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

Abstract

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 α 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 ∆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 ∆fut1− segregant (∆fut1s) 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.

An essential, kinetoplastid-specific GDP-Fuc: β-D-Gal α-1,2-fucosyltransferase is located in the mitochondrion of Trypanosoma brucei

“…Here, we report on a gene ( TbFUT1 ) encoding a mitochondrial α-1,2-fucosyltransferase protein (TbFUT1) in T. brucei that is essential to parasite survival. Similar results were obtained in the related trypanosomatid parasite L. major ( Guo et al, 2021 ), extending this unexpected finding across the trypanosomatid protozoans.…”

“…In conclusion, TbFUT1 is an essential -1,2-FUT that localizes to either outer membrane or inner compartments of the parasite mitochondrion and presents orthologues throughout the kinetoplastida. As described in Guo et al, 2021 , both activity and mitochondrial localization of the L. major homologue are required for parasite viability, whereas other putative L. major FUTs targeted to the secretory pathway are dispensable. Although no data are available so far on the enzymes from T. cruzi or other members of this group, these initial results suggest the intriguing possibility of an essential, conserved mitochondrial fucosylation pathway in kinetoplastids that might be exploitable as a common drug target.…”

“…Finally, Leishmania spp. express a family of α-1,2-arabinopyranosyltransferases (SCA1/2/L, CAZy family GT79) that decorate phosphoglycan side chains and have been suggested to act as FUTs in presence of excess fucose ( Guo et al, 2021 ).…”

“…Finally, given the propensity for TbFUT1 to transfer [ 3 H]Fuc from GDP-[ 3 H]Fuc to water, producing free [ 3 H]Fuc and presumably GDP, we set up a GDP-Glo assay similar to that used for LmjFUT1 ( Guo et al, 2021 ) to monitor the turnover by recombinant TbFUT1 of non-radioactive GDP-Fuc to GDP (see Materials and methods). In this assay, we could see TbFUT1-dependent turnover of GDP-Fuc to GDP in the absence of acceptor substrate (70 ± 2 pmol) and the stimulation of turnover in the presence of LNB acceptor substrate (265 ± 13 pmol), consistent with the results in the radiometric assay.…”

“…The essentiality and mitochondrial location of the orthologous protein in L. major , LmjFUT1, was reported in Guo et al, 2021 . Here we asked whether TbFUT1 could functionally substitute for LmjFUT1 using a plasmid shuffle approach ( Guo et al, 2017 ; Murta et al, 2009 ).…”