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J Biol Chem. 2013 Nov 8;288(45):32106-17. doi: 10.1074/jbc.M113.484840. Epub 2013 Sep 16.

Biochemical analysis of the Plasmodium falciparum erythrocyte-binding antigen-175 (EBA175)-glycophorin-A interaction: implications for vaccine design.

Author information

1
From the Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH.

Abstract

PfEBA175 has an important role in the invasion of human erythrocytes by Plasmodium falciparum and is therefore considered a high priority blood-stage malaria vaccine candidate. PfEBA175 mediates adhesion to erythrocytes through binding of the Duffy-binding-like (DBL) domains in its extracellular domain to Neu5Acα2-3Gal displayed on the O-linked glycans of glycophorin-A (GYPA). Because of the difficulties in expressing active full-length (FL) P. falciparum proteins in a recombinant form, previous analyses of the PfEBA175-GYPA interaction have largely focused on the DBL domains alone, and therefore they have not been performed in the context of the native protein sequence. Here, we express the entire ectodomain of PfEBA175 (PfEBA175 FL) in soluble form, allowing us to compare the biochemical and immunological properties with a fragment containing only the tandem DBL domains ("region II," PfEBA175 RII). Recombinant PfEBA175 FL bound human erythrocytes in a trypsin and neuraminidase-sensitive manner and recognized Neu5Acα2-3Gal-containing glycans, confirming its biochemical activity. A quantitative binding analysis showed that PfEBA175 FL interacted with native GYPA with a KD ∼0.26 μM and is capable of self-association. By comparison, the RII fragment alone bound GYPA with a lower affinity demonstrating that regions outside of the DBL domains are important for interactions with GYPA; antibodies directed to these other regions also contributed to the inhibition of parasite invasion. These data demonstrate the importance of PfEBA175 regions other than the DBL domains in the interaction with GYPA and merit their inclusion in an EBA175-based vaccine.

KEYWORDS:

Carbohydrate-binding Protein; Cell Invasion; Cell Surface Receptor; Infectious Diseases; Malaria; Protein-Protein Interactions; Surface Plasmon Resonance (SPR)

PMID:
24043627
PMCID:
PMC3820850
DOI:
10.1074/jbc.M113.484840
[Indexed for MEDLINE]
Free PMC Article

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