A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic <i>Leishmania</i>.

Abstract

Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite <i>Leishmania</i> While GDP-Fucose synthesis is essential, fucosylated glycoconjugates have not been reported in <i>Leishmania major</i> [H. Guo et al., <i>J. Biol. Chem.</i> 292, 10696-10708 (2017)]. Four predicted fucosyltransferases appear conventionally targeted to the secretory pathway; <i>SCA1/2</i> play a role in side-chain modifications of lipophosphoglycan, while gene deletion studies here showed that <i>FUT2</i> and <i>SCAL</i> were not essential. Unlike most eukaryotic glycosyltransferases, the predicted α 1-2 fucosyltransferase encoded by <i>FUT1</i> localized to the mitochondrion. A quantitative "plasmid segregation" assay, expressing <i>FUT1</i> from the multicopy episomal pXNG vector in a chromosomal null ∆<i>fut1</i>^- background, established that <i>FUT1</i> 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 ∆<i>fut1</i>^- segregant (<i>∆fut1</i>^<i>s</i> ) was obtained in rich media, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon <i>FUT1</i> reexpression. Thus, <i>FUT1</i> along with the similar <i>Trypanosoma brucei</i> 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.