Identification and characterisation of proteins from the tomato lectin binding fraction of Trypanosoma brucei

Abstract

African trypanosomes, of which Trypanosoma brucei may be considered a prototypic member, are protozoan parasites responsible for sleeping sickness in man and related diseases in cattle and other domestic animals. Although trypanosomes are obligatorily dependent on the uptake of factors from their hosts our understanding of the molecular machinery is limited. Glycans containing linear poly-N-acetyllactosamine (pNAL) are associated only with proteins from the flagellar pocket and endocytic pathway in T. brucei. These glycoproteins bind to tomato lectin which has allowed their single step isolation. This study was aimed at identifying and characterising proteins that bind to tomato lectin using a number of approaches. Several new results were obtained. Firstly, a pulse/chase labelling approach demonstrated that metabolic turnover of these proteins was rapid compared to the total pool of cellular protein. Post translational processing of these proteins was assessed and was also found to be rapid. Secondly, selected clones were isolated by screening of a cDNA library using antibodies against the tomato lectin binding fraction. A variety of molecular, biochemical, cellular and immunological approaches were employed to characterise these candidates at a functional and molecular level. These studies also employed gene-knockdown techniques based on RNA interference to establish potential functions by phenotype analysis. All of the candidate clones were expressed in vitro, and all represented likely transmembrane proteins. One of the selected cDNA clones represented a protein which was localised specifically to the flagellar pocket/endocytic pathway. Another of these clones, along with a related gene identified by screening of the genome, encoded putative type I membrane proteins. These two proteins were expressed in a stage specific manner, were essential in their respective life cycle stages and were required for proper attachment of the flagellum. Loss of these proteins caused detachment of flagellae from the cell body with the exception of a single point at the base of the flagellum. A block in cytokinesis, but not mitosis, and a corresponding increase in number of nuclei/kinetoplasts, flagellae, and other organelles per cell was observed. Severe morphological changes occurred in these cells followed by ultimately, cell death. Similar general phenotypes have frequently been reported for constituents of the trypanosome flagellum, which suggested involvement of these two proteins at this subcellular location.