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P-selectin is a host receptor for Plasmodium MSP7 ligands.

Perrin AJ, Bartholdson SJ, Wright GJ - Malar. J. (2015)

Bottom Line: Plasmodium parasites typically elicit a non-sterile but protective immune response in human host populations, suggesting that the parasites actively modulate normal immunological mechanisms.Orthologous proteins in the murine parasite Plasmodium berghei (PbMSRP1 and PbMSRP2) and mouse P-selectin also interacted.Since PfMSP7 could prevent interactions between P-selectin and its leukocyte ligands, these results provide a possible mechanism for the known immunomodulatory effects of both MSP7 and P-selectin in malaria infection models.

View Article: PubMed Central - PubMed

Affiliation: Cell Surface Signalling Laboratory and Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK. ap11@sanger.ac.uk.

ABSTRACT

Background: Plasmodium parasites typically elicit a non-sterile but protective immune response in human host populations, suggesting that the parasites actively modulate normal immunological mechanisms. P-selectin is a cell surface receptor expressed in mammals, that is a known component of the inflammatory response against pathogens and has been previously identified as a host factor that influences malaria-associated pathology both in human patients and rodent infection models.

Methods: To better understand the molecular mechanisms underlying the involvement of P-selectin in the pathogenesis of malaria, a systematic extracellular protein interaction screen was used to identify Plasmodium falciparum merozoite surface protein 7 (MSP7) as a binding partner of human P-selectin. This interaction, and those occurring between P-selectin and Plasmodium MSP7 homologues, was characterized biochemically.

Results: Plasmodium falciparum MSP7 and P-selectin were shown to bind each other directly via the N-terminus of PfMSP7 and the P-selectin C-type lectin and EGF-like domains. Orthologous proteins in the murine parasite Plasmodium berghei (PbMSRP1 and PbMSRP2) and mouse P-selectin also interacted. Finally, P-selectin, when complexed with MSP7, could no longer bind to its endogenous carbohydrate ligand, Sialyl-Lewis(X).

Conclusions: Novel interactions were identified between Plasmodium MSP7 protein family members and host P-selectin receptors. Since PfMSP7 could prevent interactions between P-selectin and its leukocyte ligands, these results provide a possible mechanism for the known immunomodulatory effects of both MSP7 and P-selectin in malaria infection models.

No MeSH data available.


Related in: MedlinePlus

PfMSP7 interacted with P-selectin at the surface of cells. a Schematic representing the experimental design, whereby recombinant, pentameric PfMSP7 proteins interact with eGFP-tagged P-selectin at the surface of transfected cells. b The interaction of P-selectin-eGFP transfected cells with FLAG-tagged pentameric PfMSP7 was detected by flow cytometry. Pre-incubation of cells with anti-Cd200R negative control mAb did not impair interaction detection. c P-selectin-GFP transfected cells incubated with CLB-thromb/6 anti P-selectin mAb prior to incubation with FLAG-tagged pentameric PfMSP7 could not replicate this interaction observed in (b)
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Fig2: PfMSP7 interacted with P-selectin at the surface of cells. a Schematic representing the experimental design, whereby recombinant, pentameric PfMSP7 proteins interact with eGFP-tagged P-selectin at the surface of transfected cells. b The interaction of P-selectin-eGFP transfected cells with FLAG-tagged pentameric PfMSP7 was detected by flow cytometry. Pre-incubation of cells with anti-Cd200R negative control mAb did not impair interaction detection. c P-selectin-GFP transfected cells incubated with CLB-thromb/6 anti P-selectin mAb prior to incubation with FLAG-tagged pentameric PfMSP7 could not replicate this interaction observed in (b)

Mentions: While the use of recombinant proteins in reductionist in vitro assays allows important experimental control over binding parameters, it does not satisfactorily replicate the complex environment of cell surfaces in which the interaction would normally occur. To replicate the elements of this environment, a P-selectin expression plasmid was constructed in which the cytoplasmic region was replaced with an eGFP reporter protein and then displayed at the surface of human cells by transiently transfecting the HEK293F cell line. Cells transfected with this construct were incubated with a pentamerized FLAG-tagged PfMSP7 prey protein and binding quantitated with an anti-FLAG fluorescent secondary antibody using flow cytometry relative to a control prey (Fig. 2a). A clear double-positive population was observed demonstrating that P-selectin-expressing (GFP-positive) cells interacted with the PfMSP7 FLAG-tagged prey (Fig. 2b). Within the sample of transfected cells used in each staining reaction, the level of PfMSP7 binding was proportional to the P-selectin-GFP expression level, and, importantly, the untransfected (GFP-negative) cells within the population did not bind PfMSP7 (Fig. 2b). The P-selectin dependency of PfMSP7 binding was further established by preventing binding by first pre-incubating the transfected cells with the anti-P-selectin antibody (Fig. 2c). These data provide additional evidence that MSP7 and P-selectin specifically interact.Fig. 2


P-selectin is a host receptor for Plasmodium MSP7 ligands.

Perrin AJ, Bartholdson SJ, Wright GJ - Malar. J. (2015)

PfMSP7 interacted with P-selectin at the surface of cells. a Schematic representing the experimental design, whereby recombinant, pentameric PfMSP7 proteins interact with eGFP-tagged P-selectin at the surface of transfected cells. b The interaction of P-selectin-eGFP transfected cells with FLAG-tagged pentameric PfMSP7 was detected by flow cytometry. Pre-incubation of cells with anti-Cd200R negative control mAb did not impair interaction detection. c P-selectin-GFP transfected cells incubated with CLB-thromb/6 anti P-selectin mAb prior to incubation with FLAG-tagged pentameric PfMSP7 could not replicate this interaction observed in (b)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4478713&req=5

Fig2: PfMSP7 interacted with P-selectin at the surface of cells. a Schematic representing the experimental design, whereby recombinant, pentameric PfMSP7 proteins interact with eGFP-tagged P-selectin at the surface of transfected cells. b The interaction of P-selectin-eGFP transfected cells with FLAG-tagged pentameric PfMSP7 was detected by flow cytometry. Pre-incubation of cells with anti-Cd200R negative control mAb did not impair interaction detection. c P-selectin-GFP transfected cells incubated with CLB-thromb/6 anti P-selectin mAb prior to incubation with FLAG-tagged pentameric PfMSP7 could not replicate this interaction observed in (b)
Mentions: While the use of recombinant proteins in reductionist in vitro assays allows important experimental control over binding parameters, it does not satisfactorily replicate the complex environment of cell surfaces in which the interaction would normally occur. To replicate the elements of this environment, a P-selectin expression plasmid was constructed in which the cytoplasmic region was replaced with an eGFP reporter protein and then displayed at the surface of human cells by transiently transfecting the HEK293F cell line. Cells transfected with this construct were incubated with a pentamerized FLAG-tagged PfMSP7 prey protein and binding quantitated with an anti-FLAG fluorescent secondary antibody using flow cytometry relative to a control prey (Fig. 2a). A clear double-positive population was observed demonstrating that P-selectin-expressing (GFP-positive) cells interacted with the PfMSP7 FLAG-tagged prey (Fig. 2b). Within the sample of transfected cells used in each staining reaction, the level of PfMSP7 binding was proportional to the P-selectin-GFP expression level, and, importantly, the untransfected (GFP-negative) cells within the population did not bind PfMSP7 (Fig. 2b). The P-selectin dependency of PfMSP7 binding was further established by preventing binding by first pre-incubating the transfected cells with the anti-P-selectin antibody (Fig. 2c). These data provide additional evidence that MSP7 and P-selectin specifically interact.Fig. 2

Bottom Line: Plasmodium parasites typically elicit a non-sterile but protective immune response in human host populations, suggesting that the parasites actively modulate normal immunological mechanisms.Orthologous proteins in the murine parasite Plasmodium berghei (PbMSRP1 and PbMSRP2) and mouse P-selectin also interacted.Since PfMSP7 could prevent interactions between P-selectin and its leukocyte ligands, these results provide a possible mechanism for the known immunomodulatory effects of both MSP7 and P-selectin in malaria infection models.

View Article: PubMed Central - PubMed

Affiliation: Cell Surface Signalling Laboratory and Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK. ap11@sanger.ac.uk.

ABSTRACT

Background: Plasmodium parasites typically elicit a non-sterile but protective immune response in human host populations, suggesting that the parasites actively modulate normal immunological mechanisms. P-selectin is a cell surface receptor expressed in mammals, that is a known component of the inflammatory response against pathogens and has been previously identified as a host factor that influences malaria-associated pathology both in human patients and rodent infection models.

Methods: To better understand the molecular mechanisms underlying the involvement of P-selectin in the pathogenesis of malaria, a systematic extracellular protein interaction screen was used to identify Plasmodium falciparum merozoite surface protein 7 (MSP7) as a binding partner of human P-selectin. This interaction, and those occurring between P-selectin and Plasmodium MSP7 homologues, was characterized biochemically.

Results: Plasmodium falciparum MSP7 and P-selectin were shown to bind each other directly via the N-terminus of PfMSP7 and the P-selectin C-type lectin and EGF-like domains. Orthologous proteins in the murine parasite Plasmodium berghei (PbMSRP1 and PbMSRP2) and mouse P-selectin also interacted. Finally, P-selectin, when complexed with MSP7, could no longer bind to its endogenous carbohydrate ligand, Sialyl-Lewis(X).

Conclusions: Novel interactions were identified between Plasmodium MSP7 protein family members and host P-selectin receptors. Since PfMSP7 could prevent interactions between P-selectin and its leukocyte ligands, these results provide a possible mechanism for the known immunomodulatory effects of both MSP7 and P-selectin in malaria infection models.

No MeSH data available.


Related in: MedlinePlus