Studies on kinetoplastid mitochondrial fucosyltransferases

  • Jose Carlos Paredes Franco

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Protozoan parasites from the Trypanosomatidae family cause terrible diseases that affect human and animal health worldwide. The causative agents of leishmaniases (Leishmania spp.), Chagas disease (Trypanosoma cruzi) and African trypanosomiases (Trypanosoma brucei subsp.) rely heavily on glycoconjugates for their survival and infectivity. Thus, the study of the glycobiology of these parasites represents an opportunity for the identification of novel and much needed therapeutic targets.

Glycosylation processes in eukaryotic cells are carried out by glycosyltransferases located mostly in the secretory pathway, and some in the cytoplasm. Consequently, most of the synthesized glycans, attached to proteins and lipids, are present on their cell surfaces, or in their endosome/lysosome systems, or are secreted.

The discovery of mitochondrial glycosyltransferases in trypanosomatids has challenged this established consensus. Previous work in our group and by collaborators showed that the protein product of an essential fucosyltransferase gene (FUT1) is located inside the mitochondrion of the parasites Leishmania major and Trypanosoma brucei. Although in vitro fucosyltransferase activity has been demonstrated for recombinant LmjFUT1 and TbFUT1, to date, the endogenous substrate(s) of these fucosyltransferases have not been identified. Furthermore, prior to this thesis, nothing was known about the orthologous fucosyltransferase (TcFUT1) in Trypanosoma cruzi.

In this study, I detail approaches to obtain an active form of TcFUT1, both by heterologous (bacteria and human cell) expression and homologous overexpression, in order to characterize its activity and substrate specificity using a panel of synthetic glycan acceptors. This defined TcFUT1 as a GDP-Fuc : βGal α1-2 fucosyltransferase. In parallel, I exploited the metabolism of the non-pathogenic trypanosomatid Crithidia fasciculata to synthesize a critical component for glycosylation activity assays (GDP-[3H]Fuc). Furthermore, initial steps were taken to characterize a high-molecular weight endogenous fucosylated substrate by biosynthetic labelling of C. fasciculata with [3H]-labelled fucose. To assist this approach, we have also generated a genetically modified C. fasciculata cell line that should be deficient in the synthesis of lipoarabinogalactan (LAG). Additionally, based on immunolocalization assays and comparison with similar reports on in T. cruzi, we propose the mitochondrial localization of TcFUT1, at least in the epimastigote form.
Date of Award2023
Original languageEnglish
SupervisorMichael Ferguson (Supervisor)

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