Identification of the human membrane receptor for a key malaria parasite protein: Collaboration with the University of Copenhagen

Background

Researchers at the University of Copenhagen have previously identified a specific malaria parasite protein sub-type (pFEMP1) associated with a form of severe childhood malaria that is responsible for around 0.5 million deaths per year in Africa. This form of pFEMP1 is expressed by parasite-infected erythrocytes and was thought to bind to blood vessel walls in the brain via an intermediary human endothelial cell receptor. The identity of this cell surface receptor was unknown. Associate Professors Louise Turner and Thomas Lavstsen of Copenhagen approached Retrogenix to use its Cell Microarray Technology to identify the receptor for pFEMP1. Endothelial receptors are well represented within Retrogenix’s Cell Microarrays which, at the start of the project, included a total of 2,500 human plasma membrane proteins. This coverage has since increased by around 70% to >4,500 individual plasma membrane proteins – around 70% of all known cell surface proteins.

The Retrogenix technology is accessed as a service, with precise experimental design determined after initial discussions with the client. Regular communication throughout the project ensures that everything progresses smoothly and that results are fed back as quickly as possible.

Materials provided to Retrogenix:

  • His-tagged form of the pFEMP1 sub-type (His-IT4var20) – the ‘test protein’
  • His-tagged positive control protein (His-IT4var13) whose receptor (ICAM1) is known

Phase 1: Optimising the detection system for His-tagged proteins

As this was the first project involving His-tagged proteins for Retrogenix, the known control His-IT4var13 / ICAM1 interaction was used to validate and optimise the detection system. Retrogenix’s custom designed expression vector containing a full length human ICAM1 cDNA was spotted on slides. Human HEK293 cells were grown on top and reverse transfected with the vector which gave rise to ICAM1 over-expression. Binding of the His-tagged IT4var13 control protein was then evaluated using a fluorescently labelled anti-His detection antibody.

Once the detection system was optimised, Retrogenix also performed a background screen with the test protein, His-IT4var20, to show that endogenous expression of its receptor by HEK293 cells was absent or low. This is typically the case, but is essential for a successful screen.

Phase 2: Screening for primary hits

The test protein, His-IT4var20, was screened against 2,500 human plasma membrane proteins individually over-expressed in HEK293 cells across Retrogenix’s Cell Microarray slides. Primary hits were identified through ‘gain of binding’. At this stage, sixteen potential receptors for IT4var20 were identified.

Phase 3: Confirmation of binding

Each of the sixteen primary hits was identified via its position in the microarray and confirmed by sequencing. To confirm the binding, each cDNA was re-spotted on a fresh slide, expressed in HEK cells and re-probed for binding with the test protein, IT4var20. In addition, identical slides were probed with the control protein (IT4var13) or no ligand, to identify which hits were specific to the test protein, IT4var20. This left one specific and reproducible hit: Endothelial Protein C Receptor (EPCR). These results were achieved within six weeks of the project commencing.

The impact of Retrogenix’s results

The finding generated significant excitement as it was immediately apparent to the lead researchers that it “made sense” biologically. EPCR plays a key role in endothelial cytoprotective and anti-coagulation pathways so it is hypothesised that binding by the pFEMP1 sub-type may interfere with these pathways by interfering with Protein C binding. The team went on to confirm EPCR as the receptor for IT4var20/pFEMP1 through recombinant proteins assays as well assessing binding of endothelial cells by live parasite-infected erythrocytes.

This extremely successful project resulted in the identification of the human receptor for an important malaria protein which furthers understanding of malaria pathogenesis and opens up new possibilities for drug and vaccine therapy. The results of this study – which involved teams from the University of Copenhagen, Seattle Biomedical Research Institute, the National Institute for Medical Research, Tanzania, and the University of Oxford – have been published in Nature.

 

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…working with you was exactly like working with an experienced and knowledgeable scientific collaborator. We had the same types of discussions and strategic planning sessions and I felt confident throughout that we were addressing the issues in the best way possible. It was fun and if I see the opportunity we would do this again.
Professor Mark Kahn, University of Pennsylvania