ABSTRACT
Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. Recently we showed that GDP-Fucose synthesis is essential, although fucosylated glycoconjugates have not been reported in Leishmania major. The Leishmania genome predicts at least five candidate fucosyltransferases, four of which appear targeted to the secretory pathway; SCA1 and SCA2 play a role in side-chain modifications of the abundant surface glycoconjugate 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. Enzymatic assays of tagged proteins expressed in vivo or of purified recombinant FUT1 showed it to have fucosyltransferase activity, with a relative broad substrate specificity including glycans and peptide substrates. A quantitative ‘plasmid segregation’ assay, expressing FUT1 from the multicopy episomal pXNG vector in a chromosomal null Δfut1-background established that FUT1 is essential. We used plasmid shuffling to confirm that both enzymatic activity and mitochondrial localization were essential for viability, comparing import-blocked or catalytically inactive enzymes respectively. Unexpectedly a single rare Δfut1s mutant was obtained, which showed severe growth defects accompanied by mitochondrial dysfunction and loss, all of which were restored upon FUT1 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1 (1) joins the eukaryotic O-GlcNAc transferases as one of the few glycosyltransferases acting within the mitochondrion. Current work is now oriented towards identifying the Leishmania fucosylated targets therein.
Significance Abundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites including Leishmania. In the course of defining their biosynthetic pathways we identified a fucosyltransferase that was localized to the parasite mitochondrion, a rare atypical compartment for such enzymes. Functional tests showed that this enzyme FUT1 was essential for normal growth, requiring both mitochondrial localization and enzymatic activity. Loss of FUT1 in a rare segregants showed extensive mitochondrial defects, all reversed upon FUT1 re-expression.
Enzymatic tests revealed an ability to fucosylate both glycan and peptide substrates, although as yet the native substrate is unknown. The essentiality of FUT1 in Leishmania and as shown elsewhere in Trypanosoma brucei suggests a potential as a chemotherapeutic target, and its mitochondrial localization in a simple genetically amenable eukaryote may provide an opportunity for probing the more complex mitochondrial isoforms of mammalian O-GlcNAc transferase.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- LPG
- lipophosphoglycan
- LmFV1
- L. major Friedlin V1
- FUT
- fucosyltransferase
- WT
- wild type
- GFP
- green fluorescent protein
- PBS
- phosphate buffer saline
- kDNA
- kinetoplast network DNA
- Fuc
- L-fucose
- D-Arap
- D-arabinopyranose
- MTP
- mitochondrial targeting peptide