Glycosylation is the most abundant post translational modification in eukaryotic cells and can be accredited to an enormous variety of functions. Yet the tools used to study rare or unusual glycosylations are limited. N-Glycosylation, glycosylation of an asparagine residue, begins in the endoplasmic reticulum where a glycan precursor is synthesised by glycosyltransferases before it is transferred to a protein by an oligosaccharide transferase. The protein is then transferred to the Golgi where the glycan is further modified by glycosidases or glycosyltransferases. During this process, the array of glycosyltransferases present in each organism serves to produce a range of glycans, many of which are organism specific. Their formation and functions provide valuable information as to how certain parasites proliferate and cause disease. Therefore, new tools to study glycans and glycan processing enzymes could pave the way towards new therapeutics. Within this thesis, three chemical methods to explore glycosylation in Trypanosoma brucei and Plasmodium falciparum are described.
Although UDP-agarose serves to enrich glycosyltransferases from complex mixtures, it is not specific towards UDP-galactosyltransferases. This particular family of enzymes forms linkages in many unique glycans of T. brucei and represent interesting drug targets due to their absence in humans. Due to their unusual nature, no homologues have been identified. The majority of this PhD project was aimed at synthesising an activity based probe to selectively enrich galactosyltransferases from T. brucei using analogues of the donor sugar nucleotide, UDP-galactose. In a newly developed synthetic route, UDP-galactose and UDP-(4F)-galactose were attached to tentagel resin. To our knowledge, these are the first resin bound sugar-nucleotides. After initial method development with a commercially available galactosyltranferase along with other proteins, the resins were proven to bind with the desired selectivity. They were then used in an assay to enrich galactosyltransferases from T. brucei lysates. UDP-Galactose was unable to enrich galactosyltransferases from this complicated mixture, most likely due to a low affinity and the complexity of the proteins it was submitted to. UDP-(4F)-Galactose showed a higher affinity but was only able to enrich one galactosyltransferase: TbGT3. The assay was only performed once, therefore with repeated experiments this result may improve.
The second tool described in this thesis is the use of the small molecule inhibiter of N-glycosylation, NGI-1. From published data, this small molecule was predicted to only inhibit the transfer of high mannose glycans in T. brucei. In doing so, the effect of reduced glycosylation could be studied without the need for RNAi knock down of STT3B. NGI-1 was toxic to T. brucei but had a very high IC50 of 75.73 μM. Therefore, at lower concentrations the effect of the drug could be observed. By lectin blots, there appeared to be an effect of the drug on N-glycosylation with an increase in complex glycans and a decrease in high mannose. However, the results were not clear so mass spectrometry analysis of T. brucei’s variant surface glycoprotein were sought to determine the exact N-glycosylation profile.
The third tool described is to enrich glycoproteins from Plasmodium falciparum lysates using various lectin based methods and mass spectrometry analysis. Initially, glycoproteins were enriched using the FASP FACE protocol and lectins GLS II and WGA. Although the mass spectrometry results indicated the presence of glycoproteins, the method used was not accurate enough to determine their nature. An Orbitrap mass analyser was then used, which improved the accuracy so that the presence of glycopeptides was confirmed. Unfortunately, these peptides were not of high enough resolution to be identified. Magnetic bead bound GSL II and WGA enrichment was tested, but there were difficulties in conjugating the lectin to the beads. Chemically modifying the glycoproteins with a galactoslytransferase so that they could be enriched with ricin (instead of GSL II and WGA) was also tested. Mass spectrometry showed that the enrichment was not successful and alternate methods must be investigated.
These new methods to study N-glycosylation in T. brucei and P. falciparum require some optimisation. However, since both parasites synthesise unique (and in the case of P. falciparum, disputed) N-glycans, tools such as the ones described will be the most effective way to profile their N-glycosylation.Part of the Marie Curie ITN funded by the EU Commission (GA. 608295
