TY - JOUR
T1 - A broadly active fucosyltransferase LmjFUT1 whose mitochondrial localization and activity are essential in parasitic Leishmania
AU - Guo, Hongjie
AU - Damerow, Sebastian
AU - Penha, Luciana
AU - Menzies, Stefanie
AU - Polanco, Gloria
AU - Zegzouti, Hicham
AU - Ferguson, Michael A. J.
AU - Beverley, Stephen M.
N1 - Funding Information:
We thank Robert Haltiwanger for providing EGFR and TSR substrates, L. Mahal and L. Zhang for anti-Glc-Fuc antibody, Giulia Bandini for discussions and encouragement, Deborah E. Dobson and Lon-Fy Lye for comments on the manuscript, Wandy Beatty of the Molecular Microbiology Imaging Facility for transmission and immuno-electron microscopy, I. C. Almeida and S. Portillo for providing IB4 lectin, and R. R. Townsend, P. E. Gilmore, and R. W. Sprung of the Washington University Proteomics Shared Resource for performing MS analysis. For single-cell sorting, we thank the staff of the high-speed sorter core, Alvin J. Siteman Cancer Center, Washington University Medical School. This work was supported by NIH Grants AI31078 and AI29646 to S.M.B., an administrative supplement to AI31078 to G.P., Berg Postdoctoral Fellowships from the Department of Molecular Microbiology (S.M. and H.G.), and a Wellcome Investigator Award (101842/Z/13/Z) to M.A.J.F.
Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/8/17
Y1 - 2021/8/17
N2 - 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 (∆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 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.
AB - 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 (∆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 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.
KW - Chemotherapy
KW - Fucose
KW - Glycobiology
KW - Glycosyltransferase
KW - Trypanosomatid protozoan parasites
UR - http://www.scopus.com/inward/record.url?scp=85112412589&partnerID=8YFLogxK
U2 - 10.1073/pnas.2108963118
DO - 10.1073/pnas.2108963118
M3 - Article
C2 - 34385330
SN - 0027-8424
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 33
M1 - e2108963118
ER -