity chromatography with a HiTrap heparin HP column (Fig. 5A). In this method, proteins weakly bound to heparin are eluted in buffer containing a lower concentration of NaCl. The collected fractions are then analyzed by SDS-PAGE, followed by silver staining (Fig. 5B). In the E2–9 fractions, clear bands were detected, and the band patterns observed in each lane were reproducible.


Analyses of interactions between heparin and the apical surface proteins of Plasmodium falciparum.

Kobayashi K, Takano R, Takemae H, Sugi T, Ishiwa A, Gong H, Recuenco FC, Iwanaga T, Horimoto T, Akashi H, Kato K - Sci Rep (2013)

Affinity chromatography of schizont proteins on a heparin column.(A) The elution profile of heparin-binding proteins of the P. falciparum HB3 clone. A schizont lysate was diluted with the binding buffer and separated by affinity chromatography on a heparin column. The proteins were washed and eluted from the column with a stepwise gradient of NaCl (0.2–1.5 M; thin line). Flow-through fractions of the lysate (FT), the wash buffer (wash), and the elution buffer (eluate) were collected (1.0 mL each) and subjected to protein quantification (thick line). (B) The eluate fractions containing proteins (E2–12) were analyzed by use of SDS-PAGE and silver staining. The molecular masses (kDa) are indicated on the right. (C) Eight fractions (FT and E3–9) were analyzed by immunoblotting. The arrowheads indicate specific bands. The molecular masses (kDa) are indicated on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3822384&req=5

f5: Affinity chromatography of schizont proteins on a heparin column.(A) The elution profile of heparin-binding proteins of the P. falciparum HB3 clone. A schizont lysate was diluted with the binding buffer and separated by affinity chromatography on a heparin column. The proteins were washed and eluted from the column with a stepwise gradient of NaCl (0.2–1.5 M; thin line). Flow-through fractions of the lysate (FT), the wash buffer (wash), and the elution buffer (eluate) were collected (1.0 mL each) and subjected to protein quantification (thick line). (B) The eluate fractions containing proteins (E2–12) were analyzed by use of SDS-PAGE and silver staining. The molecular masses (kDa) are indicated on the right. (C) Eight fractions (FT and E3–9) were analyzed by immunoblotting. The arrowheads indicate specific bands. The molecular masses (kDa) are indicated on the left.
Mentions: To isolate proteins that interact with heparin, we used affinity chromatography with a HiTrap heparin HP column (Fig. 5A). In this method, proteins weakly bound to heparin are eluted in buffer containing a lower concentration of NaCl. The collected fractions are then analyzed by SDS-PAGE, followed by silver staining (Fig. 5B). In the E2–9 fractions, clear bands were detected, and the band patterns observed in each lane were reproducible.

Bottom Line: Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion.Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles.These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, the University of Tokyo [2] Division of Stem Cell Processing, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, the University of Tokyo [3] Division of Host-Parasite Interaction, Department of Microbiology and Immunology, Institute of Medical Science, the University of Tokyo.

ABSTRACT
Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Show MeSH
Related in: MedlinePlus