The molecular architecture of the African trypanosome cell surface

African trypanosomes are representative of a group of protozoa that diverged from other eukaryotes around one billion years ago. Separate evolution over this time scale has resulted in differences to other eukaryotes that are informative about molecular and cellular processes in eukaryotes, and about the evolution of eukaryotes. One unique feature of the biology of African trypanosomes is the molecular architecture of the cell surface, which is covered by a densely packed coat of variant surface glycoprotein (VSG). The VSG coat protects the trypanosome from immune system effectors, but must also be permissive for receptor-mediated binding and uptake of macromolecular ligands. Two receptors have been characterised, one for transferrin (TfR) for iron and one for haptoglobin-haemoglobin (HpHbR) for haem. The VSG and these receptors share a conserved structural motif based on a three helical bundle. Preliminary work exploited a screen of the genome and transcriptome that identified genes encoding cell surface proteins with a predicted structure similar to the characterised receptors. The screen was successful and subsequently two novel receptors have been characterised. The aim of this project is to understand the function, mechanism and evolution of cell surface receptors in African trypanosomes by meeting the following objectives:



1. The first objective for the student will be to express and purify eight putative receptors in eukaryotic expression systems. The recombinant proteins will be used to identify any ligands in serum using pull down experiments and mass spectrometry identification of the ligand. The kinetics of the receptor-ligand interaction will be characterised and quantified. The receptors with clearly identified ligands will be taken forward. Receptor-ligand pairs will be used in crystallization trials; if any are successful, the structure will be used to inform the experiments to meet the other objectives. If not, other approaches detailed below will be used. At this point the number of receptors investigated further will be reduced to one or two depending on the outcome of the experiments above and the identity of the ligands.



2. The second objective is to characterise the orientation and accessibility of the receptor in the VSG coat. The student will use the recombinant protein to screen and isolate panels of antibodies against each of receptors. The antibodies will be binned into groups, based on competition assays, that either inhibit ligand binding in vitro and others that do not. The ligand and antibodies will be used to probe the receptor function in live cells by determining binding and uptake of fluorescently labelled ligand or antibodies. The binding site of the ligand and each antibody on the receptor will be determined through crystallisation or using hydrogen deuterium exchange depending on the results in objective 1 above. The results from objective 2 will enable a mapping of the accessibility of defined size antibodies (whole IgG or Fab fragments) to different parts of the receptor within the VSG coat. This, in turn, will enable the development of a model of how the receptor is orientated within the VSG coat.



3. The third objective is to determine the function of the receptor in cell viability and proliferation. First, either null or conditional null mutant cell lines will be made and then any growth phenotype determined. Null mutants can be readily made using targeted homologous recombination to replace a gene with a selectable marker. Conditional mutants can be made using either tet-inducible RNAi or tet-dependent expression of a transgene. Gene deletion will only work if the receptor is not essential and in these cases condition null cell lines will be made.