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Biochemical and biophysical characterisation of DBL1α1-varO, the rosetting domain of PfEMP1 from the VarO line of Plasmodium falciparum

Abstract : Rosetting of erythrocytes infected with Plasmodium falciparum is frequently observed in children with severe malaria. This adhesion phenomenon has been linked to the DBL1␣ domain of P. falciparum erythrocyte membrane protein 1 (PfEMP1) in three laboratory clones: FCR3S1.2, IT4R29 and Palo Alto varO. Here, we compare the soluble recombinant NTS-DBL1␣ 1-varO domain (NTS: N-terminal segment) obtained from E. coli, Pichia pastoris and baculovirus/insect cell expression systems. In each case, the presence of NTS was necessary for obtaining a soluble product. Successful expression in E. coli required maltosebinding protein as an N-terminal fusion partner. Each expression system produced an identical, correctly folded protein, as judged by biochemical and biophysical characterisations, and by the capacity to elicit antibodies that react with the surface of VarO-infected erythrocytes and disrupt VarO rosettes. Binding studies using surface plasmon resonance (SPR) techniques showed that NTS-DBL1␣ 1 produced in E. coli binds to heparin with micromolar affinity. IC 50 constants for other sulphated oligosaccharides were determined using SPR by measuring their competitive binding to the soluble protein in the presence of immobilized heparin. The affinity to NTS-DBL1␣ 1 was related to the degree of sulphation of the oligosaccharide, although the position of the sulphate groups on the sugar rings was also important. VarO rosettes could be disrupted by sulphated oligosaccharides with an efficacy that correlated with their binding affinity to recombinant NTS-DBL1␣ 1. Thus high yields of soluble NTS-DBL1␣ 1 with native conformation have been produced, opening novel perspectives for both structure-function studies and vaccine development.
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https://hal-pasteur.archives-ouvertes.fr/pasteur-03506823
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Submitted on : Sunday, January 2, 2022 - 8:01:52 PM
Last modification on : Thursday, April 7, 2022 - 10:10:29 AM

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Alexandre Juillerat, Sébastien Igonet, Inès Vigan‑womas, Micheline Guillotte, Stéphane Gangnard, et al.. Biochemical and biophysical characterisation of DBL1α1-varO, the rosetting domain of PfEMP1 from the VarO line of Plasmodium falciparum. Molecular and Biochemical Parasitology, 2010, 170, pp.84 - 92. ⟨10.1016/j.molbiopara.2009.12.008⟩. ⟨pasteur-03506823⟩

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