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Tue22 Mar04:55pm(5 mins)
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Poster 6 |
Where:
T/005
Session:
Speaker:
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For all eukaryotes, surface glycoproteins and glycolipids are made in the secretory pathway (i.e., the endoplasmic reticulum and the Golgi apparatus) and, therefore, this is where the vast majority of glycosyltransferases reside.
However, previous research has identified a specific glycosyltransferase enzyme, a fucosyltransferase, in the single mitochondrion of Trypanosoma brucei and of Leishmania major (Bandini G, et al., 2021, eLife; Guo H, et al., 2021, PNAS). This enzyme, called FUT1, not only has an unusual mitochondrial localization, but also it is essential for both parasites. The presence of FUT1 suggests that some novel type of glycoprotein or glycolipid glycosylation occurs in the mitochondria of trypanosomatid parasites. Nevertheless, nothing is known about the orthologous enzyme from Trypanosoma cruzi (TcFUT1) and so we decided to perform expression and characterization studies with it.
Based on the work done with TbFUT1 (Bandini G, et al., 2021, eLife), initial expression and purification attempts were done using different E. coli strains as expression systems. When eventually recombinant TcFUT1 was purified, although in low yields, we performed fucosyltransferase activity assays involving a panel of synthetic glycosidic acceptor substrates and radioactive guanosine diphosphate fucose (GDP-[3H]Fuc) as the donor substrate. However, no activity was detected, and new trials were done using eukaryotic Expi293F cells as the expression system, since HEK293-derived cells have been reported as useful for expressing many human glycosyltransferases. At the same time, we have established a procedure to synthesize our own GDP-[3H]Fuc based on previous knowledge from our group on using the monoxenous parasite Crithidia fasciculata as an enzyme source for the generation of this and other GDP nucleotide sugars (Schneider P, et al., 1995, Biochem. J.; Mengeling B J, et al., 1999, Analytical Biochem.). Combining our home-made radioactive donor, and recombinant TcFUT1 obtained from the culture medium of our eukaryotic expression system, we aim to define the substrate specificity of this fucosyltransferase and potentially characterize the fine chemical structure of its product(s).
