4.8 Article

A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania

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NATL ACAD SCIENCES
DOI: 10.1073/pnas.2108963118

关键词

trypanosomatid protozoan parasites; glycobiology; fucose; glycosyltransferase; chemotherapy

资金

  1. NIH [AI31078, AI29646]
  2. Department of Molecular Microbiology
  3. Wellcome Investigator Award [101842/Z/13/Z]
  4. Wellcome Trust [101842/Z/13/Z] Funding Source: Wellcome Trust

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This study identified a mitochondrial fucosyltransferase, FUT1, in the trypanosomatid parasite Leishmania, which is essential for cell viability. The enzyme showed a broad fucosyltransferase activity and its mitochondrial localization was crucial for normal growth and mitochondrial function. Mitochondrial FUT1s in trypanosomatids may be used to study mitochondrial glycosylation and could be a potential target for chemotherapy against these serious human pathogens.
Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in Leishmania major [H. Guo et al., J. Biol. Chem. 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; SCA1/2 play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that FUT2 and SCAL were not essential. Unlike most eukaryotic glycosyltransferases, the predicted alpha 1-2 fucosyltransferase encoded by FUT1 localized to the mitochondrion. A quantitative plasmid segregation assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null Delta fut1- background, established that FUT1 is essential. Similarly, plasmid shuffling confirmed that both enzymatic activity and mitochondrial localization were required for viability, comparing import-blocked or catalytically inactive enzymes, respectively. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it had a broad fucosyltransferase activity including glycan and peptide substrates. Unexpectedly, a single rare Delta fut1(-) segregant (Delta fut1(s)) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 reexpression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 [G. Bandini et al., bioRxiv, https://www.biorxiv.org/content/10.1101/726117v2 (2021)] joins the eukaryotic O-GlcNAc transferase isoform as one of the few glycosyltransferases acting within the mitochondrion. Trypanosomatid mitochondrial FUT1s may offer a facile system for probing mitochondrial glycosylation in a simple setting, and their essentiality for normal growth and mitochondrial function renders it an attractive target for chemotherapy of these serious human pathogens.

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