A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and catalytic activity is essential in the parasitic protozoan Leishmania

TL;DR: In this article, the authors identified a fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical compartment for glycosyltransferases.

Abstract: Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite Leishmania. While GDP-Fucose synthesis is essential (Guo et al 2017), fucosylated glycoconjugates have not been reported in Leishmania major. 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 Δfut1- 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 Δfut1-s segregant (Δfut1s) was obtained in rich media, 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 (Bandini et al 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 renders it an attractive target for chemotherapy of these serious human pathogens. Significance Statement Abundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites including Leishmania. Through defining biosynthetic pathways we identified a fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical compartment for glycosyltransferases. FUT1 was essential for normal growth, requiring both mitochondrial localization and enzymatic activity. Loss of FUT1 in a unique segregant showed extensive mitochondrial defects. Enzymatic tests showed FUT1 could fucosylate glycan and peptide substrates in vitro, although as yet the native substrate is unknown. 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 these deadly parasites.

Summary

Introduction

• 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.

Results

• 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).

Discussion

• 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.

Figures

Figure 5. LmjFUT1 is a fucosyltransferase.

Figure 7. Recovery of a single FUT1-null mutant segregant (∆fut1s) .

Figure 3. LmjFUT1 is localized to the parasite mitochondrion

Figure 4. Expression of WT or mutant FUT1s in E. coli or within Leishmania.

Figure 1. Comparison of mitochondrial Leishmania major FUT1 relative to related fucosyltransferases.

Figure 2. Plasmid segregation tests show FUT1 is essential.

Figure 8. ∆fut1s shows multiple mitochondrial abnormalities.

Full text

May 13 2021
Title:
A broadly active fucosyltransferase LmjFUT1 whose
mitochondrial localization and catalytic activity is essential in
the parasitic protozoan Leishmania
Authors: Hongjie Guo
1,
, Sebastian Damerow
2,
, Luciana Penha
1,
, Stefanie
Menzies
1,
, Gloria Polanco
1
, Hicham Zegzouti
3
, Michael A. J. Ferguson
2
,
and Stephen M. Beverley
1,
*
Affiliations:
1
Dept. of Molecular Microbiology, Washington University School
of Medicine, St. Louis, MO 63110, USA;
2
Division of Biological
Chemistry & Drug Discovery, Wellcome Trust Biocentre, College of Life
Science, University of Dundee, Dundee DD1 5EH, Scotland, United
Kingdom;
3
R&D Department, Promega Corporation, 2800 Woods
Hollow Road, Madison, WI 53711, USA.
Corresponding author: Department of Molecular Microbiology, Campus Box
8230, Washington University School of Medicine, 660 S. Euclid Ave., St.
Louis MO 63110 USA. Telephone 314-747-2630, FAX 314-747-2634,
stephen.beverley@wustl.edu
.
Author Contributions: HG, SD, SM, LP GP MAJF and SMB designed research;
HG, SD, LP, GP, and SM performed the work; HZ contributed key reagents
and methods; HG, SD, LP, GP, SM, MAJF and SMB analyzed data; and HG
MAJF and SMB wrote the paper.
Competing Interest Statement: The authors disclose no competing interests.
Classification: Biological Sciences, Microbiology
Keywords: trypanosomatid protozoan parasites, glycobiology,
glycosyltransferase, fucose, chemotherapy
This PDF file includes: Main Text including Figures 1-8 and Table 1;
Supplemental Figures 1-6 and Supplemental Tables 1-2
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ABSTRACT (246 words)
Glycoconjugates play major roles in the infectious cycle of the trypanosomatid parasite
Leishmania. While GDP-Fucose synthesis is essential (Guo et al 2017), fucosylated
glycoconjugates have not been reported in Leishmania major. 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
-
s
segregant (∆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 re-expression. Thus, FUT1 along with the similar Trypanosoma brucei enzyme TbFUT1
(Bandini et al 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 renders it an attractive target for chemotherapy of these serious human pathogens.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.10.443387doi: bioRxiv preprint

Significance Statement (120 words)
Abundant surface glycoconjugates play key roles in the infectious cycle of protozoan parasites
including Leishmania. Through defining biosynthetic pathways we identified a
fucosyltransferase FUT1 that was localized to the parasite mitochondrion, an atypical
compartment for glycosyltransferases. FUT1 was essential for normal growth, requiring both
mitochondrial localization and enzymatic activity. Loss of FUT1 in a unique segregant showed
extensive mitochondrial defects. Enzymatic tests showed FUT1 could fucosylate glycan and
peptide substrates in vitro, although as yet the native substrate is unknown. 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 these deadly parasites.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 13, 2021. ; https://doi.org/10.1101/2021.05.10.443387doi: bioRxiv preprint

Introduction
Leishmania is a widespread human pathogen, with more than 1.7 billion people at risk,
several hundred million infected, and with 12 million showing active disease ranging from mild
cutaneous lesions to severe disfiguring or ultimate lethal outcomes (2-4). The Leishmania
infectious cycle alternates between extracellular promastigote in the midgut of sand flies and
intracellular amastigote residing within macrophages of the mammalian host, where it survives
and proliferates in highly hostile environments. These parasites have evolved specific
mechanisms enabling them to endure these adverse conditions, including a dense cell surface
glycocalyx composed of lipophosphoglycan (LPG), glycosylphosphatidylinositol (GPI) anchored
proteins (GP63 and GP46), glycosylinositolphospholipids (GIPLs) and secreted glycoconjugates
such as proteophosphoglycan (PPG) and secreted acid phosphatase (sAP) (reviewed in 5; 6-8).
One prominent feature of LPG, PPGs and sAPs is the presence of disaccharide phosphate
repeating units ([6Gal(β)1,4)Man(α1)-PO
4
]), also termed phosphoglycan or PG repeats.
Our lab has focused on both forward and reverse genetic approaches to map out
glycoconjugate synthesis in Leishmania, emphasizing genes impacting the glycocalyx (9, 10) as
well as ether lipids and sphingolipids (11, 12). 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).
In several Leishmania species modifications of the dominant PG repeats of LPG and PPG
play key roles in the insect stages in mediating both the attachment and release of promastigotes
and metacyclics, respectively, from the sand fly midgut via binding to midgut receptors there
(20). In L. major strain FV1 (LmjF), β1-3 galactosyl modifications of the PG repeating units
enable replicating promastigotes to bind to the midgut lectin PpGalec, while addition of D-
Arabinopyranose (D-Arap) to the side-chain galactosyl residues block this interaction and allow
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release of parasites for subsequent transmission (20-22). Accordingly, we used forward genetic
analysis to identify a large family of LPG side chain galactosyltransferases (SCG1-7) and D-
Arabinopyranosyltransferases (SCA1/2) mediating these key modifications (22-27).
As D-Arap is relatively uncommon in nature (28), we were motivated to explore its
synthetic pathway. Previously we identified two genes showing strong homology to the
bifunctional Bacteroides protein FKP mediating synthesis of GDP-L-Fucose (GDP-Fuc) through
successive kinase and pyrophosphorylase steps (29, 30). Many enzymes using L-Fucose will
also accept D-Arap (which differ only by the 6-methyl group), and assays of the two
recombinant Leishmania proteins showed that indeed one could synthesize both GDP-D-Arap
and GDP-Fuc (AFKP; LmjF.16.0480), while the second could only synthesize GDP- Fuc (FKP;
LmjF.16.0440; (30)). These data were consistent with studies showing the presence of both
GDP-Fuc and GDP-Arap in Leishmania (31). Correspondingly, genetic studies showed that
knockouts of AFKP completely abrogated GDP-Arap and arabinosylated LPG synthesis, while
knockouts of FKP showed little effect (30). However, we were unable to knockout both genes
simultaneously, suggesting an unanticipated role for GDP-Fuc. That the essential role of
A/FKPs depended on GDP-Fucose was established when GDP-Fuc but not GDP-Arap was
provided through expression of the two de novo GDP-Fucose enzymes from Trypanosoma
brucei, GDP-mannose 4,6-dehydratase (GMD) and GDP-Fuc synthetase, also known as GDP-4-
dehydro-6-deoxy-D-mannose epimerase/reductase (GMER) (32). Similarly the loss of de novo
GDP-fucose synthesis was also lethal in T. brucei, which lacks the FKP salvage pathway (32).
The essentiality of GDP-Fuc was unexpected since there are few reports of fucosylated
molecules in Leishmania. One is a “Fucose Mannose Ligand” from L. donovani (33) for which a
definitive structure is lacking. Several proteins were predicted to be fucosylated from mass
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Frequently asked questions

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" ?

Was not certified by peer review ) is the author/funder. 

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 ).