Receptor-binding studies of the DBL? domain of Plasmodium falciparum erythrocyte membrane protein 1 from a placental isolate - Analytical Methods & Tools for Lyophilization

Receptor-binding studies of the DBL? domain of Plasmodium falciparum erythrocyte membrane protein 1 from a placental isolate
Received 30 August 2006; revised 18 October 2006; accepted 20 October 2006. Available online 7 November 2006.
Cyril Badauta, 1, Grazyna Faurea, 1, Nicaise G. Tuikue Ndamb, Gwladys Bertinb, Alain Chaffottec, Ayman Khattabd, Mo-Quen Klinkertd, Philippe Deloronb and Graham A. Bentleya
Molecular and Biochemical Parasitology
Volume 151, Issue 1 , January 2007
ScienceDirect
Copyright ? 2006 Elsevier B.V. All rights reserved
aUnit? d?Immunologie Structurale, CNRS URA 2185, D?partement de Biologie Structurale et Chime, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris cedex 15, France
bInstitut de Recherche pour le D?veloppement, UR010, Laboratoire de Parasitologie, Universit? Paris Descartes, IFR 71, 4 av. de l?observatoire, 75006 Paris, France
cUnit? de R?sonance Magn?tique Nucl?aire des Biomol?cules, CNRS URA 2185, D?partement de Biologie Structurale et Chimie, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris cedex 15, France
dBernhard-Nocht-Institut f?r Tropenmedizin, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
Abstract
We have previously identified a number of DBL? domains in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) transcripts obtained from placental parasite isolates, showing that they bind specifically to chondroitin sulfate A (CSA) (Khattab A, Kun J, Deloron P, Kremsner PG, Klinkert MQ. Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A. J Infect Dis 2001;183:1165?9). Here we give a more detailed physico-chemical and binding characterisation of the soluble, recombinant DBL? domain derived from one of these isolates. Results from circular dichroism and limited proteolysis experiments are consistent with the recombinant domain being expressed with the native fold. Specific binding of DBL? to placental cryosections was demonstrated by labeling with antibodies raised against the recombinant domain; binding was diminished after treatment of the cryosections with chondroitinase or by blocking with anti-CSA antibody, showing that CSA mediates the interaction. Binding of the DBL? domain to purified placental chondroitin sulfate proteoglycan (CSPG) was also studied using surface plasmon resonance techniques, with DBL? as analyte and CSPG immobilised on the sensor chip; these quantitative measurements gave an affinity constant in the ?-molar range under the conditions used. The native conformation of the DBL? domain is essential for CSPG recognition since binding to the sensor chip is abolished when the protein is irreversibly reduced. As with the placental cryosections, association was significantly reduced after treating the immobilised CSPG with chondroitinase. Together, these results demonstrate specific interaction between the DBL? domain and the placental receptor.
Keywords: Plasmodium falciparum erythrocyte membrane protein 1; Duffy binding-like domain; Placental malaria; Receptor binding; Surface plasmon resonance; Circular dichroism
Abbreviations: CD, circular dichroism; CSA, chondroitin sulfate A; CSPG, chondroitin sulfate proteoglycan; DBL, Duffy-binding like; DLS, dynamic light scattering; MALDI-TOF, matrix-assisted laser desorption/ionisation time-of-flight; mAb, monoclonal antibody; PE, parasitised erythrocytes; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; SPR, surface plasmon resonance

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1 These authors contributed equally.
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