PLoS Negl Trop Dis plos plosntds PLOS Neglected Tropical Diseases 1935-2735 Public Library of Science San Francisco, CA USA 10.1371/journal.pntd.0009132 PNTD-D-20-01075 Research Article Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoansTrypanosoma Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoansTrypanosomaTrypanosoma bruceiTrypanosoma brucei gambiense Biology and life sciencesBiochemistryNucleotides Biology and life sciencesBiochemistryBiosynthesis Research and analysis methodsMicroscopyLight microscopyFluorescence microscopyImmunofluorescence microscopy Biology and life sciencesCell biologyCellular structures and organellesCell membranesMembrane proteins Biology and life sciencesCell biologyCellular structures and organellesCell membranes Biology and life sciencesOrganismsEukaryotaProtozoansParasitic protozoansTrypanosomaTrypanosoma brucei Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei Glycosomal nucleotide sugar synthesis in T. brucei Sampaio Guther Maria Lucia Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Supervision Validation Visualization Writing – original draft Writing – review & editing 1 https://orcid.org/0000-0002-0747-7317 Prescott Alan R. Data curation Formal analysis Investigation Methodology Resources Software Validation Visualization Writing – original draft 2 Kuettel Sabine Methodology Resources 1 https://orcid.org/0000-0002-0051-017X Tinti Michele Data curation Formal analysis Resources Software Validation Visualization Writing – original draft 1 https://orcid.org/0000-0003-1321-8714 Ferguson Michael A. J. Conceptualization Formal analysis Funding acquisition Project administration Supervision Writing – original draft Writing – review & editing 1 * Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, United Kingdom Jardim Armando Editor McGill university, CANADA

The authors have declared that no competing interests exist.

 * E-mail: m.a.j.ferguson@dundee.ac.uk 16 2 2021 2 2021 15 2 e0009132 16 6 2020 12 1 2021 2021 Sampaio Guther et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.

 Author summary

All eukaryotes add sugar chains to proteins to make glycoproteins, most of which decorate the cell surface and play central roles in how cells interact with their environment and with other cells. These sugar chains are built up using nucleotide sugars to donate the individual sugars. The nucleotide sugars themselves are generally made in the cytoplasm of cells but in Trypanosoma brucei, the causative agent of human African trypanosomiasis and nagana in cattle, they are made inside small organelles called glycosomes. This very unusual arrangement in parasite metabolism is notable and may offer therapeutic opportunities.

 http://dx.doi.org/10.13039/100004440 Wellcome Trust 101842/Z13/Z https://orcid.org/0000-0003-1321-8714 Ferguson Michael A. J. This work was funded by The Wellcome Trust through an Investigator Award (10842/Z/13/Z) to MAJF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stage vor-update-to-uncorrected-proof Publication Update 2021-02-26 Data Availability Immunofluorescence microscopy localisation of nucleotide sugar biosynthetic enzymes in procyclic form T. brucei (wide-field images) are available at DOI: 10.6084/m9.figshare.13124909. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD023124. Introduction

The tsetse-fly transmitted protozoan parasite Trypanosoma brucei is responsible for human and animal African trypanosomiasis. The bloodstream form (bsf) of this organism depends on a surface coat made of glycosylphosphatidylinositol (GPI) anchored and N-glycosylated variant surface glycoprotein (VSG) to evade the host innate immune system and the acquired immune system through antigenic variation [1]. The bsf parasite also expresses many lower abundance glycoproteins, such as a novel transferrin receptor (TfR) [2–4], a lysosomal/endosomal protein called p67 [5], the invariant surface (ISG) and endoplasmic reticulum (IGP) glycoproteins [6,7], the Golgi/lysosomal glycoprotein tGLP-1 [8], the membrane-bound histidine acid phosphatase TbMBAP1 [9], the flagellar adhesion zone glycoproteins Fla1-3 [10,11], the flagellar pocket/endosomal system haptoglobin-hemoglobin receptor (HpHbr) [12] and serum resistance antigen (SRA) [13], the complement factor H receptor (FHR) [14] and others, such as the metacyclic trypomastigote-specific ISG [15]. Some of these are metacyclic and/or bsf specific glycoproteins (eg. VSG, TfR, ISG, TbMAP1, HpHbr, SRA, FHR) while others are also common to the tsetse midgut-dwelling procyclic form (pcf) of the parasite. Further, pcf parasites also express unique glycoproteins, notably the abundant GPI-anchored procyclins, some of which are N-glycosylated [16,17], and a high-molecular weight glycoconjugate [18,19]. Many of the N-glycan structures expressed by bsf T. brucei have been solved and these include conventional oligomannose and biantennary complex structures as well as paucimannose and extremely unusual ‘giant’ poly-N-acetyl-lactosamine (poly-LacNAc) containing complex structures [20–23]. In contrast, only oligomannose N-glycans have been structurally described in wild type pcf trypanosomes [16,24]. The GPI anchor structures of several bsf VSGs [25–28] and of the TfR [5,29] have also been solved, as have those of pcf procyclins [16]. Both bsf and pcf GPI anchors contain the canonical conserved GPI core structure but they are the most divergent among the eukaryotes in terms of their carbohydrate sidechains, containing up to 1 βGal and 5 αGal residues in the bsf GPI sidechains and multiple, branched, N-acetyllactosamine and lacto-N-biose repeats capped with α2–3 sialic acid in the pcf GPI sidechains [16,27,30,31]. Recently, the O-glycosylation (via novel Glcα1-O-Ser linkages) of certain VSG variants has also been described [32]. The entire repertoire of known T. brucei N-linked, O-linked and GPI glycans is composed exclusively of the monosaccharides mannose, galactose, glucose, N-acetylglucosamine, glucosamine and sialic acid. In addition, a trace of fucose has been reported in the pcf high-molecular weight glycoconjugate [19], and recent data suggest that fucosylation may occur inside the parasite mitochondrion [33]. These combined monosaccharide compositions are consistent with the nucleotide sugar biosynthetic capacity of bsf and pcf trypanosomes [34] (Fig 1), apart from the absence of CMP-sialic acid. However, this apparent discrepancy is because sialic acid is added to the procyclin GPI sidechains via cell-surface transialidase enzymes that transfer α2–3 sialic acid from host glycoconjugates in a nucleotide sugar-independent reaction [35,36]. The non-N-acetylated glucosamine in the GPI anchors originates from UDP-GlcNAc and is the product of the GPI pathway GlcNAc-PI de-N-acetylase [37,38]. Although mature T. brucei N-glycans do not contain glucose, the trypanosome N-glycans undergo reversible glucosylation from UDP-Glc in the endoplasmic reticulum (ER) as part of a UDP-Glc: unfolded glycoprotein glucosyltransferase (UGGT) / α-glucosidase II / calreticulin ER quality control system [23,39]. In T. brucei UDP-Glc is also the donor for the biosynthesis of base J [40], the obligate precursor of UDP-Gal (via UDP-Glc 4’-epimerase (TbGALE) [41]), and presumed to be the donor for VSG O-glycosylation [32].

10.1371/journal.pntd.0009132.g001 Scheme of nucleotide sugar biosynthesis in <italic>T</italic>. <italic>brucei</italic>.

Nucleotide sugar biosynthesis in T. brucei according to [34], showing the 14 biotransformations and 13 enzymes involved. No dedicated phospho-glucose mutase (PGM) gene exists in T. brucei and the interconversion of Glc6P and Glc1P is performed by PAGM and/or PMM [42]. The enzyme abbreviations (in italics) appear in the Introduction. The terminal nucleotide sugars used for glycosylation reactions are in bold and underlined. Information on enzyme essentiality, protein and antibody production, and sub-cellular localisation appear in (Table 1). The main source of all of the nucleotide sugars is Glc but both Man and GlcN can enter their respective pathways via hexokinase (HK1).

Nucleotide sugar biosynthesis in T. brucei is quite conventional, in so far as homologues of most of the necessary enzymes can be found by BLASTp searches with corresponding prokaryotic and/or eukaryotic amino acid sequences [34]. The only exception to this is the absence of a canonical phosphoglucose mutase (PGM) enzyme, the function of which is redundantly replaced by the parasite phosphomannose mutase (TbPMM) and phospho-N-acetylglucosamine mutase (TbPAGM) enzymes [42]. Further, the following trypanosome enzymes of nucleotide sugar biosynthesis have been expressed in E. coli and shown, where determined, to have typical kinetic properties and conserved three dimensional structures (Table 1): UDP-glucose 4’-epimerase (TbGALE) [43,44]. UDP-glucose pyrophosphorylase (TbUGP) [45]. Phosphomannose isomerase (TbPMI) [46]. Phosphomannose mutase (TbPMM) [42,47]. Mannose phosphate guanyltransferase (TbMPGT) [47,48]. Glucosamine-6-phosphate N-acetyltransferase (TbGNA) [49]. Phospho-N-acetylglucosamine mutase (TbPAGM) [42]. UDP-N-acetylglucosamine pyrophosphorylase (TbUAP) [50,51]. GDP-mannose dehydratase (TbGMD) [52]. GDP-4-dehydro-6-deoxy-D-mannose epimerase/reductase (TbGMER) [52]. However, where nucleotide sugar biosynthesis in T. brucei diverges most dramatically from the norm is with respect to its subcellular location. Thus, whereas eukaryotic nucleotide sugar biosynthesis is generally either known or assumed to occur in the cytoplasm, for T. brucei many of these reactions appear to occur in the glycosomes [42,44–46,49,50,52–54] (Table 1). Glycosomes are kinetoplastid peroxisome-like membrane-bound organelles, named such because they contain enzymes of glycolysis [55–59,66–68], including hexokinase (TbHK1) and phospho-glucose isomerase (TbPGI) that are also components of the de novo pathways to the nucleotide sugars.

10.1371/journal.pntd.0009132.t001 Summary of data on <italic>T</italic>. <italic>brucei</italic> nucleotide sugar biosynthetic enzymes.

Data are from the literature [#] or from this paper [TP]. GL = glycosomal; CP = cytoplasmic; IFM = immunofluorescence microscopy; *indicates that IFM was performed using an epitope tag rather than an antibody to the whole protein; PTS = peroxisome targeting sequence (type 1 or type 2) for the respective T. brucei (Tb), T. cruzi (Tc) and L. major (Lm) enzymes [50,65]. Enzyme abbreviations are listed in the Introduction, with the exception of TbGFAT, glutamine: fructose-6-phosphate aminotransferase.

Enzyme Gene Essentialfor cellsurvival Protein expression,crystallography andantibodies IFM Digitonin Latency Sub-cellularFractionation Glycosomeproteome PTS
bsf pcf Prot Xtal Ab bsf pcf bsf pcf bsf pcf bsf pcf Tb Tc Lm
TbHK1 Tb927.10.2010 [62][63] [60] [59] GL[59] GL[59] GL[58][59] GL[57][59] GL[55][56] GL[57][58] GL[53] GL[54][TP] 2 2 2
TbPGI Tb927.1.3830 [64] [64] GL[59] GL[55][56] GL[57] GL[53][54][TP] 1 1 1
TbUGP Tb927.10.13130 [45] [45] [45] [45] GL[45] GL[TP] GL[TP] GL[TP] GL[TP] GL[54] 1
TbGALE Tb927.11.2730 [41] [44] [41] [43] [44][TP] GL[44] GL[44][TP] GL[TP] GL[TP] GL[44] GL[54] 1 2
TbGFAT Tb927.7.5560
TbGNA Tb927.11.11100 [49] [49] [49] [49] GL[49] GL[TP]
TbPAGM Tb927.8.980 [42] [42] [42] GL[42] GL+CP[TP] GL+CP[42] GL+CP[TP]
TbUAP Tb927.11.2520 [50] [50] [50] [50][TP] GL[50] GL[TP] GL[TP] GL[TP] GL[54] 1 1 1
TbPMI Tb927.11.14780 [46] [46] [46] GL[46] GL[TP] GL[TP] GL[TP] GL[TP] GL[53][54][TP] 1 1
TbPMM Tb927.10.6440 [42] [42] [42] [42] GL[42] GL[TP] GL[42][TP] GL[TP] 1 1
TbMPGT Tb927.8.2050 [47] [47][48][TP] [TP] CP[48] GL[TP] GL[TP] GL[TP] GL[TP]
TbGMD Tb927.10.15490 [52] [52] [52] GL[52]*
TbGMER Tb927.11.13990 [52] [TP] GL[TP] GL[TP] GL[TP]

Here, we analyse the subcellular locations of nine nucleotide sugar biosynthetic enzymes in pcf trypanosomes by immunofluorescence microscopy using mono-specific mouse polyclonal antibodies raised to purified recombinant, soluble and enzymatically active proteins. Further, we use eight of these nine antibodies to determine the digitonin latency of the corresponding enzymes in both pcf and bsf trypanosomes. In addition, we localise three representative NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP) to the glycosome-containing small granular fraction by subcellular fractionation, and demonstrate heterogeneity in glycosome composition by density gradient centrifugation and proteomics.

 Results Subcellular localization by immunofluorescence microscopy

In previous work, we have purified the following soluble, enzymatically active, recombinant nucleotide sugar biosynthetic enzymes: TbUGP, TbGALE, TbGNA, TbPAGM, TbUAP, TbPMI and TbPMM (Table 1). With these, we have prepared either rabbit (TbGALE) or mouse polyclonal antibodies for immunofluorescence microscopy (IFM) localization studies against bsf trypanosomes (Table 1). To these we have added new polyclonal mono-specific mouse antibodies to recombinant TbGALE, TbGNA, TbUAP, TbMPGT and TbGMER (see Methods). In all cases, the mouse antibodies were affinity-purified against immobilized immunogen and/or demonstrated to be mono-specific by Western blotting (Table 2; S2 Fig). These antibodies were used in immunofluorescence microscopy against fixed and permeabilized pcf trypanosomes, along with rabbit antibodies to definitive glycosomal (TbGAPDH) and cytosolic (TbEnolase) markers (Figs 2, 3, and 4). Wider field images containing several cells are available in (10.6084/m9.figshare.13124909). In all cases, except for TbPAGM (Fig 2, middle panels), the mono-specific mouse antibodies produced punctate staining that was coincident, at least in part, with the rabbit anti-TbGAPDH antibody staining, suggesting glycosomal locations. In contrast, there was no overlap with cytoplasmic anti-TbEnolase immunostaining. In the case of TbPAGM, the immunostaining suggested both glycosomal and cytosolic localization. Interestingly, the punctate co-staining with anti-NS biosynthetic enzyme and anti-TbGAPDH antibodies was imperfect, in so far as some puncta were more red or green than yellow. This implies heterogeneity in glycosomal contents, which is discussed later. However, although the ratios of TbGAPDH (red) to NS enzyme (green) signals vary widely, quantitative analysis (Table 3) shows that the majority of the NS enzyme signals colocalize with punctate TbGAPDH signals, with the exception of TbPAGM.

10.1371/journal.pntd.0009132.g002 Immunofluorescence microscopy localisation of UDP-GlcNAc biosynthetic enzymes in procyclic form <italic>T</italic>. <italic>brucei</italic>.

Enzymes of UDP-GlcNAc biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

 10.1371/journal.pntd.0009132.g003 Immunofluorescence microscopy localisation of GDP-Man biosynthetic enzymes in procyclic form <italic>T</italic>. <italic>brucei</italic>.

Enzymes of GDP-Man biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

 10.1371/journal.pntd.0009132.g004 Immunofluorescence microscopy localisation of UDP-Glc, UDP-Gal and GDP-Fuc biosynthetic enzymes in procyclic form <italic>T</italic>. <italic>brucei</italic>.

Enzymes of UDP-Glc, UDP-Gal and GDP-Fuc biosynthesis were localised with mono-specific mouse antibodies (green channels). Authentic glycosome and cytosol markers TbGAPDH and TbEnolase, respectively, were localised with specific rabbit antisera (red channels). Merged green and red channels and phase-contrast images are also shown.

 10.1371/journal.pntd.0009132.t002 Antibodies to nucleotide sugar biosynthetic enzymes used in this study.

[TP] = this paper.

Enzyme Antibody species Affinity purified Mono-specific by Western Reference
TbUGP Mouse Yes Yes [45]
TbGALE Mouse No Yes [TP]
TbGNA Mouse Yes Yes [TP]
TbPAGM Mouse No Yes [42]
TbUAP Mouse Yes Yes [TP]
TbPMI Mouse No Yes [46]
TbPMM Mouse No Yes [42]
TbMPGT Mouse No Yes [TP}
TbGMER Mouse No Yes [TP]
10.1371/journal.pntd.0009132.t003 Quantitative analysis of anti-NS enzyme and anti-TbGAPDH colocalization by IFM in pcf cells.
NS Enzyme na Mander’s colocalization coefficientb
TbGNA 6 0.85 ± 0.05
TbPAGM 5 0.33 ± 0.06
TbUAP 5 0.89 ± 0.02
TbPMI 9 0.96 ± 0.01
TbPMM 4 0.73 ± 0.06
TbMPGT 4 0.89 ± 0.04
TbUGP 8 0.89 ± 0.03
TbGalE 4 0.88 ± 0.05
TbGMER 7 0.88 ± 0.05

a n is number of cells uses in the analysis.

b The Mander’s colocalization coefficient (± standard deviation of the mean) is a measure of the amount of green (anti-NS enzyme) signal above background that colocalizes with red (anti-TbGAPDH) stained compartments.

 Subcellular localization by digitonin latency

Digitonin latency is a powerful adjunct technique to assess whether soluble proteins are either cytosolic or sequestered in membrane-bound intracellular organelles. Digitonin is a steroidal-saponin detergent-like natural product that first permeabilizes and subsequently solubilizes biological membranes. Digitonin has a preference for high sterol content membranes. Thus, cytosolic soluble proteins are liberated from cells at very low digitonin concentrations, as the sterol-rich plasma membrane is the first affected, and organellar contents are subsequently liberated at significantly higher digitonin concentrations. The requirement for a higher digitonin concentration to effect protein release is known as ‘digitonin latency’. This method has proved extremely useful in discriminating cytosolic versus glycosomal proteins in trypanosomatids, for example [42,58–60,61,69]. In our studies, we used the aforementioned mono-specific polyclonal mouse antibodies to eight nucleotide biosynthetic enzymes, along with rabbit antibodies to authentic glycosome lumen (TbGAPDH or TbAldolase), glycosome membrane (TbPEX13.1) and soluble cytosolic (TbEnolase) markers to assess digitonin latency of these enzymes in pcf and bsf trypanosomes (Fig 5A and 5B, respectively). As expected, the cytosolic marker, TbEnolase, was released from both lifecycle stages at very low digitonin concentrations whereas TbAldolase and TbGAPDH were not quantitatively released until 0.1% TX100 was used. This was not because the glycosome membrane was not being gradually solubilized by the digitonin, since the gradual release of membrane protein TbPEX13 was evident in these experiments, but because of the possibly aggregated nature of these very abundant proteins in what has been proposed to be a dense crystalloid core inside of glycosomes [56]. In contrast, TbPAGM showed a biphasic release pattern, consistent with dual cytoplasmic and glycosomal location. All the other nucleotide sugar biosynthetic enzymes showed release starting around 0.04 mg digitonin / mg protein, consistent with them being predominantly or exclusively glycosomal proteins. Interestingly, in all cases, the proteins seem to be slightly harder to release with digitonin from pcf cells than from bsf cells.

10.1371/journal.pntd.0009132.g005 Digitonin latencies of nucleotide sugar biosynthetic enzymes in bloodstream and procyclic forms of <italic>T</italic>. <italic>brucei</italic>.

Trypanosomes (bsf, panel A; pcf, panel B) were treated with increasing concentrations of digitonin (0.01 to 0.1 mg digitonin per mg trypanosome protein) and with no detergent and 0.1% TX100 as controls for no cell permeabilization and total cell and glycosome membrane permeabilization, respectively. The release of the cytoplasmic marker TbEnolase at the lowest digitonin concentration is consistent with the selective permeabilization of the plasma membrane over the glycosome membrane. The release of TbAldolase (panel A) or TbGAPDH (panel B) at higher digitonin concentrations, or only with 0.1% TX100, i.e., showing latency, is consistent with their known glycosome lumen locations. From the patterns of digitonin latency of the nucleotide sugar biosynthetic enzymes, we conclude that all shown, except TbPAGM, are principally or exclusively protected against release by digitonin by being sequestered in a low-sterol intracellular membrane-bound compartment.

 Subcellular localization by subcellular fractionation and evidence for glycosome heterogeneity by density gradient centrifugation and proteomics

Procyclic cells were lysed using a high-pressure homogenizer [54] and the lysate was subjected to differential centrifugation according to [70]. This procedure results in a nuclear fraction, a large granular fraction (LG), a small granular fraction (SG) and a cytoplasmic fraction (C), with the latter also containing microsomes of the plasma membrane, Golgi apparatus and endoplasmic reticulum (ER). These fractions were subjected to SDS-PAGE and Western blotting and the blots were probed with antibodies to authentic glycosomal, mitochondrial, cytoplasmic and ER markers (TbGAPDH, TbGAP1 [71], TbENO and calreticulin, respectively) which confirmed that the majority of mitochondrial TbGAP1 was in the LG fraction, that the majority of cytoplasmic TbENO and ER calreticulin was in the C fraction and that the majority of glycosomal TbGAPDH was in the SG fraction, as expected [70] (Fig 6A). Antibodies to three representative NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP) showed that these enzymes are found exclusively to the glycosome-containing SG fraction (Fig 6A, Table 1), consistent with the immunofluorescence microscopy and digitonin latency data described above.

10.1371/journal.pntd.0009132.g006 Subcellular localization by subcellular fractionation and evidence for glycosome heterogeneity by density gradient centrifugation and proteomics.

Panel A: Pcf trypanosome lysate was submitted to differential centrifugation [70] and aliquots of the large granular (LG), small granular (SG) and cytosolic plus microsomal fractions (C) were subjected to SDS-PAGE and Western blotting with antibodies to authentic glycosomal (TbGAPDH), mitochondrial (TbGAP1), cytoplasmic (TbENO) and ER (calreticulin) markers and to NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP), as indicated. Panel B: The SG fraction was further fractionated by iodixanol density gradient centrifugation and two opalescent bands (fractions 1 and 2) were isolated. Panel 3: Aliquots of fractions 1 and 2 from panel B were subjected to proteomic analysis by LC-MS/MS. The relative abundances (iBAQ values over three scales, y-axes) are plotted against the identified proteins (TriTrypDB identifiers, x-axes).

The same SG fraction was further subjected to iodixanol density gradient centrifugation. Two opalescent bands (fractions 1 and 2) were observed (Fig 6B) at densities of 1.15 and 1.18 g/mL, similar to that reported for rat liver peroxisomes using iodixanol (1.175 g/mL) [72]. Each was isolated and, following reduction, alkylation and trypsin digestion of the protein content, subjected to proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein groups identified in both fractions were highly-enriched for glycosomal proteins, as expected. Intensity-based absolute quantitation (iBAQ) values were extracted for the protein groups belonging to a reference high-confidence glycosomal proteome [54]. These protein groups (x axis) are plotted against three iBAQ scales, from low abundance (iBAQ values <1 x104) to high-abundance (iBAQ values >3 x 106), in (Fig 6C). Consistent with its higher density, fraction 2 contains more glycosomal protein than fraction 1. However, the relative abundances of glycsomal proteins between the two fractions are different, suggesting compositional heterogeneity between them.

 Discussion

Evidence that NS biosynthetic enzymes might be glycosomal first came from the sequencing of the gene encoding TbGALE, the enzyme interconverts UDP-Glc and UDP-Gal. The predicted amino acid sequence contained a C-terminal peroxisomal targeting sequence type 1 (PTS1) of -TKL [41]. The glycosomal location of TbGALE was later confirmed experimentally in bsf and pcf cells [44]. A comprehensive screen of kinetoplastid genes for predicted PTS1 and N-terminal PTS2 sequences [65] added TbHK1 (PTS2), TbPGI (PTS1) and TbPMI (PTS1). TbUAP was later found to possess an atypical PTS1 sequence (-SNM) [50]. Interestingly, the HK1, GPI, UAP, and PMM enzymes of T. cruzi and Leishmania major also possess PTS motifs, as do the PMI and UGP enzymes of T. cruzi and L. major, respectively (Table 1). While many glycosomal components possess PTS signals for glycosomal import, many others do not. Internal targeting sequences have been proposed for T. cruzi phosphoglucomutase and T. brucei triosephosphate isomerase [73,74] but these are not amenable to bioinformatic prediction. Other peroxisomal proteins are imported by a ‘piggyback’ mechanism through association with PTS-bearing proteins [75]. In this context, analysis of the protein complex database for pcf cells, using the cluster explorer function, suggests that TbPMM may piggyback on PTS1-containing TbPGI since both appear in cluster 314 in SEC300 gel-filtration and cluster 274 in SEC1000 gel-filtration [76]. The mapping of PTS1- and/or PTS2 -containing proteins to the high-confidence proteome for pcf glycosomes suggested a sensitivity and specificity of about 40% and 50%, respectively, for the bioinformatic prediction of glycosomal location [54]. Consequently, experimental confirmations of helpful PTS1/2 glycosomal location predictions are highly desirable.

Previously, the majority of immunofluorescence microscopy (IFM) localisation data for T. brucei NS biosynthetic enzymes were for bsf trypanosomes, with only TbGALE and TbGMD localised by IFM in pcf cells (Table 1). Now, all of the NS biosynthetic enzymes in pcf cells, except for TbPGI (that contains a PTS1 sequence and that is known to be glycosomal from sub-cellular fractionation) and for TbGFAT (which has proven difficult to express as a soluble protein for antibody production) have been successfully localised to the glycosome by IFM. The majority of these localisations, both for bsf and pcf cells, are further strongly supported here by digitonin latency experiments (Fig 5; Table 1). Further, subcellular fractionation and Western blotting for three representative NS biosynthetic enzymes (TbPMI, TbMPGT and TbUGP) show that these are found in the glycosome-containing small granular (SG) fraction (Fig 6A; Table 1).

Some of the IFM images (Figs 2, 3, and 4) show imperfect colocalization with the classical glycosomal marker in that the ratios of green NS enzyme- to red TbGAPDH-signals are variable. For example, some of the NS enzymes visually appear to occupy a subset of the TbGAPDH positive glycosomes (eg. for TbGNA, TbPMM, TbMPGT, TbUGP and TbGMER) whereas others show (green) glycosomes that contain less TbGAPDH (eg. for TbUAP, TbPMI, TbGALE). Similar results have been noted for bsf cells for TbPMM and TbPMI [42,46]. Nevertheless, quantitative analysis of the red and green channel signals across several cells (Table 3) shows that the majority of NS enzyme (green) signals colocalize with TbGAPDH (red) signals. Collectively, these data suggest some heterogeneity in glycosomal content, whereby the ratios of NS enzymes to TbGAPDH in individual glycosomes can vary quite widely. Whether this represents a mixture of immature and mature glycosomes or a mixture of different mature glycosomes, or a combination of both, is unclear from these data. However, glycosome biogenesis by growth and fission and by de novo biogenesis of nascent glycosomes that bud from the ER seems likely, as reviewed in [77]. Evidence for the latter can be found in our label-chase proteomic studies [54]. In this work, cells expressing GFP-tagged Pex13.1 were labelled to steady-state over 8 cell divisions with heavy isotope lysine and arginine and then placed into medium containing light lysine and arginine to perform a 5 h isotopic chase. Immediately after steady-state (heavy) labelling and after (light) chase, magnetic beads bearing anti-GFP antibodies were used to capture GFP-tagged Pex13.1 containing organelles which were taken for stable isotope in cell culture (SILAC) quantitative proteomics. At the steady-state label time point, Pex13.1 was found associated with several ER resident proteins as well as glycosome components. However, after the light isotopic chase period, the apparent association of Pex13.1 with ER proteins was greatly diminished, suggesting that Pex13.1 and ER resident proteins part company during the biogenesis of glycosomes. Consistent with this, Bauer et al. showed by IFM and sub-cellular fractionation that Pex13.1 accumulates in the ER in pcf cells grown in low glucose [78]. The same group has also demonstrated the induction of glycosome heterogeneity in response to extracellular glucose levels [79], suggesting that the de novo and growth and division pathways of glycosome biogenesis are dynamic and responsive to changing environmental factors [77].

Also consistent with the aforementioned concept of glycosome heterogeneity, we found that we could resolve two glycosomal fractions by density gradient centrifugation (Fig 6B) and that the glycosomal protein contents of these two fractions were different (Fig 6C). In this analysis, TbPMI was identified in both fractions but was enriched in fraction 2, consistent with the presence of TbPMI-rich (green and yellow) and TbPMI-poor (red) glycosomes in the TbPMI immunolocalization images (Fig 2B). Interestingly, proteins likely to be involved in protein import (glycosomal HSP70 and Pex11) are enriched in fraction 1, suggesting immature and actively importing glycosomes may be enriched in this lower density (lower protein content) fraction.

Only one of the NS biosynthetic enzymes (TbPAGM) appears to have dual location, being found in the cytoplasm as well as the glycosomes by IFM and digitonin latency (Fig 2, Table 3 and Fig 5). Whether TbPAGM has some role in cytoplasmic metabolism or whether this PTS-less protein is simply imported inefficiently into the glycosome remains to be determined.

Our localisation of TbMPGT, which converts Man1P to GDP-Man, to the glycosomes is at variance with a previous report using bsf cells that suggested a cytoplasmic location [48]. However, in that case the antibody used was raised to L. major MPGT and it is also possible that different growth conditions or other factors might account for this discrepancy. Several IFM localisations reported here do not agree well with those reported in the high-throughput TrypTag database [80]. In that case, eight epitope-tagged proteins, TbPGI, TbUGP, TbPAGM, TbUAP, TbPMI, TbPMM, TbGMD and TbGMER were variously described as flagellar cytoplasmic and/or nucleoplasmic and/or mitochondrial and/or cytoplasmic. However, C-terminal tagging of PTS1-containing TbPGI and TbPMI would likely cause their mislocalisation, and visual inspection of the IFM micrographs for N-terminally tagged TbUGP and TbPAGM suggests punctate staining reminiscent of glycosomes.

In summary, together with previously published sub-cellular fractionation data for TbHK1, TbPGI and TbGALE and glycosome proteome data for TbHK, TbPGI, TbUAP, TbGALE, TbUAP and TbPMI (Table 1), the data presented here provide a compelling case for the presence of the entire NS biosynthetic machinery in the glycosomes of both bsf and pcf T. brucei. This is radically different from other eukaryotes, where NS biosynthesis is either known or assumed to be located in the cytoplasm. The glycosomal location of NS biosynthesis in T. brucei, in turn, strengthens our postulate that an NS transporter (NST) or transporters is/are likely to exist in the bsf and pcf glycosome membranes to transport these large and negatively charged metabolites from the lumen of the glycosome into the cytoplasm. Once in the cytoplasm, NSs can be imported into the Golgi apparatus and ER by canonical SLC35-family NSTs [81] to perform glycosylation reactions in glycoprotein and glycolipid biosynthesis. The putative glycosomal NSTs may be quite different from the canonical SLC35-family NSTs since the direction of transport of the glycosomal NSTs (organelle lumen to cytosol) is in the opposite direction to that of the SLC35-family NSTs (cytosol to organelle lumen). It is possible that glycosmal NST(s) may provide a selective therapeutic target(s) for human and animal African trypanosomiasis.

 Methods Cell culture

Procyclic form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein genes, respectively under control of G418 and hygromycin (clone 29.13.6 cells, kindly provided by George Cross) were grown at 28°C without CO2 in original SDM-79 medium [82] containing hygromycin B (Roche) at 50 μg/ mL, G418 (Invitrogen) at 15 μg/ mL, 15% (v/v) heat inactivated fetal bovine serum (FBS), 2 g/L sodium bicarbonate, fresh 2 mM Glutamax I (Invitrogen) and 22.5 mg / ml haemin (added from a 0.05 M stock in NaOH), adjusted to pH 7.3. Bloodstream form T. brucei 427 strain containing T7 RNA polymerase and Tet repressor protein, under control of G418, known as single markers cells, kindly provided by George Cross, were grown at 37°C with 5% CO2 in HMI-9T medium [83] containing fresh 2mM Glutamax I (Invitrogen) and 2.5 μg/ mL G418 (Invitrogen).

 Production of recombinant TbMPGT

The TbMPGT ORF (gene Tb927.8.2050) was amplified from genomic DNA by PCR and cloned into pET28a, containing a 6-His N-terminal tag with a thrombin cleavage site, and overexpressed in BL21 (DE 3) Gold E. coli grown at 37°C in LB medium supplemented with kanamycin (50 μg / ml) and tetracycline (10 μg / ml). When the OD600 reached about 2.0, the temperature was lowered to 16°C and protein expression was induced with 1 mM IPTG (Formedium) for 22 h. Cells were harvested, resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0), supplemented with DNAse I and EDTA-free protease inhibitors (Roche), disrupted using a French press and centrifuged at 30,000 g for 30 min at 4°. The supernatant was filtered (0.2 μm) and applied to a 5 ml HisTrap HP chelating column (GE healthcare), pre-equilibrated in lysis buffer, and eluted with a linear imidazole gradient from 96% buffer A (20 mM Tris, 500 mM NaCl, pH 8) to 100% buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8). Fractions containing TbMPGT (which eluted around 300 mM imidazole) were pooled, concentrated, digested with thrombin (NEB) to remove the His-tag and subsequently purified on a Superose 12 10/300 column (GE healthcare) equilibrated with 10 mM Hepes, 100mM NaCl, pH 7.4. The yield, estimated by BCA assay, was about 10 mg of soluble, enzymatically active and highly purified TbMPGT per litre of culture (S1 Fig). The identity of the recombinant TbMPGT was confirmed by proteomics.

 Production of mouse antibodies against recombinant nucleotide sugar biosynthetic enzymes

Aliquots of recombinant purified TbMPGT (S1 Fig), TbGALE [41], TbGNA [49], TbUAP [50] and TbGMER [52] were submitted to David’s Biotech (Germany) for the immunization of two mice per protein, according to their recommended protocol using Freund’s complete adjuvant for the primary immunisation with 0.1 mg protein per mouse and two secondary boosts with 0.05 mg per mouse in Freund’s incomplete adjuvant. Pooled mouse anti-TbMPGT, anti-TbUAP and anti-TbGNA sera were immunopurified by affinity chromatography on Sepharose4B beads coupled to recombinant TbMPGT, TbUAP and TbGNA, respectively, at 3–5 mg / ml gel. The affinity chromatography beads were prepared using CNBr-Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The coupled beads were stored in phosphate buffered saline (PBS) containing 0.05% (w/v) sodium azide. For affinity purification, pooled mouse sera were centrifuged at 16,000 g for 15 min at 4°C and the supernatants mixed with an equal volume of PBS. These were incubated with the corresponding affinity chromatography beads for 2 h with rotation at 4°C. The beads were washed with PBS until the optical density at 280 nm (OD280) of the supernatant was below 0.05 and the specifically bound antibodies were then eluted with 50 mM sodium citrate pH 2.8. The eluates were immediately neutralized with 1M Tris-HCl pH 8.8 and quantitated by OD280.

 Immunoprecipitation

T. brucei bsf cells were washed three times with trypanosome dilution buffer (TBD; 20 mM Na2HPO4, 2 mM NaH2PO4, 80 mM NaCl, 5 mM KCl, 1 mM MgSO4, 20 mM glucose pH 7.7) and lysed at 1 x 109 cells/ml in 1% (w/v) SDS in 20 mM Tris-HCl pH 6.8 containing 0.1 M DTT and heated at 50°C for 15 min. The SDS lysate was diluted to 0.03% SDS with 1% (w/v) Triton X-100 in 20 mM Tris-HCl pH 6.8, 0.15 M NaCl, 0.1 mM TLCK, 1 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM PMSF. Insoluble material was removed by centrifugation at 16,000 g for 10 min at 4°C and an aliquot of the supernatant (equivalent to 2 x 108 cells) was incubated with 5 μL of rabbit anti-TbGalE antiserum [44] for 1 h at 4°C and immunoprecipitated with 50μL of Protein A magnetic beads (Invitrogen). A similar procedure was used for TbGMER immunoprecipitation, but instead mouse antisera anti-GMER and protein G magnetic beads (Invitrogen) were used. Samples were run in SDS-PAGE for Western blotting.

 Western blotting

Samples were run on 4–12% gradient NuPAGE gels (Invitrogen) and Western blotted onto nitrocellulose using an iBlot system (Invitrogen). The pieces of nitrocellulose were either stained with Ponceau red for protein or blocked with 30 ml pre-filtered (20 μm) blocking buffer (50 mM Tris-HCl pH 7.4 containing 0.15 M NaCl, 0.25% BSA (w/v), 2% (w/v) fish skin gelatin and 0.05% (w/v) Tween 20) using a SNAPid system (Millipore). The latter were incubated with 0.5–1 μg / ml affinity purified antibody or with 1:1,000 diluted mouse antiserum (both diluted in blocking buffer) against the respective nucleotide sugar biosynthetic enzymes for 1 h at room temperature. Using the SNAPid system, the blots were washed 3 times 30 ml PBS, 0.1% Tween 20 and then incubated with 5 ml anti-mouse HRP (Invitrogen) diluted 1:10,000 in blocking buffer without sodium azide. The blots were subsequently washed with PBS, 0.1% Tween 20, removed from the SNAPid system and developed with ECL (ThermoFisher). Rabbit anti-TbGAPDH, rabbit anti-TbEnolase, rabbit anti-TbPEX13 and rabbit anti-TbAldolase (kind gifts from Prof. Paul Michels, Univ. of Edinburgh, UK) were used at 1:5,000, 1:5,000, 1:10,000 and 1:5,000 dilutions in blocking buffer, respectively. In addition, developments with mitochondrial marker [rabbit anti-TbGAP1 (a kind gift from Prof. Julius Luke, Institute of Parasitology, Czech Republic)] were performed at a dilution of 1:1,500 and developments with ER marker [immunopurified rabbit anti-calreticulin (Stressgen, Enzo LifeSci, USA)] were performed at a dilution of 1:1,000. Blots were developed with 1:10,000 anti-rabbit HRP (Invitrogen) and ECL reagents according to the manufacturer’s instructions (ThermoFisher). The blots were exposed onto ECL film (GE Healthcare Amersham), developed and scanned in an Epson office scanner.

 Immunofluorescence microscopy

Trypanosomes were washed in ice-cold phosphate buffer saline (PBS) for pcf cells or ice-cold trypanosome dilution buffer (TDB; 20 mM Na2HPO4, 2 mM NaH2PO4, 80 mM NaCl, 5 mM KCl, 1 mM MgSO4, 20 mM glucose, pH 7.7) for bsf cells and resuspended in the same buffer. Equal volumes of 8% paraformaldehyde (PFA) in PBS or TDB, respectively, were added and cells were fixed at 4° C. Subsequently, suspensions of fixed cells were spotted on cover slips, allowed to air dry, permeablized with 0.1% TX100 in PBS for 10 min and blocked in PBS, 5% fish skin gelatin, 10% normal goat serum and 0.05% TX100. Affinity purified anti-TbMPGT, anti-TbUGP and anti-TbGNA mouse antibodies were used at 0.5–1.0 μg / ml diluted in PBS, 1% fish skin gelatin, 0.05% TX100 and incubated for 1 h at room temperature in a humid chamber. Anti-TbGALE, anti-TbPAGM, anti-TbPMI, anti-TbPMM and anti-TbGMER mouse sera were diluted at 1:500 or 1:1,000 in the same buffer. Rabbit anti-GAPDH and rabbit anti-TbEnolase sera (generous gifts of Dr Paul Michels) were used at 1:2,000 and 1:4,000 dilution, respectively. Coverslips were washed in PBS, 1% fish skin gelatin, 0.05% TX100 and incubated for 1 h at room temperature with 50 μL goat anti-mouse IgG Alexa 488 conjugated mixed goat anti-rabbit IgG Alexa 594 conjugated, both diluted at 1:500 in same buffer. Coverslips were washed and mounted on glass slides using Prolong Gold (Invitrogen). Microscopic images were obtained in a Zeiss LSM 710 META confocal microscope.

Colocalisations were quantified using Volocity software (Quorum Technologies). Zeiss.lsm images were imported into the Volocity library and processed as follows: The thresholds for the red and green channels were calculated from a region of interest outside the cell images. Individual cells were selected using the region of interest (ROI) tool and colocalization statistics were calculated and recorded for each cell. Mander’s colocalisation coefficients (the fraction of green signal colocalising with red compartments) are reported in (Table 3), according to the recommendations of [84].

 Digitonin latency

Digitonin latency was performed as previously described in [42]. Briefly T.brucei bloodstream or procyclic form cells (2 x 109 cells) were washed twice with 10 ml 250 mM sucrose, 25 mM Tris-HCl pH 7.4 and 1 mM EDTA (STE buffer) and resuspended in 1.5 ml STEN (STE containing 0.15 M NaCl). Aliquots (0.15 ml) were treated with equal volume of digitonin at various concentrations in the presence of 0.1 mM TLCK, 1 μg / ml leupeptin, 1 μg / ml aprotinin and 1 mM PMSF. Digitonin stock was prepared at 10 mg / ml in DMSO and diluted in STEN to the required concentrations. Complete extraction was obtained in parallel by treating an equivalent number of cells with 0.1% Triton X-100 in STEN. The lysates were incubated for 5 min at room temperature and the insoluble materials removed by centrifugation at 16,000 g for 2 min. The pellets were discarded and samples of the supernatants were run on a reducing 4–12% gradient NuPage gel (Invitrogen) and transferred to nitrocellulose for Western blotting using antisera produced in mouse and at the dilutions described in the immunofluorescence section. Different amounts of same samples were loaded onto separate gels for each antibody Western blot, in order to keep within the linear range of film exposure. For TbPMM, TbGALE and TbGMER 1x107 cell equivalents per lane, for TbPAGM, TbMPGT, TbUGP, TbUAP, TbPMI, 5 x 106 cell equivalents per lane, for TbGAPDH, TbAldolase (Aldo), for TbEnolase (Eno) 1x105 cell equivalents per lane and for TbPEX13 5x106 cell equivalents per lane.

 Subcellular fractionation by differential centrifugation followed by density gradient centrifugation

The pcf cells used for differential and density gradient centrifugation came from a stable isotope in cell culture (SILAC) labelling experiment. However, the isotopic labelling is not relevant to the specific experiments and results presented here. The starting pcf cell preparation (a total of 5.6 x 109 cells washed in STE buffer) was a 1: 1 mixture of (i) GFP-tagged PEX13.1 pcf mutants labelled to steady state over 8 cell divisions, and to a final cell density of 1.1.x 107 cells / ml, in R6K6 SDM-79 medium containing heavy isotopes of L-Arginine (R6) and L-Lysine (K6), and (ii) double marker cells grown in normal SDM-79 medium (R0K0) and also harvested at 1.1 x 107 cells / ml.

The mixed cells were lysed using a One-shot high-pressure cell disruptor (Constant Systems, UK) operated at 10,000 psi as described in [54]. The lysate was submitted to differential centrifugation as described in [70]: After spinning at 1,500 g to the remove nuclei and any remaining intact cells and cell ghosts, the supernatant was submitted to centrifugation at 5,000 g to yield a Large Granular fraction (LG) pellet, enriched in mitochondria. Subsequent centrifugation of the supernatant at 15,000 g pelleted the Small Granular fraction (SG), enriched in glycosomes, with concomitant generation of a supernatant (C) enriched in cytosol and ER, Golgi and plasma membrane microsomes.

The SG fraction was subsequently submitted to density gradient centrifugation using iodixanol (Optiprep, Axis Shield, UK) according to [53], with some modifications: A discontinuous step gradient of iodixanol was prepared by layering 20, 25, 30, 35, 40 and 45% (w/v) iodixanol in STE buffer inside an Optiseal polyallomer tube (Beckman) using a peristaltic pump to apply each layer to the bottom of the tube, starting with the least dense layer and displacing it with next dense layer and so on. The SG fraction resuspended in STE buffer was loaded carefully on top of the gradient and centrifuged at 45,000 rpm using VTi-50 vertical rotor in a Beckman ultracentrifuge for 1h at 4°C. After ultracentrifugation, the tube was punctured with needle and syringe to collect 2 distinct opalescent bands, fraction 1 (top band) and fraction 2 (lower band). The refractive index of each fraction was measured using a refractometer (Abbe refractometer model NAR-1T) and plotted against a standard curve of 20, 25, 30, 35, 40 and 45% (w/v) iodixanol in STE. The density was calculated according to published tables of iodixanol percentage versus density (g/mL).

 Proteomic analyses

Samples of fraction 1 and fraction 2 were reduced with dithiothreitol and alkylated with iodoacetamide and subsequently digested with trypsin and processed using FASP II (filter-aided sample processing), as described previously in [54]. Samples were desalted using a custom made Porous R2 columns packed with 1.5 mg Porous R2 reverse phase beads (ABI). The columns were pre-conditioned by washing with 90% CH3CN containing 0.1% formic acid followed by equilibration in 0.1% formic acid in water. Samples were applied in 0.1% formic acid and the columns washed with 0.1% formic acid then subsequently eluted three times with 90% CH3CN containing 0.1% formic acid. The eluates were combined, dried in a Speedvac vacuum concentrator, resuspended in 35% CH3CN containing 0.1% formic acid and fractionated using strong cation exchange (SCX) chromatography: The combined eluates were applied into a custom made SCX column made by adding Poros 50 HS SCX beads (ABI) to a SCX Ziptip (Millipore). Bound peptides were eluted sequentially with 20, 100, 150, and 200 mM NaCl in 35% CH3CN containing 0.1% formic acid, followed by final elutions with 0.5% NH4OH in 50% isopropanol and 50% isopropanol (IPA sample). Each of these five sub-fractions were dried and re-suspended in 50 μL 1% formic acid and 15 μL aliquots were subjected to liquid chromatography on Ultimate 3000 RSLCnano system (Thermo Scientific) fitted with an Acclaim PepMap 100 (C18, 100 μM x 2 cm) trap cartridge. Peptides were separated on an Easy-Spray PepMap RSLC C18 column (75 μM x 50 cm) (Thermo Scientific) using a linear gradient from 2 to 40% buffer B (80% acetonitrile, 0.1% formic acid) in buffer A (0.1% formic acid) over 70 min. The HPLC system was coupled to a Orbitrap Velos Mass Spectrometer (Thermo Scientific) with a source voltage of 1.2 kV.

 Proteomic data analysis

LC-MS/MS data were analysed for protein identification using MaxQuant 1.6.14 [85,86] with the in-built Andromeda search engine [87]. The raw files were searched against the T. brucei brucei 927 annotated protein sequences from TriTrypDB release 46 [88] supplemented with the T. brucei brucei 427 VSG221 (Tb427.BES40.22) protein sequence. The mass tolerance was set to 4.5 ppm for precursor ions and trypsin was set as proteolytic enzyme with two missed cleavage allowed. Carbamidomethyl on cysteine was set as fixed modifications. SILAC labelling [5] of heavy arginine (Arg-6) and Lysine (Lys-6) were specified. Oxidation of methionine and Acetylation of Protein N-term were set as variable modifications. The false-discovery rate for protein and peptide level identifications was set at 1%, using a target-decoy based strategy. The minimum peptide length was set to seven amino acids and protein quantification was performed on unique plus razor peptides [89]. “Reverse Hits”, “Only identified by site” and “Potential contaminant” identifications were filtered out. Only protein groups with at least two unique peptide sequences and Andromeda protein score greater than 5 were selected for further analysis. The high confidence glycosomal resident proteins were extracted from Güther et al., 2014 [54]. Gene abbreviations were retrieved from the TriTrypDB database. The iBAQ values [89,90] were extracted from the MaxQuant output and visualised as bar graphs. The analysis pipeline was implemented in python using the SciPy packages (https://www.scipy.org/) and Jupyter notebook (https://jupyter.org/).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [91] partner repository with the dataset identifier PXD023124. Reviewer account details: Username: reviewer_pxd023124@ebi.ac.uk. Password: qqm7vZg3. The analysis pipeline is available in GitHub (https://github.com/mtinti/nucleotide_sugar) and it is archived in Zenodo (https://doi.org/10.5281/zenodo.4289929). The analysis pipeline is reproducible using the mybinder app with the link reported in the GitHub repository.

 Supporting information Recombinant TbMPGT.

Analysis by SDS-PAGE and Coomassie blue staining of TbMPGT expressed in E.coli, purified by nickel chromatography, digested with thrombin to remove the N-terminal 6-His tag and then purified by FPLC gel-filtration. Molecular weight standards are shown on the left.

(DOCX)

 Mono-specificity of antibodies to nucleotide sugar biosynthetic enzymes used in this study.

The mouse polyclonal antibodies raised against TbMPGT, TbGNA, TbUAP, TbGALE and TbGMER (Tables 1 and 2) were used in Western blotting with an anti-mouse-HRP secondary antibody. The lanes contained either 5 x 106 cell equivalents of T. brucei bsf total cell lysates (lanes 1, 2, 3, 5, and 7) or, when no signal was recorded against whole lysate due to low abundance, the lanes contained an immunoprecipitate from 2 x 108 cell equivalents of total bsf lysate using rabbit anti-TbGALE [44] (lane 4) or mouse anti-TbGMER (lane 6). In each case, a single band* with an apparent molecular weight consistent with the target antigen was recorded, demonstrating the mono-specificity of the antibodies. *Note: the two additional bands marked by asterixis in (lane 6) are due to mouse IgG heavy and light chains from the immunoprecipitation. A Ponceau red stain of a Western blot lane containing 5 x 106 cell equivalents of T. brucei bsf total cell lysate is shown in (lane 8). The positions of MW standards are shown on the right.

(DOCX)

We thank Paul Michels, University of Edinburgh, for helpful discussions and for supplying rabbit anti-TbGAPDH, rabbit anti-TbEnolase, rabbit anti-TbAldolase, rabbit anti-Pex13. We also thank former members of MAJF lab: Karina Marino for supplying TbGNA, TbUGP and TbPAGM proteins and Matt Stokes for TbUAP in order to raise antibodies. We thank Fred Opperdoes for pointing out that TbGALE has a PTS1 signal many years ago.

 References Schwede A, Macleod OJS, MacGregor P, Carrington M. (2015) How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins? PLoS Pathog 11, e1005259 doi: 10.1371/journal.ppat.1005259 26719972 Salmon D., Geuskens M., Hanocq F., Hanocq-Quertier J., Nolan D., Ruben L., and Pays E. (1994) A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei. Cell 78, 75–86 doi: 10.1016/0092-8674(94)90574-6 8033214 Trevor CE, Gonzalez-Munoz AL, Macleod OJS, Woodcock PG, Rust S, Vaughan TJ, et al. Structure of the trypanosome transferrin receptor reveals mechanisms of ligand recognition and immune evasion. Nat Microbiol. 2019;4(12):2074–81. doi: 10.1038/s41564-019-0589-0 31636418 Mehlert A., Wormald M. R., and Ferguson M. A. J. (2012) Modeling of the N-glycosylated transferrin receptor suggests how transferrin binding can occur within the surface coat of Trypanosoma brucei. PLoS Pathog 8, e1002618 doi: 10.1371/journal.ppat.1002618 22496646 Peck R. F., Shiflett A. M., Schwartz K. J., McCann A., Hajduk S. L., and Bangs J. D. (2008) The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes. Mol Microbiol 68, 933–946 doi: 10.1111/j.1365-2958.2008.06195.x 18430083 Jackson A. P., Allison H. C., Barry J. D., Field M. C., Hertz-Fowler C., and Berriman M. (2013) A cell-surface phylome for African trypanosomes. PLoS Negl Trop Dis 7, e2121 doi: 10.1371/journal.pntd.0002121 23556014 Allison H., O’Reilly A. J., Sternberg J., and Field M. C. (2014) An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids. Microb Cell 1, 325–345 doi: 10.15698/mic2014.10.170 26167471 Lingnau A., Zufferey R., Lingnau M., and Russell D. G. (1999) Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes. J Cell Sci 112 Pt 18, 3061–3070 10462522 Engstler M., Weise F., Bopp K., Grunfelder C. G., Gunzel M., Heddergott N., and Overath P. (2005) The membrane-bound histidine acid phosphatase TbMBAP1 is essential for endocytosis and membrane recycling in Trypanosoma brucei. J Cell Sci 118, 2105–2118 doi: 10.1242/jcs.02327 15855239 LaCount D. J., Barrett B., and Donelson J. E. (2002) Trypanosoma brucei FLA1 is required for flagellum attachment and cytokinesis. J Biol Chem 277, 17580–17588 doi: 10.1074/jbc.M200873200 11877446 Woods K, Nic a’Bhaird N, Dooley C, Perez-Morga D, Nolan DP. Identification and characterization of a stage specific membrane protein involved in flagellar attachment in Trypanosoma brucei. PLoS One. 2013;8(1):e52846. doi: 10.1371/journal.pone.0052846 23335957 Higgins MK, Tkachenko O, Brown A, Reed J, Raper J, Carrington M. Structure of the trypanosome haptoglobin-hemoglobin receptor and implications for nutrient uptake and innate immunity. Proc Natl Acad Sci U S A. 2013;110(5):1905–10. doi: 10.1073/pnas.1214943110 23319650 Zoll S, Lane-Serff H, Mehmood S, Schneider J, Robinson CV, Carrington M, et al. The structure of serum resistance-associated protein and its implications for human African trypanosomiasis. Nat Microbiol. 2018;3(3):295–301. doi: 10.1038/s41564-017-0085-3 29358741 Macleod OJS, Bart JM, MacGregor P, Peacock L, Savill NJ, Hester S, et al. A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies. Nat Commun. 2020;11(1):1326. doi: 10.1038/s41467-020-15125-y 32165615 Casas-Sánchez A, Perally S, Ramaswamy R, Haines LR, Rose C, Yunta C, et al. bioRxiv 477737; https://doi.org/10.1101/477737 Treumann A., Zitzmann N., Hulsmeier A., Prescott A. R., Almond A., Sheehan J., and Ferguson M. A. (1997) Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J Mol Biol 269, 529–547 doi: 10.1006/jmbi.1997.1066 9217258 Acosta-Serrano A., Cole R. N., Mehlert A., Lee M. G., Ferguson M. A., and Englund P. T. (1999) The procyclin repertoire of Trypanosoma brucei. Identification and structural characterization of the Glu-Pro-rich polypeptides. J Biol Chem 274, 29763–29771 doi: 10.1074/jbc.274.42.29763 10514452 Guther M. L., Lee S., Tetley L., Acosta-Serrano A., and Ferguson M. A. (2006) GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat. Mol Biol Cell 17, 5265–5274 doi: 10.1091/mbc.e06-08-0702 17035628 Guther M. L., Beattie K., Lamont D. J., James J., Prescott A. R., and Ferguson M. A. (2009) Fate of glycosylphosphatidylinositol (GPI)-less procyclin and characterization of sialylated non-GPI-anchored surface coat molecules of procyclic-form Trypanosoma brucei. Eukaryot Cell 8, 1407–1417 doi: 10.1128/EC.00178-09 19633269 Zamze S. E., Wooten E. W., Ashford D. A., Ferguson M. A., Dwek R. A., and Rademacher T. W. (1990) Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins. Eur J Biochem 187, 657–663 doi: 10.1111/j.1432-1033.1990.tb15350.x 2303059 Zamze S. E., Ashford D. A., Wooten E. W., Rademacher T. W., and Dwek R. A. (1991) Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins. J Biol Chem 266, 20244–20261 1939085 Atrih A., Richardson J. M., Prescott A. R., and Ferguson M. A. J. (2005) Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains. J Biol Chem 280, 865–871 doi: 10.1074/jbc.M411061200 15509560 Izquierdo L., Atrih A., Rodrigues J. A., Jones D. C., and Ferguson M. A. J. (2009) Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase has unusual substrate specificity and protects the parasite from stress. Eukaryotic cell 8, 230–240 doi: 10.1128/EC.00361-08 19114500 Acosta-Serrano A., O’Rear J., Quellhorst G., Lee S. H., Hwa K. Y., Krag S. S., and Englund P. T. (2004) Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant. Eukaryot Cell 3, 255–263 doi: 10.1128/ec.3.2.255-263.2004 15075256 Ferguson M. A., Homans S. W., Dwek R. A., and Rademacher T. W. (1988) Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science 239, 753–759 doi: 10.1126/science.3340856 3340856 Guther M. L., and Ferguson M. A. (1993) The microanalysis of glycosyl-phosphatidylinositol glycans. Methods Mol Biol 14, 99–117 doi: 10.1385/0-89603-226-4:99 8348247 Mehlert A., Richardson J. M., and Ferguson M. A. (1998) Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei. J Mol Biol 277, 379–392 doi: 10.1006/jmbi.1997.1600 9514751 Mehlert A., Sullivan L., and Ferguson M. A. (2010) Glycotyping of Trypanosoma brucei variant surface glycoprotein MITat1.8. Mol Biochem Parasitol 174, 74–77 doi: 10.1016/j.molbiopara.2010.06.007 20558211 Mehlert A., and Ferguson M. A. (2007) Structure of the glycosylphosphatidylinositol anchor of the Trypanosoma brucei transferrin receptor. Mol Biochem Parasitol 151, 220–223 doi: 10.1016/j.molbiopara.2006.11.001 17140675 Izquierdo L., Nakanishi M., Mehlert A., Machray G., Barton G. J., and Ferguson M. A. (2009) Identification of a glycosylphosphatidylinositol anchor-modifying beta1-3 N-acetylglucosaminyl transferase in Trypanosoma brucei. Mol Microbiol 71, 478–491 doi: 10.1111/j.1365-2958.2008.06542.x 19040631 Ferguson M. A. J., Hart G. W., and Kinoshita T. (2015) Glycosylphosphatidylinositol Anchors. in Essentials of Glycobiology (rd, Varki A., Cummings R. D., Esko J. D., Stanley P., Hart G. W., Aebi M., Darvill A. G., Kinoshita T., Packer N. H., Prestegard J. H., Schnaar R. L., and Seeberger P. H. eds.), Cold Spring Harbor (NY). pp 137–150 Pinger J, Nesic D, Ali L, Aresta-Branco F, Lilic M, Chowdhury S, et al. African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat. Nat Microbiol. 2018;3(8):932–8. doi: 10.1038/s41564-018-0187-6 29988048 Bandini G, Damerow S, Güther MLS, Mehlert A, Guo H, Beverley SM, Ferguson MAJ(2019) bioRxiv 726117; https://doi.org/10.1101/726117 Turnock D. C., and Ferguson M. A. (2007) Sugar nucleotide pools of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Eukaryot Cell 6, 1450–1463 doi: 10.1128/EC.00175-07 17557881 Pontes de Carvalho L. C., Tomlinson S., Vandekerckhove F., Bienen E. J., Clarkson A. B., Jiang M. S., Hart G. W., and Nussenzweig V. (1993) Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor. J Exp Med 177, 465–474 doi: 10.1084/jem.177.2.465 8426115 Engstler M., Reuter G., and Schauer R. (1993) The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein. Mol Biochem Parasitol 61, 1–13 doi: 10.1016/0166-6851(93)90153-o 8259122 Doering T. L., Masterson W. J., Englund P. T., and Hart G. W. (1989) Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J Biol Chem 264, 11168–11173 2525555 Urbaniak M. D., Crossman A., Chang T., Smith T. K., van Aalten D. M., and Ferguson M. A. (2005) The N-acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol biosynthesis is a zinc metalloenzyme. J Biol Chem 280, 22831–22838 doi: 10.1074/jbc.M502402200 15817455 Jones D. C., Mehlert A., Guther M. L., and Ferguson M. A. (2005) Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein. J Biol Chem 280, 35929–35942 doi: 10.1074/jbc.M509130200 16120601 Bullard W, Lopes da Rosa-Spiegler J, Liu S, Wang Y, Sabatini R. Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome. J Biol Chem. 2014;289(29):20273–82. doi: 10.1074/jbc.M114.579821 24891501 Roper J. R., Guther M. L., Milne K. G., and Ferguson M. A. (2002) Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei. Proc Natl Acad Sci U S A 99, 5884–5889 doi: 10.1073/pnas.092669999 11983889 Bandini G., Marino K., Guther M. L., Wernimont A. K., Kuettel S., Qiu W., Afzal S., Kelner A., Hui R., and Ferguson M. A. (2012) Phosphoglucomutase is absent in Trypanosoma brucei and redundantly substituted by phosphomannomutase and phospho-N-acetylglucosamine mutase. Mol Microbiol 85, 513–534 doi: 10.1111/j.1365-2958.2012.08124.x 22676716 Shaw M. P., Bond C. S., Roper J. R., Gourley D. G., Ferguson M. A., and Hunter W. N. (2003) High-resolution crystal structure of Trypanosoma brucei UDP-galactose 4’-epimerase: a potential target for structure-based development of novel trypanocides. Mol Biochem Parasitol 126, 173–180 doi: 10.1016/s0166-6851(02)00243-8 12615316 Roper J. R., Guther M. L., Macrae J. I., Prescott A. R., Hallyburton I., Acosta-Serrano A., and Ferguson M. A. (2005) The suppression of galactose metabolism in procylic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number. J Biol Chem 280, 19728–19736 doi: 10.1074/jbc.M502370200 15767252 Marino K., Guther M. L., Wernimont A. K., Amani M., Hui R., and Ferguson M. A. (2010) Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase. Glycobiology 20, 1619–1630 doi: 10.1093/glycob/cwq115 20724435 Kuettel S., Wadum M. C., Guther M. L., Marino K., Riemer C., and Ferguson M. A. (2012) The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei. Mol Microbiol 84, 340–351 doi: 10.1111/j.1365-2958.2012.08026.x 22375793 Zmuda F, Shepherd SM, Ferguson MAJ, Gray DW, Torrie LS, De Rycker M. Trypanosoma cruzi Phosphomannomutase and Guanosine Diphosphate-Mannose Pyrophosphorylase Ligandability Assessment. Antimicrob Agents Chemother. 2019;63(10). doi: 10.1128/AAC.01082-19 31405854 Denton H., Fyffe S., and Smith T. K. (2010) GDP-mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei. Biochem J 425, 603–614 doi: 10.1042/BJ20090896 19919534 Marino K., Guther M. L., Wernimont A. K., Qiu W., Hui R., and Ferguson M. A. (2011) Characterization, localization, essentiality, and high-resolution crystal structure of glucosamine 6-phosphate N-acetyltransferase from Trypanosoma brucei. Eukaryot Cell 10, 985–997 doi: 10.1128/EC.05025-11 21531872 Stokes M. J., Guther M. L., Turnock D. C., Prescott A. R., Martin K. L., Alphey M. S., and Ferguson M. A. (2008) The synthesis of UDP-N-acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation. J Biol Chem 283, 16147–16161 doi: 10.1074/jbc.M709581200 18381290 Urbaniak M. D., Collie I. T., Fang W., Aristotelous T., Eskilsson S., Raimi O. G., Harrison J., Navratilova I. H., Frearson J. A., van Aalten D. M., and Ferguson M. A. (2013) A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei. ACS Chem Biol 8, 1981–1987 doi: 10.1021/cb400411x 23834437 Turnock D. C., Izquierdo L., and Ferguson M. A. (2007) The de novo synthesis of GDP-fucose is essential for flagellar adhesion and cell growth in Trypanosoma brucei. J Biol Chem 282, 28853–28863 doi: 10.1074/jbc.M704742200 17640865 Colasante C, Ellis M, Ruppert T, Voncken F. Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei. Proteomics. 2006;6(11):3275–93. doi: 10.1002/pmic.200500668 16622829 Guther ML, Urbaniak MD, Tavendale A, Prescott A, Ferguson MA. High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics. J Proteome Res. 2014;13(6):2796–806. doi: 10.1021/pr401209w 24792668 Opperdoes F. R., and Borst P. (1977) Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett 80, 360–364 doi: 10.1016/0014-5793(77)80476-6 142663 Opperdoes F. R., Baudhuin P., Coppens I., De Roe C., Edwards S. W., Weijers P. J., and Misset O. (1984) Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei. J Cell Biol 98, 1178–1184 doi: 10.1083/jcb.98.4.1178 6715405 Aman R. A., and Wang C. C. (1986) An improved purification of glycosomes from the procyclic trypomastigotes of Trypanosoma brucei. Mol Biochem Parasitol 21, 211–220 doi: 10.1016/0166-6851(86)90126-x 3807943 Heise N., and Opperdoes F. R. (1999) Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei. Mol Biochem Parasitol 99, 21–32 doi: 10.1016/s0166-6851(98)00176-5 10215021 Visser N., and Opperdoes F. R. (1980) Glycolysis in Trypanosoma brucei. Eur J Biochem 103, 623–632 doi: 10.1111/j.1432-1033.1980.tb05988.x 6766864 Joice A.C., Lyda T.L., Sayce A.C., Verplaetse E., Morris M.T., Michels P.A., et al. Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2. Int J Parasitol. 2012;42(4):401–9. doi: 10.1016/j.ijpara.2012.02.008 22619756 Morris MT, DeBruin C, Yang Z, Chambers JW, Smith KS, Morris JC. Activity of a second Trypanosoma brucei hexokinase is controlled by an 18-amino-acid C-terminal tail. Eukaryot Cell. 2006;5(12):2014–23. doi: 10.1128/EC.00146-06 17028241 Albert MA, Haanstra JR, Hannaert V, Van Roy J, Opperdoes FR, Bakker BM, et al. Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei. J Biol Chem. 2005;280(31):28306–15. doi: 10.1074/jbc.M502403200 15955817 Chambers JW, Fowler ML, Morris MT, Morris JC. The anti-trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1. Mol Biochem Parasitol. 2008;158(2):202–7. doi: 10.1016/j.molbiopara.2007.12.013 18262292 Arsenieva D., Appavu B. L., Mazock G. H., and Jeffery C. J. (2009) Crystal structure of phosphoglucose isomerase from Trypanosoma brucei complexed with glucose-6-phosphate at 1.6 A resolution. Proteins 74, 72–80 doi: 10.1002/prot.22133 18561188 Opperdoes F. R., and Szikora J. P. (2006) In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes. Mol Biochem Parasitol 147, 193–206 doi: 10.1016/j.molbiopara.2006.02.010 16546274 Gualdron-Lopez M., Brennand A., Hannaert V., Quinones W., Caceres A. J., Bringaud F., Concepcion J. L., and Michels P. A. (2012) When, how and why glycolysis became compartmentalised in the Kinetoplastea. A new look at an ancient organelle. Int J Parasitol 42, 1–20 doi: 10.1016/j.ijpara.2011.10.007 22142562 Haanstra J. R., Gonzalez-Marcano E. B., Gualdron-Lopez M., and Michels P. A. (2016) Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites. Biochim Biophys Acta 1863, 1038–1048 doi: 10.1016/j.bbamcr.2015.09.015 26384872 Allmann S., and Bringaud F. (2017) Glycosomes: A comprehensive view of their metabolic roles in T. brucei. Int J Biochem Cell Biol 85, 85–90 doi: 10.1016/j.biocel.2017.01.015 28179189 Galland N, Demeure F, Hannaert V, Verplaetse E, Vertommen D, Van der Smissen P, et al. Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei. Biochim Biophys Acta. 2007;1773(4):521–35. doi: 10.1016/j.bbamcr.2007.01.006 17320990 Steiger RF, Opperdoes FR, Bontemps J. Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases. Eur J Biochem. 1980;105(1):163–75. doi: 10.1111/j.1432-1033.1980.tb04486.x 6245876 Ammerman ML, Hashimi H, Novotna L, Cicova Z, McEvoy SM, Lukes J, Read LK. 2011. MRB3010 is a core component of the MRB1 complex that facilitates an early step of the kinetoplastid RNA editing process. RNA 17:865–877. doi: 10.1261/rna.2446311 21451155 Van Veldhoven PP, Baumgart E, Mannaerts GP. Iodixanol (Optiprep), an improved density gradient medium for the iso-osmotic isolation of rat liver peroxisomes. Anal Biochem. 1996;237(1):17–23. doi: 10.1006/abio.1996.0194 8660531 Penha LL, Sant’Anna CB, Mendonca-Previato L, Cunha-e-Silva NL, Previato JO, Lima AP. Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain. Glycobiology. 2009;19(12):1462–72. doi: 10.1093/glycob/cwp121 19696235 Galland N, de Walque S, Voncken FG, Verlinde CL, Michels PA. An internal sequence targets Trypanosoma brucei triosephosphate isomerase to glycosomes. Mol Biochem Parasitol. 2010;171(1):45–9. doi: 10.1016/j.molbiopara.2010.01.002 20138091 Titorenko VI, Nicaud JM, Wang H, Chan H, Rachubinski RA. Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica. J Cell Biol. 2002;156(3):481–94. doi: 10.1083/jcb.200111075 11815635 Crozier TWM, Tinti M, Larance M, Lamond AI, Ferguson MAJ. Prediction of Protein Complexes in Trypanosoma brucei by Protein Correlation Profiling Mass Spectrometry and Machine Learning. Mol Cell Proteomics. 2017;16(12):2254–67. doi: 10.1074/mcp.O117.068122 29042480 Bauer S, Morris MT. Glycosome biogenesis in trypanosomes and the de novo dilemma. PLoS Negl Trop Dis. 2017;11(4):e0005333. doi: 10.1371/journal.pntd.0005333 28426655 Bauer ST, McQueeney KE, Patel T, Morris MT. Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum. J Eukaryot Microbiol. 2017;64(1):97–105. doi: 10.1111/jeu.12343 27339640 Bauer S, Morris JC, Morris MT. Environmentally regulated glycosome protein composition in the African trypanosome. Eukaryot Cell. 2013;12(8):1072–9. doi: 10.1128/EC.00086-13 23709182 Dean S, Sunter JD, Wheeler RJ. TrypTag.org: A Trypanosome Genome-wide Protein Localisation Resource. Trends Parasitol. 2017;33(2):80–2. doi: 10.1016/j.pt.2016.10.009 27863903 Liu L, Xu YX, Caradonna KL, Kruzel EK, Burleigh BA, Bangs JD, et al. Inhibition of nucleotide sugar transport in Trypanosoma brucei alters surface glycosylation. J Biol Chem. 2013;288(15):10599–615. doi: 10.1074/jbc.M113.453597 23443657 Brun R, Schonenberger M. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979;36(3):289–92. 43092 Greig N, Wyllie S, Patterson S, Fairlamb AH. A comparative study of methylglyoxal metabolism in trypanosomatids. FEBS J. 2009;276(2):376–86. doi: 10.1111/j.1742-4658.2008.06788.x 19076214 Dunn KW, Kamocka MM, McDonald JH. A practical guide to evaluating colocalization in biological microscopy. Am J Physiol Cell Physiol. 2011;300(4):C723–42. doi: 10.1152/ajpcell.00462.2010 21209361 Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008;26(12):1367–72. doi: 10.1038/nbt.1511 19029910 Tyanova S, Temu T, Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc. 2016;11(12):2301–19. doi: 10.1038/nprot.2016.136 27809316 Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment. J Proteome Res. 2011;10(4):1794–805. doi: 10.1021/pr101065j 21254760 Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38(Database issue):D457–62. doi: 10.1093/nar/gkp851 19843604 Sury MD, Chen JX, Selbach M. The SILAC fly allows for accurate protein quantification in vivo. Mol Cell Proteomics. 2010;9(10):2173–83. doi: 10.1074/mcp.M110.000323 20525996 Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc. 2009;4(5):698–705. doi: 10.1038/nprot.2009.36 19373234 Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019; 47(D1):D442–D450. doi: 10.1093/nar/gky1106 30395289 10.1371/journal.pntd.0009132.r001 Decision Letter 0 Jardim Armando Associate Editor Madison-Antenucci S Deputy Editor 2021 Jardim, Madison-Antenucci This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

10 Jul 2020

Dear Prof. Ferguson,

Thank you very much for submitting your manuscript "Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Associate Editor

PLOS Neglected Tropical Diseases

S Madison-Antenucci

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: In this paper, the authors demonstrate that enzymes in the nucleotide sugar biosynthetic pathway are localized ot glycosomes. Methods include immunofluorescence assays (IFA) and digitonin fractionations. Sufficient information is provided for the analysis of IFA. I presume that the gels in figure 3 are representative of three biological replicates but I could not find this explicitly stated. Can the authors include the number of replicates in either the Materials and methods or the figure legends? In lines 710-712 the authors state that a portion of the figure has been reproduced from another manuscript. I've not seen this done before and am unsure how to evaluate this data. Perhaps the authors could state why they are using previously published work instead of data from their own experiments. Also, it would be helpful if the authors identified these data visually in the figure as I wasn't able to discern exactly which parts were previously published. Is this portion of the figure is necessary to show that digitonin is solubilizing cell membrane but that glycosome components are still protected? If so, the enolase and aldolase panels are essential for those controls. Can the authors clarify this issue? In lines 164-166 the authors state that aldolase and GAPDH are not quantitatively released until addition of Triton because of the "aggregated nature of these very abundant proteins in the dense crystalloid glycosome core" While many have speculated that the dense nature of the glycosomes is responsible for some of the unique behaviors of glycosome proteins, this hasn't been experimentally demonstrated. For this reason, it may be desirable to qualify the statement to "it has been proposed that ....". I agree that this "non-quantitive" release is common but I don't know that the cause of this is understood. I am always uncomfortable when proteins are not detectable at all in digitonin experiments e.g. TbGMER, GAPDH, PMM. While not likely, it is possible that the proteins are cytosolic but digitonin-sensitive. Appearance of the proteins in the digitonin-insoluble fraction would be comforting. However, I understand that this is not commonplace for these assays and it is not fair for me to ask the authors to include that experiment. Because the authors provide two independent methods to demonstrate localization (IFA and digitonin fractionations), I am comfortable with the conclusion that the proteins are glycosomal.

Reviewer #2: Methods are acceptable.

Reviewer #3: See comments below

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The data does match the analysis plan. With the exception of issues described above in which previously published data was included, the figures are clear and suitable for publication.

Reviewer #2: See my overall review in the Editorial and Data Presentation Modifications section.

Reviewer #3: See comments below

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: It is my opinion that the conclusions are supported by the data, that the authors discuss how these data can be helpful to advance our understanding of parasite metabolism, and that public health relevance is addressed (see summary and general comments). The authors provide a compelling explanation for conflicting data in regards to the localization of several of the proteins that are included in the Tryptag database. The explanation regarding the previous finding of TbMPGT in the cytoplasm is less compelling as the authors suggest the discrepancy is due to using antibodies raised against Leishmania MPGT (Denten et al. PMID:19919534). While the IFA signal in the Denten et al paper may have been a result of non-specific interactions (there is no control with MPGT-deficient parasites), the fractionations indicate that most of the signal was found in the cytosolic fraction with a small amount found in microsomes. While there does appear to be a difference in size between the cytosolic and microsomal species the westerns yield a single, well-defined band. Is there another explanation? Is it possible that this protein could exhibit dual localization under different growth conditions or in different strains where perhaps import of the protein is inefficient?

Reviewer #2: See my overall review in the Editorial and Data Presentation Modifications section.

Reviewer #3: See comments below

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor editorial suggestions: Qualify the statement in lines 163-166 to highlight that people have hypothesize that the non-quantitative release often observed with glycosome proteins is due to the aggregated nature of these proteins.

--Ln 286 should read "His-tag"?

--Lines 308,310: OD280 formatting

--Line 314-TBD buffer composition is not given

--Line 682 T. brucei should be italicized

--Lines 710-712 and Figure 3: clarify which regions of the figure were reproduced from previous work

--Figure 3--red box around text

Reviewer #2: Many proteins are modified by the addition of different sugars. Sugar addition is accomplished by different enzymes that use different activated nucleotide sugars to distinctively modify proteins. In most eukaryotic cells, these enzymes are localized to the cytosol; however, in the trypanosomatids, including the trypanosome Trypanosoma brucei that causes African sleeping sickness, some of these enzymes have been localized to the glycosome, a specialized member of the peroxisome family of membrane-enclosed organelles. In this manuscript, Güther and colleagues perform a comprehensive analysis of the subcellular localization of all the sugar transferases in T. brucei in both its insect (procyclic) and bloodstream forms. Using primarily immunofluorescence microscopy and latency of protein release following treatment of cells with different amounts of the detergent digitonin, the authors provide evidence that the sugar transferase enzymes of T. brucei are localized preferentially to glycosomes. However, this evidence is preliminary and essentially descriptive, and would be greatly strengthened by additional data and analysis.

Major points:

1) The images in Figure 2 appear to be maximum intensity projections. Planar slices should be shown. Moreover, quantification should be performed and the quantification data presented to show the amount of colocalization between a particular enzyme and the glycosomal marker GAPDH.

2) Subcellular fractionation must be done to show that a particular enzyme cofractionates with a known glycosomal marker and not with markers of other organelles such as the mitochondrion or ER.

3) Fig. 3A. As stated on lines 710-712, images in Figure 3A are taken from a previous publication. This is not acceptable. The authors should repeat the experiments.

Minor points:

1) Add scale bars to Figure 2.

2) Figure S1 can be removed.

3) Line 76. “... data suggest...” NOT “...data suggests...”.

4) Lines 158 and 159. There is some controversy that TbPEX13.1 is transmembrane. Best to rephrase simply as ‘membrane’.

5) Line 175. The word ‘notion’ is inappropriate. Replace with ‘evidence’ and remove the ‘The’.

6) Line 215. ‘pulse-chase’ NOT ‘label-chase’.

7) Line 217. ‘chase away’ is not scientific. Rephrase.

8) Line 219. “The same group has...” NOT “the same group have...”.

9) Line 229. “...which converts...” NOT “...that converts...”.

10) Line 681. ‘mutase’ NOT ‘mutate’.

11) Line 698. “...forms of T. brucei.” NOT “...form T. brucei.”

Reviewer #3: See comments below

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: My interpretation of the authors conclusions include two significant findings. First, this work adds to the growing literature suggesting that glycosomes are heterogeneous as many of the glycosome proteins exhibit distinct localization patterns and do not exhibit complete colocalization. While the appreciation of glycosome variation is growing, findings such as these add to the list of proteins that may exhibit distinct localization patterns and are essential to understanding the basis of such heterogeneity. Second, the authors conclude that the NS pathway resides in glycosomes. Such localization necessitates a membrane transporter to facilitate the transfer of these metabolites to the cytoplasm and such a transporter would be an excellent drug target. The next step, identification of the NS transporter, is essential and challenging as there are no clear homologs in the genome and proteomics of the glycosome membrane has revealed few candidates. However, the evidence here that the NS pathway is glycosomal provides additional findings to support premise that such as transporter exists and that the search to identify it is worthwhile.

Reviewer #2: See my overall review in the Editorial and Data Presentation Modifications section.

Reviewer #3: In this original research article, Güther et al., building on a substantial body of previous work, have explored the subcellular location of nucleotide sugar biosynthetic enzymes in trypanosomes. Using IFA against procyclic form trypanosomes (previous worked explored the localization of these proteins by IFA in bloodstream form parasites) they have shown that the majority of these NS biosynthetic enzymes have a punctate distribution that colocalizes, in part with the glycosome marker protein GAPDH. In a separate experiment using differential solubilization of cellular membranes by digitonin, the location of these proteins to an intracellular subcompartment was confirmed. Thus, it is highly likely based upon these studies, as well as the substantial body of previously published work that includes glycosome proteomics, bioinformatic analyses, IFA, and digitonin latency experiments, that the NS biosynthetic enzymes colocalize to the glycosome. While the observations in this manuscript are not particularly novel, since 9 of the 13 NS biosynthetic enzymes had previously been observed to have a glycosome location, this paper does supply important confirmatory evidence for the unusual compartmentalization of these enzymes, which makes them an outlier in comparison to other eukarya.

The manuscript is fairly limited in terms of the types of the types of experiments and the results produced, but does contain a significant body of experimental work when one considers that the authors produced 5 de novo antibodies, and conducted IFA and digitonin latency experiments on 9 different antisera. Most of the experiments were executed with the appropriate controls (see comment below) and the data generated were relatively straight-forward in their interpretation, though there are a few concerns and minor comments for the authors to address below.

1) Since the bulk of the evidence in this manuscript relies on the immunofluorescence localization studies, the inclusion of more representative images is a must. The evaluation of subcellular distribution and colocalization with GAPDH based on just one image per NS biosynthetic enzyme is inadequate. Moreover, given the considerable amount of non-overlap between GAPDH and the nucleotide sugar enzymes, this seems particularly important.

2) Along those lines, it appears from the images that some of the NS biosynthetic enzymes show a minor distribution adjacent to or even along the flagellum, and this appears distinct from GAPDH. Can the authors comment on whether this is the case or perhaps an artifact of the images shown here?

3) I agree that the data certainly indicate glycosome colocalization for the NS biosynthetic enzymes, however, the distinct distribution from GAPDH is intriguing. While the authors indicate this is likely due to glycosome heterogeneity, I am curious whether the authors have looked at other markers? Either glycosomal or perhaps other subcellular compartments?

4) For the digitonin latency studies with BSF and PCF parasites described in Figure 3 A and B, the distribution of TbPAGM seems more distinctly glycosomal in PCF parasites. Also, I may be interpreting the figure legend incorrectly, but it suggests that the bottom two control panels for the BSF digitonin experiments, aldolase and enolase, were included from previous work? I don't consider data generated in a previous experiment to be an adequate control for use in this experiment.

5) Typographical error ln 300, TbUGP should read TbUAP.

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Reviewer #3: No

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 10.1371/journal.pntd.0009132.r002 Author response to Decision Letter 0 Submission Version 1

27 Oct 2020

Submitted filename: PLoS NTD paper replies to reviewers_261020.docx

 10.1371/journal.pntd.0009132.r003 Decision Letter 1 Jardim Armando Associate Editor Madison-Antenucci S Deputy Editor 2021 Jardim, Madison-Antenucci This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

9 Nov 2020

Dear Prof. Ferguson,

Thank you very much for submitting your manuscript "Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

  

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***********************

Dear Professor Ferguson,

We thank you for the revisions addressing many of the reviewers issues. However, at this time it will not be possible to accept the manuscript in its current state since a critical point raised by by two reviewers with extensive cell biology and organelle biogenesis were not addressed. This is an important issue since the major thrust of the manuscript is to assign the localization of NS biosynthesis enzymes. Given the fact that the localization of some NS biosynthesis enzymes have been previously reported to have an incorrect localization based on IFM studies ist is critical to validate the findings that you report in this manuscript using subcellular localization as recommended by Reviewer #2 and that additional marker enzymes for ER and mitochondria should be included. The need for these additional experiments is further emphasized by the issue raised by Reviewer # 3, "Along those lines, it appears from the images that some of the NS biosynthetic enzymes

show a minor distribution adjacent to or even along the flagellum, and this appears distinct from GAPDH. Can the authors comment on whether this is the case or perhaps an artifact of the images shown here?" The rebuttal to this comment

">>>We think this is an artefact of the wide range of NS enzyme : TbGAPDH signal ratios (evident from Table 3) and this is now discussed." is a little concerning as does not sufficiently address the reviewer's concerns. It is acknowledge that IFM and digitonin latency is frequently used (right or wrong), a more robust approach would be to include a subcellular fractionation experiment as indicate by the Reviewers. A careful examination of the Reviewer comment indicate that a subcellular fractionation experiment was a MUST.

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Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

 10.1371/journal.pntd.0009132.r004 Author response to Decision Letter 1 Submission Version 2

24 Dec 2020

Submitted filename: PLoS NTD paper replies to reviewers_241220.docx

 10.1371/journal.pntd.0009132.r005 Decision Letter 2 Jardim Armando Associate Editor Madison-Antenucci S Deputy Editor 2021 Jardim, Madison-Antenucci This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 2

12 Jan 2021

Dear Prof. Ferguson,

We are pleased to inform you that your manuscript 'Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Armando Jardim, PhD

Associate Editor

PLOS Neglected Tropical Diseases

S Madison-Antenucci

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

 10.1371/journal.pntd.0009132.r006 Acceptance letter Jardim Armando Associate Editor Madison-Antenucci S Deputy Editor 2021 Jardim, Madison-Antenucci This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

10 Feb 2021

Dear Prof. Ferguson,

We are delighted to inform you that your manuscript, "Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei.," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases