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

  1. Giulia Bandini
  2. Sebastian Damerow
  3. Maria Lucia Sempaio Guther
  4. Hongjie Guo
  5. Angela Mehlert
  6. Jose Carlos Paredes Franco
  7. Stephen Beverley
  8. Michael AJ Ferguson

  • Curated by eLife

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    Evaluation Summary:

    This study demonstrates for the first time that the single annotated fucosyltransferase (TbFUT1) in the parasitic protist, Trypanosoma brucei is specifically targeted to the mitochondrion, rather than in the secretory pathway or cytoplasm, as in other eukaryotes. TbFUT1 utilizes glycan substrates in vitro and conditional down-regulation of TbFUT1 expression causes a severe growth defect in the two major developmental stages of these pathogens, indicating that it is essential for pathogenesis and a potential drug target.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Fucose is a common component of eukaryotic cell-surface glycoconjugates, generally added by Golgi-resident fucosyltransferases. Whereas fucosylated glycoconjugates are rare in kinetoplastids, the biosynthesis of the nucleotide sugar GDP-Fuc has been shown to be essential in Trypanosoma brucei . Here we show that the single identifiable T. brucei fucosyltransferase (TbFUT1) is a GDP-Fuc: β-D-galactose α-1,2-fucosyltransferase with an apparent preference for a Galβ1,3GlcNAcβ1-O-R acceptor motif. Conditional null mutants of TbFUT1 demonstrated that it is essential for both the mammalian-infective bloodstream form and the insect vector-dwelling procyclic form. Unexpectedly, TbFUT1 was localized in the mitochondrion of T. brucei and found to be required for mitochondrial function in bloodstream form trypanosomes. Finally, the TbFUT1 gene was able to complement a Leishmania major mutant lacking the homologous fucosyltransferase gene (Guo et al., 2021). Together these results suggest that kinetoplastids possess an unusual, conserved and essential mitochondrial fucosyltransferase activity that may have therapeutic potential across trypanosomatids.

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  1. eLife

    Author Response:

    Reviewer #2 (Public Review):

    The authors used a nice combination of biochemical methodologies, including NMR and mass spectrometry, to work out the substrate specificity of this activity; they concluded that it mainly transfers alpha1-2Fuc to Galbeta1-3GlcNAc but not Galbeta1-4GlcNAc dissacharides. Interestingly, Galbeta1-3GlcNAc residues are highly abundant on surface glycoconjugates expressed by the procyclic (midgut) stage and also as terminal sugars on N-glycans from flagellar pocket glycoproteins expressed in the blood stages, but their mitochondrial presence has never been found in any organism. It remains to be determined what is the natural substrate of this enzyme and whether fucosylation indeed occurs in the mitochondrion of kinetoplastid organisms.

    I only have few comments and no further experiments are requested.

    1. Did the authors try to determine the exact location of TbFUT1 within the parasite mitochondrion, for instance using TEM immunogold? This could help understanding access to GDP-Fuc (although I believe the enzyme is facing the cytosol) and also the possible location of the natural acceptor molecule(s).

    No, we did not. Cryo-immunoEM performed by Guo et al. suggests that the LmjFUT1 localises to the mitochondrial lumen. The assumption would be that TbFUT1 also localises to this mitochondrial compartment, but this indeed needs to be experimentally confirmed. A localisation in the mitochondrial lumen or the inter-membrane space would not suggest easy access to cytosolic GDP-Fuc pools and the need for transporters, since T. brucei sugar nucleotide biosynthetic enzymes are predominantly localised in the glycosomes. Consequently, these sugar precursors would need to be transported out of these organelles to the cytosol and then into ER, Golgi and, as we suggest here, mitochondrion.

    1. In relation with the previous point, given the challenges in trying to localise fucosylated glycans using fucose-specific lectins, I wonder if there is a precedent for detecting terminal beta-Gal residues on the trypanosome mitochondrion using lectins.

    Thank you for the suggestion. This is definitely an experiment worth trying in the search for TbFUT1 endogenous substrates. We concentrated our efforts on the fucose-specific lectins, although unsuccessfully, and did not look at staining of either wild type or TbFUT1-deficient cells with beta-Gal specific lectins. Previously published IFA with the beta-Gal specific lectin RCA120 labelled the flagellar pocket of bloodstream form trypanosomes, where the N-glycans carrying poly-LacNAc repeats are abundant. However, no permeabilisation step was performed and so any organelle labelling would have been missed (Atrih et al., 2005, JBC, 280:865-871)

    1. I found interesting that N-terminal tagging of TbTUF1 sends the protein to the Golgi apparatus. This seems like a great coincidence for a protein that normally would be predicted to be Golgi-resident, so I wonder if there is any identifiable Golgi targeting sequence within TbTUF1. Also, there was any attempt to localise the protein after deletion of the mitochondrial signal (no tagging)?

    We agree with the reviewer that the localization of the N-terminally tagged TbFUT1 to the Golgi is an odd coincidence. It is worth noting that L. major FUT1 tagged at the N-terminus localizes to the cytosol as described in Guo et al., 2021. The same is observed of a tagged LmjFUT1 lacking the N-terminal mitochondrial targeting sequence. Furthermore, data from the TrypTag project suggests N-terminal tagging of TbFUT1 in PCF results in cytosolic and nucleoplasm localisation (tryptag.org, Dean et al., 2017, Trends Parasitol, 33:80-82). As briefly mentioned in the results section, the same algorithms that suggested untagged TbFUT1 was likely to localize to the mitochondrion, confirm this prediction for the C-terminally MYC3-tagged protein, but not the N-terminal HA3-tagged versions. However, the predictions for either HA3-TbFUT1 or HA3-TbFUT1-MYC3 do not support the observed Golgi localisation and are in better agreement with the cytosolic staining observed for the comparable LmjFUT1 construct or PCF expression. In the case of mammalian and yeast Golgi-resident glycosyltransferases (GTs), retention of these type II membrane proteins to the Golgi seems to be dependent on a combination of features in the cytosolic tail, TM and stem domains however no specific sequence or motif has been identified so far (Tu & Banfield 2010, Cell Mol Life Sci, 67:29-41). There is no predicted signal peptide for TbFUT1 and the prediction of a type II topology is weak (see Reviewer 1, point 3) and thus it does not fit with what is known about retention in the Golgi.

    We did not try to localize TbFUT1 lacking the putative mitochondrial targeting sequence, but the experiment is very much worth performing to better understand this fucosyltransferase behaviour (see also Reviewer 3, point 1).

  2. eLife

    Evaluation Summary:

    This study demonstrates for the first time that the single annotated fucosyltransferase (TbFUT1) in the parasitic protist, Trypanosoma brucei is specifically targeted to the mitochondrion, rather than in the secretory pathway or cytoplasm, as in other eukaryotes. TbFUT1 utilizes glycan substrates in vitro and conditional down-regulation of TbFUT1 expression causes a severe growth defect in the two major developmental stages of these pathogens, indicating that it is essential for pathogenesis and a potential drug target.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In 2007 the authors published a paper demonstrating that GDP-fucose, the donor substrate for fucosyltransferases (FUTs), is essential for Trypanosoma brucei growth in procyclic and bloodstream forms (Turnock et al., 2007). At that time, no FUTs or fucosylated proteins were known to exist in T. brucei. Here, using bioinformatic analysis, the authors identified a single FUT homologue in the T. brucei genome, TbFUT1, which belongs to the GT11 CAZy family of a1,2-FUTs. They expressed TbFUT1 in E. coli and demonstrated that it is a FUT that transfers fucose to acceptor substrates, preferentially with a terminal Galb1,3GlcNAc. Glycosidase digestions, mass spectral and NMR analyses demonstrated addition of fucose to the 2'-OH of the galactose in alpha-linkage. They generated conditional null (with a tetracycline-inducible promoter) alleles of TbFUT1 in both bloodstream (BSF) and procyclic (PCF) forms of the parasite. Deletion of TbFUT1 resulted in a significant growth defect in both forms when grown in vitro. Surprisingly, TbFUT1 localized to the single mitochondrion in T. brucei, and deletion of TbFUT1 disrupted mitochondrial activity. A homolog exists in Leishmania major (LmjFUT1), which has recently been reported to be essential for L. major growth (Guo et al. 2021). The authors showed that TbFUT1 rescued the growth defect in LmjFUT1 mutants, and also localized to the mitochondrion in L. major.

    This study is the first to identify a FUT in T. brucei, and the first to identify a FUT localized to mitochondria in any species. FUTs are typically localized in the Golgi, ER, or as more recently described, in the cytoplasm, but this is the first demonstration of a FUT in mitochondria. Only one glycosyltransferase has been reported in mitochondria, O-GlcNAc transferase (OGT). OGT transfers GlcNAc directly to protein, so no additional glycosylation machinery is necessary. The fact that TbFUT1 prefers a Galb1,3GlcNAc acceptor substrate strongly suggests additional glycosyltransferases will also be localized to the mitochondria in T. brucei and L. major. Thus, these are highly significant and novel studies. They are well done and rigorously performed. A few minor suggestions for clarity and presentation of the data are needed.

  4. eLife

    Reviewer #2 (Public Review):

    The authors used a nice combination of biochemical methodologies, including NMR and mass spectrometry, to work out the substrate specificity of this activity; they concluded that it mainly transfers alpha1-2Fuc to Galbeta1-3GlcNAc but not Galbeta1-4GlcNAc dissacharides. Interestingly, Galbeta1-3GlcNAc residues are highly abundant on surface glycoconjugates expressed by the procyclic (midgut) stage and also as terminal sugars on N-glycans from flagellar pocket glycoproteins expressed in the blood stages, but their mitochondrial presence has never been found in any organism. It remains to be determined what is the natural substrate of this enzyme and whether fucosylation indeed occurs in the mitochondrion of kinetoplastid organisms.

    I only have few comments and no further experiments are requested.

    1. Did the authors try to determine the exact location of TbFUT1 within the parasite mitochondrion, for instance using TEM immunogold? This could help understanding access to GDP-Fuc (although I believe the enzyme is facing the cytosol) and also the possible location of the natural acceptor molecule(s).

    2. In relation with the previous point, given the challenges in trying to localise fucosylated glycans using fucose-specific lectins, I wonder if there is a precedent for detecting terminal beta-Gal residues on the trypanosome mitochondrion using lectins.

    3. I found interesting that N-terminal tagging of TbTUF1 sends the protein to the Golgi apparatus. This seems like a great coincidence for a protein that normally would be predicted to be Golgi-resident, so I wonder if there is any identifiable Golgi targeting sequence within TbTUF1. Also, there was any attempt to localise the protein after deletion of the mitochondrial signal (no tagging)?

  5. eLife

    Reviewer #3 (Public Review):

    A great strength of the manuscript is the meticulous, very convincing biochemical analysis of the recombinant trypanosomal fucosyltransferase. No surprise since it has been done by leaders in the field. Also the immunofluoresecence localization is convincing, within the limits of the technique. The authors went through the great effort of producing a monospecific antiserum against the fucosyltransferase which allows them to localize the natively expressed untagged protein. The conditional knock out cell lines for both cell cycle stages unambiguously demonstrate that the enzyme is essential for cell growth.
    The manuscript is well written, has clearly composed figures and the results are presented in a logical way. I agree with the authors general conclusions. My major criticism is that I think both the localization of the enzyme as well as its physiological properties could have been analyzed in some more detail using the great tools the authors produced in their study (cell lines, antiserum). Having said that I am aware that characterizing the exact function of the protein might be beyond the scope of this study.

  6. Version published to 10.7554/elife.70272 on eLife
  7. Version published to 10.1101/726117 on bioRxiv