Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry

Malaria Journal

Research

S Josefin Bartholdson¹  Gavin J Wright¹  Abigail J Perrin¹  Madushi Wanaguru¹  Phoonthawee Saetear²  Darryl J Bornhop³  Amanda Kussrow³ 

[1] Cell Surface Signalling Laboratory and Malaria Programme, Wellcome Trust Sanger Institute, CB10 1SA, Hinxton, Cambridge, UK;Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama VI Road, 10400, Bangkok, Thailand;Department of Chemistry and the Vanderbilt Institute for Chemical Biology, Vanderbilt University, 4226 Stevenson Center, 37235, Nashville, Tennessee, USA;

关键词: Backscattering interferometry;    Biosensors;    Glycophorin A;    PfEBA175;    Basigin;    PfRH5;    Plasmodium falciparum;    Merozoite;    Recombinant proteins;    Malaria;   

DOI  :  10.1186/s12936-015-0553-2

 received in 2014-10-02, accepted in 2015-01-06,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundInvasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes.MethodsThe extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes.ResultsFindings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, KDs of 1.1 μM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions.ConclusionsThese results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

【 授权许可】

Unknown   
© Saetear et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.