A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania
Summary (2 min read)
- Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania.
- 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.
- These studies have provided powerful tools leading to new insights on the requirements for LPG and related phosphoglycan -bearing molecules in both parasite stages within the mammalian and sand fly hosts (6, 7, 13-19).
- The essentiality of GDP-Fuc was unexpected since there are few reports of fucosylated molecules in Leishmania.
- Unexpectedly, FUT1 localizes within the parasite mitochondrion, an uncommon observation for glycosyltransferases.
- Database mining for fucosyltransferase (FUTs)/ arabinopyranosyltransferase candidates in L. major Using a diverse collection of FUTs compiled from GenBank or CAZY databases (36), the authors searched the predicted L. major proteome for proteins showing significant similarities and/or conserved motifs.
- To confirm the surprising prediction of mitochondrial localization, the authors expressed a C-terminal HA-tagged FUT1 protein using the pIR1NEO vector as an episome; this construct was introduced into the ∆fut1- / +pXNGPHLEO-FUT1 line, yielding ∆fut1--/ +pXNGPHLEO-FUT1 / pIR1NEO-FUT1-HA (Fig. 2E).
- Similarly, 67 % of the dim cells also grew out, and subsequent tests showed that 96% (26/27) tested lacked GFP and were phleomycin sensitive, while retaining tagged FUT1-HA, yielding ∆fut1-/+ pIR1NEO-FUT1-HA (Fig. 2F).
- The authors confirmed the fucosyltransferase activity of recombinant FUT using enzyme expressed within Leishmania.
- The authors next tested whether the essential function of FUT1 required fucosyltransferase activity, perhaps through a structural role (52).
- Previous studies have shown that GDP-Fucose synthesis is essential in trypanosomatids, yet for Leishmania and Trypanosoma brucei fucosylated molecules that might account for this requirement remained unknown.
- These data established that both mitochondrial localization and catalytic activity were required for essential FUT1 function.
- The authors were able to obtain only a single mutant completely lacking FUT1 entirely (∆fut1s; Figs. 7, 8).
- While two substrates for mammalian protein O-fucosyltransferase were inactive with LmjFUT1, two peptides predicted to be fucosylated in L. donovani functioned as acceptors, and MS/MS of the one tested confirmed fucosylation (Figs. 5B, S5).
- Proteomic surveys have revealed approximately 100 O-GlcNAc modified mitochondrial proteins (67-69), mostly encoded by nuclear genes raising the possibility of glycosylation prior to mitochondrial import (70).
Materials and Methods.
- L. major strain FV1 (LmjF or WT; WHO code MHOM/IL/80/Friedlin) was grown at 26°C in M199 medium (U.S. Biologicals) containing 10% heat-inactivated fetal bovine serum and other supplements and transfected by electroporation using a high voltage protocol as described (73, 74).
- Molecular methods were performed as described (30).
- For plasmid shuffling experiments, Δfut1-/ +pXNG-FUT1 was further transfected with episomal pIR1NEO (B6483) into which test FUT1 genes had been inserted (Table S1).
- Samples were viewed with a JEOL 1200EX transmission electron microscope (JEOL USA Inc., Peabody, MA).
- For biorthogonal labeling, parasites were inoculated into M199 medium at 1 x 105/mL (WT or Δlpg2-) or 3.8 x 106/ml (Δfut1s) and grown for three days in the presence/absense of 100µM alkyne fucose (Invitrogen C10264), after which parasites were washed in PBS.
Legend to Figure 6.
- CAT-MUT-HA has an R297A substitution; MTP-MUT-HA replaces two Glu residues in the MTP with Arg ; BLOCK-MUT-HA has the cytoplasmic protein PTR1 fused to the N-terminus.
- The results of mitochondrial localization tests (Panel D) and plasmid shuffling tests (Panel C) are summarized on the right of the illustrations.
- Growth in the absence of phleomycin and FACS sorting of ‘dim’ and ‘bright’ cells was performed as described in Fig. Δfut1-pXNG-FUT1/pIR-MUT-HA lines was grown for 24 h in the absence of phleomycin, and analyzed by GFP flow cytometry.
- Indirect immunofluorescence of HA tagged FUT1 expressed from pIRNEO in the Δfut1-/ +pXNGPHLEO-FUT1 background, also known as Panel D.
- The average and standard deviation of three preparations is shown.
Legend to Figure 8
- M, mitochondria; k, kinetoplast; black arrow, normal mitochondrial cristae; white arrows, aggregates inside mitochondria; star, bloated cristae.
- BglII forward primer to amplify ORF of LmjFUT1 SMB3956 CCCAGATCTTCAGATGAGTATCCATCCAGGG.
- L-SAT FUT1 primer a = l-PAC = l-WT (Fig. S3) SMB3990 CCCAGATCTCCACCATGCCTGATAATAGATACGGC.
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Q1. What contributions have the authors mentioned in the paper "A broadly active fucosyltransferase lmjfut1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan leishmania" ?
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Q2. What have the authors stated for future works in "A broadly active fucosyltransferase lmjfut1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan leishmania" ?
Future studies will address this possibility. Future studies will be necessary to resolve the nature of the GDP-Fucose and FUT1dependent product ( s ) in trypanosomatids, which genetic and biochemical data strongly predict must nonetheless exist. Potentially, trypanosomatid mitochondrial FUT1s may offer a facile system in the future for probing mitochondrial glycosylation in a setting uncomplicated by multiple isoforms targeted to diverse compartments, and its essentiality renders it an attractive target for chemotherapy of trypanosomatid parasites. L. donovani expresses a mannose-fucose conjugate whose structure has not been definitively established ( 33 ), and several L. donovani proteins exhibited MS/MS signatures suggestive of fucosylation ( 34 ).