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Originally published In Press as doi:10.1074/jbc.M502370200 on March 14, 2005

J. Biol. Chem., Vol. 280, Issue 20, 19728-19736, May 20, 2005
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The Suppression of Galactose Metabolism in Procylic Form Trypanosoma brucei Causes Cessation of Cell Growth and Alters Procyclin Glycoprotein Structure and Copy Number*

Janine R. Roper{ddagger}, M. Lucia S. Güther, James I. MacRae§, Alan R. Prescott, Irene Hallyburton, Alvaro Acosta-Serrano, and Michael A. J. Ferguson||

From the Division of Biological Chemistry and Molecular Microbiology, The School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom

Received for publication, March 2, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Galactose metabolism is essential in bloodstream form Trypanosoma brucei and is initiated by the enzyme UDP-Glc 4'-epimerase. Here, we show that the parasite epimerase is a homodimer that can interconvert UDP-Glc and UDP-Gal but not UDP-GlcNAc and UDP-GalNAc. The epimerase was localized to the glycosomes by immunofluorescence microscopy and subcellular fractionation, suggesting a novel compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T. brucei single-allele knockouts, and conditional (tetracycline-inducible) null mutants were constructed. Under non-permissive conditions, conditional null mutant cultures ceased growth after 8 days and resumed growth after 15 days. The resumption of growth coincided with constitutive re-expression epimerase mRNA. These data show that galactose metabolism is essential for cell growth in procyclic form T. brucei. The epimerase is required for glycoprotein galactosylation. The major procyclic form glycoproteins, the procyclins., were analyzed in TbGALE single-allele knockouts and in the conditional null mutant after removal of tetracycline. The procyclins contain glycosylphosphatidylinositol membrane anchors with large poly-N-acetyl-lactosamine side chains. The single allele knockouts exhibited 30% reduction in procyclin galactose content. This example of haploid insufficiency suggests that epimerase levels are close to limiting in this life cycle stage. Similar analyses of the conditional null mutant 9 days after the removal of tetracycline showed that the procyclins were virtually galactose-free and greatly reduced in size. The parasites compensated, ultimately unsuccessfully, by expressing 10-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is responsible for human sleeping sickness and the cattle disease Nagana in sub-Saharan Africa. The organism undergoes a complex life cycle between the mammalian host and the insect vector. The bloodstream (trypomastigote) form of the parasite lives in the blood, lymph, interstitial fluids and, ultimately, the cerebrospinal fluid of the host. It avoids the innate immune system of the host through the expression of a dense monolayer of 107 variant surface glycoprotein molecules, and it avoids specific immune responses through antigenic variation (1, 2). Thus, each parasite expresses only one of a repertoire of several hundred variant surface glycoprotein genes at a time. The bloodstream form parasites exist as dividing "slender" forms and non-dividing "stumpy" forms that are preadapted for survival in the tsetse fly. Following ingestion in a blood meal, the stumpy trypomastigote form differentiates into the dividing procyclic form that colonizes the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat made up of about 3 x 106 procyclin glycoproteins (3–6) and a smaller number of free glycoinositol phospholipids (GIPLs)1 (7–9). The procyclins are polyanionic, rod-like (6, 10) proteins encoded by four procyclin genes: GPEET, which encodes a protein with five or six Gly-Pro-Glu-Glu-Thr repeats, and EP1, EP2, and EP3, which encode proteins with 18–30 Glu-Pro repeats (11). In T. brucei strain 427, used in this study, the parasites contain (per diploid genome) two copies of the GPEET1 gene encoding six Gly-Pro-Glu-Glu-Thr repeats, one copy each of the EP1-1 and EP1-2 genes, encoding EP1 procyclins with 30 and 25 Glu-Pro repeats, respectively, two copies of the EP2-1 gene, encoding EP2 procyclin with 25 Glu-Pro repeats, and two copies of the EP3-1 gene, encoding EP3 procyclin with 22 Glu-Pro repeats (12). The EP1 and EP3 procyclins contain a single N-glycosylation site, occupied exclusively by a conventional Man5GlcNAc2 oligosaccharide, at the N-terminal side of the Glu-Pro repeat domain (6, 11). Although neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on 6 out of 7 Thr residues (13–15). In culture, the procyclin expression profile depends on the carbon source (16) and metabolic state of the cells (17, 18), and in the tsetse fly, there appears to be a program of procyclin expression such that GPEET procyclin is expressed early, giving way to EP1 and EP3 procyclin expression (16, 19). GPEET and EP procyclins contain similar glycosylphosphatidylinositol (GPI) membrane anchors. These are the largest and most complex known and are characterized by the presence of large poly-disperse branched poly-N-acetyl-lactosamine (Gal{beta}1-4GlcNAc)-containing side chains (with an average of about 8–12 repeats, depending on the preparation) that can terminate with {alpha}2–3-linked sialic acid residues (6, 20). Sialic acid is transferred from serum sialoglycoconjugates to terminal {beta}-galactose residues by the action of a cell surface trans-sialidase enzyme (21–23) and trans-sialylation of surface components plays a role in the successful colonization of the tsetse fly (9). In vivo, the N termini of the procyclins are removed by tsetse fly gut proteases, and it is thought that the underlying (protease-resistant) anionic repeat units and associated GPI anchor side chains might protect the parasite from the approach tsetse fly gut hydrolases (19).

In this study, we describe the nature and subcellular location of T. brucei UDP-Glc 4'-epimerase, an essential enzyme required for galactose metabolism in bloodstream form T. brucei (24), and show that it is also essential for the in vitro growth of procyclic form T. brucei. We also describe the changes in the cell surface molecular architecture of procyclic form T. brucei when they undergo partial and complete galactose starvation by genetic manipulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Procyclic form T. brucei cells (strain 29:13; a gift from G. A. M. Cross), referred to herein as wild-type cells, were grown in SDM-79 medium (25) in the presence of 50 µg/ml hygromycin and 15 µg/ml G418 to maintain selection for the constitutively expressed T7RNAP and TETR genes, respectively. Bloodstream form T. brucei cells (strain 427, variant 221) were grown in HMI-9 medium (26) supplemented with 10% fetal calf serum at 37 °C and 5% CO2 as described in Ref. 24.

Gel Filtration and Analytical Ultracentrifugation of Recombinant T. brucei UDP-Glc 4'-Epimerase—Bacterial expression and purification of recombinant epimerase have been described previously (24). Recombinant protein (100 µg at 1 mg/ml in phosphate-buffered saline (PBS)) was loaded onto a Superdex 200 HR30 HPLC column (Amersham Biosciences). Proteins were eluted at 0.5 ml/min in PBS and monitored by absorbance at 280 nm. Fractions (2 ml) were collected, and the presence of the epimerase in peak fractions was verified by SDS-PAGE. Molecular mass standards (cytochrome c, 12.4 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 43 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase, 150 kDa; {beta}-amylase, 200 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa) were run under the same conditions to calibrate the column.

Recombinant T. brucei epimerase (1 mg/ml PBS) was analyzed by sedimentation velocity using a Beckman Optima XL-I analytical Ultracentrifuge with an AN50-Ti rotor at 32,000 rpm at 20 °C. Absorbance data (72 scans at 280 nm) were collected and analyzed using the SEDFIT program (27). The epimerase was assumed to be globular, and its density was predicted from its amino acid composition.

HPLC Assay of T. brucei UDP-Glc 4'-Epimerase—Epimerase reactions were performed with UDP-Gal, UDP-Glc, UDP-GalNAc, or UDP-GlcNAc (0.8 mM final concentration) for 16 h at 37 °C in 1 ml of 100 mM glycine buffer (pH 8.7), 1 mM {beta}-NAD+ containing 500 ng (2.95 mU) recombinant epimerase. Samples containing 10 nmol of sugar nucleotide from each reaction were loaded onto a Partisil P10 strong anion exchange column (25 x 0.46 cm; HiChrom) and analyzed under conditions that separate UDP-hexoses (28). The column was eluted at 1.5 ml/min with 5 mM Na2B4O7 for 2 min and then with a linear gradient to 200 mM Na2B4O7 over 40 min, held for 10 min. The eluate was monitored for absorbance at 262 nm. Standards of NAD+, UMP, UDP, UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc (10 nmol) were run under the same conditions to calibrate the system.

Fluorescence Microscopy—Cultured procyclic form and bloodstream form T. brucei cells were harvested by centrifugation and washed and resuspended in ice-cold trypanosome dilution buffer (25 mM KCl, 400 mM NaCl, 5 mM MgSO4, 100 mM Na2HPO4, 10 mM NaH2PO4, and 100 mM glucose) at a final concentration of 2 x 107 cells/ml. The parasites were air-dried on coverslips (13 mm) at room temperature, fixed in 4% paraformaldehyde in trypanosome dilution buffer for 30 min at 4 °C, and permeabilized with 1% Nonidet P-40 in PBS. After washing four times with PBS, fixed parasites were treated with 0.5% fish skin gelatin in PBS for 1 h to block nonspecific binding and incubated for 1 h with preimmune rabbit serum or rabbit antiserum (diluted 1/500 with 0.1% bovine serum albumen in PBS) raised against T. brucei glyceraldehyde-3-phosphate dehydrogenase or against recombinant T. brucei UDP-Glc 4'-epimerase (24). The coverslips were washed six times with PBS and incubated for 1 h with Alexa 488 conjugated to anti-rabbit secondary antibody (Molecular Probes) diluted 1/500 with 0.1% bovine serum albumen in PBS. After six washes with PBS, specimens were mounted in Hydromount, and single optical sections were collected on a Zeiss 510 META confocal microscope (alpha-Plan-Fluor x100).

Subcellular Fractionation and Western Blotting—Bloodstream form T. brucei (strain 427, variant 117) were isolated from infected rats, purified over DEAE-cellulose (29), and fractionated into large granular, small granular, microsomal, and cytosolic fractions according to Ref. 30. Aliquots of these fractions (equivalent to 2.9 x 108 cells) were subjected to SDS-PAGE on 10% NuPage (Invitrogen) gels and transferred to nitrocellulose, and Western blotting was performed using rabbit polyclonal antibodies raised against recombinant UDP-Glc 4'-epimerase and affinity-purified on immobilized recombinant UDP-Glc 4'-epimerase. Blots were incubated for 1 h at room temperature with 0.01 µg/ml antibody in Tris-buffered saline (pH 7.4), 0.05% Nonidet P-40, 2.5 mg/ml bovine serum albumen, washed three times with the same buffer, incubated 1 h with anti-rabbit conjugated to horseradish peroxidase (Scottish Antibody production Unit) diluted 1/5000 with the same buffer, washed three times, and developed with ECL (Amersham Biosciences) according to the manufacturer's instructions.

Generation of TbGALE Single-allele Knock-out and Conditional Null Mutant Procyclic Form T. brucei Clones—Puromycin acetyl transferase (PAC) and blasticidin (BSR) antibiotic resistance genes were cloned into TbGALE-targeted gene replacement plasmids as described in Ref. 24. The tetracycline-inducible expression plasmid (pLew100) containing the TbGALE open reading frame has been described previously (24). Linearized plasmids (10 µg) were introduced into mid-log (4–8 x 106 cells/ml) procyclic cells that had been washed and resuspended in cytomix (31) at 4 x 107 cells/ml, by electroporation at 1.7 kV (three pulses) using a BTX830 square-wave electroporator with a 630B shocking chamber and 0.4-cm gap cuvettes. Antibiotic selection (1 µg/ml for puromycin and 10 µg/ml for blasticidin) was added after overnight recovery in 10 ml of SDM-79. From this stock, aliquots of 2 ml were plated in each of 4 wells of a 24-well plate, and a series of doubling-dilutions in SDM-79 was made across the remaining rows. The remaining 2 ml was retained in a T10 flask and diluted 1:5 with SDM-79.

Epimerase expression from the inducible pLew100 vector was maintained by the addition of 1 µg/ml tetracycline. To test whether TbGALE was essential, conditional epimerase-null cells were washed three times in medium without tetracycline, and cultures (with and without 1 µg/ml tetracycline) were inoculated at 1 x 106 cells/ml. Cells were counted daily, and cultures were diluted to 1 x 106 cells/ml when densities were around 1 x 107 cells/ml.

Southern and Northern Blotting—Genomic DNA for Southern blotting was prepared from 1–2 x 108 cells using DNAzol (Helena Biosciences). BSR, PAC, TbGALE, and TbGALE 5'-untranslated region probes were PCR-amplified, purified, dUTP-fluorescein-labeled by random priming (Gene Images Kit, Amersham Biosciences), and used to probe PstI digests of 5 µg of DNA.

Total RNA for Northern blots was prepared using Qiagen RNeasy Midi kits. Samples of RNA (5 µg) were run on formaldehyde-agarose gels and transferred to Hybond N nylon membrane (Amersham Biosciences) for hybridization with [{alpha}-32P]dCTP-labeled T. brucei TbGALE probe (Stratagene, Prime-It RmT random primer labeling kit). The control {beta}-tubulin probe was used after the TbGALE probe had decayed.

Procyclin Extraction and Analysis by MALDI-TOF Mass Spectrometry—Procyclins were extracted from batches of ~5 x 107 cells, as described in Ref. 12. Aliquots of 9% butanol-extracted procyclins (from the equivalent of 3 x 107 cells) were freeze-dried and treated with 50 µl of ice-cold 50% aqueous hydrogen fluoride (aq.HF) for 24 h at 0 °C to cleave the GPI anchor ethanolamine-phosphate bond. After freeze-drying, aliquots equivalent to 3 x 106 cells were further treated with 50 µl of 40 mM trifluoroacetic acid, 100 °C for 20 min to cleave Asp-Pro bonds and remove N-glycosylated N termini. The samples were dried and redissolved in 5 µl of 0.1% trifluoroacetic acid. Aliquots (0.5 µl) of each sample were mixed with 0.5 µl of 20 mg/ml sinapinic acid in 70% acetonitrile, 0.1% trifluoroacetic acid and analyzed by negative ion MALDI-TOF. Data collection was in linear mode on a Voyager-DE STR instrument. The accelerating voltage was 2500 V, and grid voltage was set at 94% with an extraction time delay of 700 ns. Data were collected manually at 100 shots/spectrum with the laser intensity set at 2500.

SDS-PAGE and Western Blotting of Procyclins—Procyclic form T. brucei cells were washed and resuspended in PBS and lysed by the addition of an equal volume of 2x concentrated SDS sample buffer. Aliquots, from either total parasite lysates or butanol extracts, equivalent to 5 x 106 cells, were subjected to SDS-PAGE on 4–12% NuPAGE (Invitrogen) gels and transferred to polyvinylidene difluoride Hybond-P membranes (Amersham Biosciences) in a semidry transfer apparatus at 40 mA for 1 h. In the case of the data in Fig. 7A, butanol extracts (2 x 107 cell equivalents) were submitted to mild trifluoroacetic acid hydrolysis, as described above, before Western blotting. After blocking overnight with 5% milk powder in PBS, the membranes were washed three times with PBS, incubated for 1 h with anti-EP-procyclin mouse monoclonal antibody 247 (5) diluted 1:1000 in PBS, washed three times with PBS, and incubated for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma catalog number A2179) diluted 1:1000 in PBS. The membranes were washed four times with PBS and once with water and then incubated in 10 ml water containing one nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate detection tablet (Roche Applied Science) until bands developed. The membranes were rinsed with water and dried.



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  		FIG. 1.
Estimation of the molecular mass (MW) of recombinant T. brucei UDP-Glc 4'-epimerase by analytical ultracentrifugation. SEDFIT analysis of sedimentation velocity data from recombinant T. brucei enzyme in PBS at 1 mg/ml.

 
GC-MS Composition Analyses of Procyclins—Samples of extracted procyclins (from 2 x 107 cells) were mixed with 200 pmol of scyllo-inositol internal standard and subjected (in triplicate) to total GPI quantification according to protocol A of Ref. 32 and/or to monosaccharide analysis according to Ref. 33.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
T. brucei UDP-Glc 4'-Epimerase Is a Dimer in Aqueous Solution—The purification and kinetic properties of recombinant T. brucei epimerase have been described previously (24). To determine its oligomeric state, gel filtration and analytical ultracentrifugation of T. brucei recombinant epimerase were performed. The purified recombinant protein was applied to a calibrated Superdex 200 gel filtration column. A major protein and enzyme activity peak corresponding to 85 kDa was observed (data not shown). Analytical ultracentrifugation of the same material showed a main component of 79 kDa and a very minor component of 162 kDa (Fig. 1). These molecular masses are similar to those predicted for recombinant T. brucei UDP-Glc 4'-epimerase homodimer (87 kDa) and homotetramer (174 kDa), respectively. We conclude that T. brucei epimerase is predominantly a homodimer in aqueous solution, consistent with x-ray crystallographic data (34).

Substrate Specificity of T. brucei UDP-Glc 4'-Epimerase— The recombinant enzyme was incubated with UDP-Glc, UDP-Gal, UDP-GlcNAc, or UDP-GalNAc, and the products were analyzed by strong anion exchange-HPLC in the presence of borate ions, a chromatographic system that allows resolution of UDP-Glc from UDP-Gal and UDP-GlcNAc from UDP-GalNAc (28, 35). The HPLC profiles showed the interconversion of UDP-Glc and UDP-Gal (Fig. 2, A and B). However, the T. brucei enzyme was unable to interconvert UDP-GlcNAc and UDP-GalNAc (Fig. 2, C and D).

T. brucei UDP-Glc 4'-Epimerase Is Located in the Glycosome—Polyclonal rabbit antisera were raised to recombinant T. brucei epimerase and used to stain fixed bloodstream form and procyclic form T. brucei cells (Fig. 3, A and B). The punctate staining pattern throughout the cell body is similar to that observed using polyclonal rabbit antibodies to the glycosome marker enzyme glyceraldehyde-3-phosphate dehydrogenase (Fig. 3, C and D). Preimmune sera did not show immunoreactivity against fixed parasites (Fig. 3, E and F). These data suggest that the T. brucei epimerase is located in the glycosomes in both life cycle stages. This conclusion was supported by subcellular fractionation of bloodstream form parasites. The post-nuclear supernatant from mechanically disrupted trypanosomes was fractionated into a large granular (mitochondria-enriched), small granular (glycosome-enriched), microsomal (endoplasmic reticulum- and Golgi apparatus-enriched), and cytosolic fractions by differential centrifugation according to Ref. 30. Aliquots of these fractions were analyzed by SDS-PAGE and Western blotting with affinity-purified anti-UDP-Glc 4'-epimerase (Fig. 3G). The results clearly show that the UDP-Glc 4'-epimerase is located predominantly in the small granular, glycosome-enriched fraction.



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  		FIG. 2.
HPLC assay of sugar nucleotide interconversion by recombinant T. brucei UDP-Glc 4'-epimerase. The T. brucei epimerase was incubated with UDP-Glc (A), UDP-Gal (B), UDP-GlcNAc (C), or UDP-GalNAc (D), and the products were resolved by strong anion exchange-HPLC in borate buffer and detected using a UV monitor at 262 nm. The elution positions of authentic UDP-sugar standards are indicated above the chromatograms. The small peaks at 19 and 32 min are UMP and UDP, respectively, from partial decomposition of the UDP-sugar substrates.

 
UDP-Glc 4'-Epimerase Is Essential for the Growth of Procyclic Form T. brucei—It was possible to replace one TbGALE allele in procyclic form T. brucei by homologous recombination with either PAC or BSR. However, several attempts to replace the second allele with the complementary drug resistance gene failed, suggesting that TbGALE might be an essential gene in procyclic form T. brucei. To investigate this, a conditional null mutant was created. A trypanosome cell line that constitutively expresses T7 RNA polymerase and the tetracycline repressor (TETR) protein under hygromycin and G418-neomycin selection, respectively, was used, and an ectopic epimerase gene was introduced into the trypanosome rDNA locus using the pLew100 expression vector (36). Four conditional null mutant clones (TbGALETi {Delta}TbGALE::PAC/{Delta}TbGALE::BSR) were obtained, and Southern blots confirmed the replacement of both chromosomal TbGALE alleles with antibiotic resistance genes (Fig. 4).

To test whether the TbGALE gene is essential, conditional null mutant cells were washed three times in medium without tetracycline, and cultures (with and without 1 µg/ml tetracycline) were inoculated. Cells were counted daily, and cultures were diluted when densities were around 1 x 107 cells/ml. In the presence of tetracycline, the cells continued to grow with normal (wild-type) kinetics (Fig. 5A), whereas in the absence of tetracycline, the cells grew normally for 8 days followed by a cessation of cell division and some cell death (Fig. 5B) until day 15, when the cultures spontaneously started to grow once more at normal rates (Fig. 5B). Northern blot analysis showed that TbGALE mRNA was more abundant in the conditional mutant than in wild-type cells (Fig. 5C, compare lanes 1 and 2) but that it became undetectable within 6 h of tetracycline removal (Fig. 5C, lane 5). Northern blot analysis of the RNA of cells that spontaneously grew after 15 days showed that they had undergone genetic rearrangement to escape tetracycline control and to constitutively express the ectopic TbGALE gene (Fig. 5C, lane 7). The inclusion of 10 mM Gal in the medium did not alter the results (data not shown).



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  		FIG. 3.
Subcellular localization of T. brucei UDP-Glc 4'-epimerase in bloodstream form and procyclic form T. brucei. Merged phase-contrast (green) and fluorescein isothiocyanate-fluorescence (red) microscopy images of fixed bloodstream form (A, C, and E) and procyclic form (B, D, and F) trypanosomes. The cells were stained for immunofluorescence with rabbit polyclonal antibodies to recombinant T. brucei UDP-Glc 4'-epimerase (A and B) or to the T. brucei glycosome marker glyceraldehyde-3-phosphate dehydrogenase (C and D). Control incubations with preimmune sera are also shown (E and F). G, Western blot of subcellular fractions of bloodstream form trypanosomes (equivalent to 2.9 x 108 cells/lane) using rabbit polyclonal antibodies to recombinant T. brucei UDP-Glc 4'-epimerase. The subcellular fractions are large granular (LG), small granular (SG), microsomal (M), and cytosolic (C) (lanes 1–4, respectively). A positive control of His6-tagged recombinant epimerase (rec) is shown in (lane 5).

 



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  		FIG. 4.
Southern blot characterization of procyclic single-allele knockout and conditional null mutants. Southern blots of genomic DNA digested with PstI were probed with TbGALE open reading frame (GALE), the 5'-untranslated region of TbGALE (5'-untranslated region (5'-UTR)), the blasticidin resistance selectable marker gene (BSR), or the puromycin resistance selectable marker gene (PAC). Lanes 1, wild-type cells; lanes 2, {Delta}TbGALE::BSR; lanes 3, {Delta}TbGALE::PAC; lanes 4, TbGALETi {Delta}TbGALE::PAC (clone 1); lanes 5, TbGALETi {Delta}TbGALE::PAC (clone 2); lanes 6–9, TbGALETi {Delta}TbGALE::BSR/{Delta}TbGALE::PAC (clones 1–4). Note: TbGALETi {Delta}TbGALE::PAC clone 2 (lane 5) was used to create all four conditional null mutant clones (lanes 5–9).

 
T. brucei UDP-Glc 4'-Epimerase Single-allele Knock-out Clones Exhibit Haploid Insufficiency with Respect to Procyclin Galactosylation—We analyzed whole cell lysates from wild-type cells and from two independent single-allele knock-out clones ({Delta}TbGALE::PAC) and ({Delta}TbGALE::BSR) by SDS-PAGE and Western blotting with anti-EP procyclin antibodies. This revealed a reduction in procyclin apparent molecular weight in the single-allele knock-out cells (Fig. 6A). We also extracted procyclins from the same cells and analyzed them by negative ion MALDI-TOF following aq.HF dephosphorylation and mild acid treatment (Fig. 6B). This showed that only EP1-1, EP1-2, and EP3 procyclins were being expressed.

Although the higher proportion of the shorter EP3 procyclin in {Delta}TbGALE::BSR cells would account for a slight decrease in procyclin average molecular mass (about 0.3 kDa), it seemed likely that the majority of the observed reduction in procyclin apparent molecular mass would be due to changes in the GPI anchor side chains, the only site of galactosylation in procyclins (6, 20). Therefore, we measured the Man and Gal content of the procyclins by GC-MS following methanolysis and trimethylsilane derivatization (33) and normalized the figures to Man = 7.0 (Table I). EP1-1, EP1-2, and EP3 procyclins contain 7 measurable Man residues/molecule because all three contain the same single Man5GlcNAc2 N-linked glycan (6, 12, 37) and because only 2 out of the 3 Man residues of their GPI anchors can be liberated as free mannose by methanolysis (38). The data suggest that wild-type procyclins contain an average of 11.8 ± 1.4 Gal residues, whereas the single-allele UDP-Glc 4'-epimerase knock-out mutants contain an average of 8.4 ± 0.6 Gal residues (Table I). This 30% reduction in Gal content suggested that procyclic form T. brucei suffer from haploid insufficiency with respect to procyclin galactosylation but that this insufficiency has negligible effects on growth rate in vitro.


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  		TABLE I
Procyclin compositional data All figures are the means of triplicate analyses ± 1 standard deviation. ND, not determined; KO, knockout.

 



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  		FIG. 5.
TbGALE is essential for the growth of procyclic form T. brucei. A, continuous growth of a TbGALETi {Delta}TbGALE::BSR/{Delta}TbGALE::PAC conditional null mutant in the presence of tetracycline (Tet). B, continuous growth followed by retarded growth and, eventually, recovery of the same clone in the absence of tetracycline (no Tet). C, Northern blot of RNA isolated from wild-type cells (lane 1), the mutant cells 0, 2, 4, 6, and 24 h after removal of tetracycline (lanes 2–6, respectively,) and the mutant cells after growth had resumed after 15 days without tetracycline (lane 7).

 
Procyclic Form T. brucei TbGALE Conditional Null Mutant Cells Express a High Copy Number of Hypo-galactosylated Procyclin Molecules under Non-permissive Conditions—Procyclin samples were extracted from TbGALE conditional null mutant cells 0, 5, and 9 days following the removal of tetracycline from the medium. SDS-PAGE and Western blot analysis with anti-EP procyclin antibodies revealed that the day 5 and day 9 procyclins had significantly lower apparent molecular weights than the day 0 material (data not shown). The samples were reanalyzed following mild acid treatment, a procedure that cleaves EP procyclins at the Asp-Pro bonds present before the (EP)n-GPI domain in all EP procyclins and that simplifies the SDS-PAGE pattern of these molecules (12). A similar reduction in apparent molecular weight was observed (Fig. 7A), suggesting that the molecular weight shift was due to changes in the C-terminal portion of the molecule. Negative ion MALDI-TOF analysis of the aq.HF dephosphorylated/mild acid-treated procyclins from day 0 and day 9 showed that only EP1-1, EP1-2, and EP3 procyclins were present and that the relative proportions of these isoforms were very similar (Fig. 7B). An analysis of aq.HF dephosphorylated material without mild acid treatment revealed the presence of the same Man5GlcNAc2 N-linked oligosaccharide on all procyclin species (data not shown). The mass spectrometric data, therefore, confirmed that changes in the apparent molecular weight observed in (Fig. 7A) were indeed due to changes in the C-terminal portion of the EP procyclins.



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  		FIG. 6.
Gene replacement of one allele of TbGALE results in the expression of smaller procyclin molecules. A, anti-procyclin Western blot of cell lysates from wild type and two independent single allele knock-out clones of procyclic form T. brucei. Molecular mass markers (MW) are shown on the left. B, MALDI-TOF mass spectra of aq.HF dephosphorylated and mild acid-treated procyclins from wild type (top), {Delta}TbGALE::BSR single allele knock-out cells (middle), and {Delta}TbGALE::PAC single allele knock-out cells (bottom). The diagnostic peptide ions (12) for EP1-1, EP1-2, and EP3 procyclins are indicated.

 



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  		FIG. 7.
Galactose starvation causes an up-regulation in the expression of hypo-galactosylated procyclin. A, SDS-PAGE and anti-procyclin Western blot of mild acid-treated procyclins from the UDP-Glc 4'-epimerase conditional (tetracycline-inducible) null mutant 0, 5, and 9 days after withdrawal of tetracycline (Tet, lanes 1–3) and following the resumption of cell growth by "revertants" (rev) after 15 days (lane 4). B, MALDI-TOF mass spectra of aq.HF dephosphorylated and mild acid-treated procyclins from the UDP-Glc 4'-epimerase conditional (tetracycline-inducible) null mutant 0 and 9 days after withdrawal of tetracycline. The diagnostic peptide ions (12) for EP1-1, EP1-2, and EP3 procyclins are indicated.

 
The anti-EP procyclin Western blot also suggested a substantial increase in the amount of (lower molecular weight) EP procyclin at day 9 (Fig. 7A, compare lane 1 with lane 3). To analyze this directly, we measured the absolute molar quantity of procyclins in the day 0 and day 9 samples using a GC-MS method that quantifies the GPI component of a sample by measuring the non-N-acetylated glucosamine content (32). The results showed that procyclin expression was ~10-fold higher in the day 9 cells when compared with the day 0 cells (Table I). We further analyzed the Man:Gal ratio in these samples by GC-MS and found that the day 9 procyclins were hypo-galactosylated and contained, on average, 27.5-fold less Gal than day 0 procyclins, i.e. an average of 0.3 Gal residues/molecule (Table I). Finally, we noted that cells that resumed growth after day 15, due to constitutive expression of the ectopic TbGALE gene, restored their expression of higher molecular weight procyclin (Fig. 7A, lane 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
T. brucei UDP-Glc 4'-epimerase is a typical dimeric (Fig. 1) NADH-dependent oxidoreductase (EC 5.1.3.2 [EC] ) that interconverts UDP-Glc and UDP-Gal. However, unlike the human epimerase (39), the T. brucei enzyme is unable to interconvert UDP-GlcNAc and UDP-GalNAc (Fig. 2). The same is true for the Trypanosoma cruzi enzyme (35), and this almost certainly explains why GalNAc has not been found in any trypanosome glycoconjugates.

The epimerase appears to be located in glycosomes in the bloodstream and procyclic form of T. brucei according to immunofluorescence microscopy (Fig. 3, A–F) and in bloodstream forms by subcellular fractionation and Western blotting (Fig. 3G). Subcellular fractionation and Western blotting were also attempted with procyclic cells, but there was insufficient epimerase in these cells to allow Western blot detection. This low level of epimerase expression in procyclic form T. brucei is consistent with the haploid insufficiency data, described below. The glycosomal location of the epimerase is consistent with the presence of a C-terminal peroxisome targeting sequence type 1 (PTS1) of -TKL in the epimerase amino acid sequence (40). Glycosomes are peroxisome-related microbodies found in all kinetoplastids that contain (and thus compartmentalize) the enzymes of glycolysis, fatty acid {beta}-oxidation, ether lipid synthesis, purine salvage, and pyrimidine and sterol synthesis (41, 42). The presence of UDP-Glc 4'-epimerase in the glycosome suggests a complex compartmentalization of sugar nucleotide biosynthesis such that the product of hexokinase and phosphoglucose mutase, glucose-1-phosphate, is presumably transported out of the glycosome and into the cytoplasm to react with UTP via UTP:glucose-1-phosphate uridylyltransferase to form UDP-Glc. The putative T. brucei UTP:glucose-1-phosphate uridylyltransferase gene sequence (Tb10.389.0330) predicts neither a PTS1-type nor a PTS2-type glycosomal import signal and is presumed to be cytoplasmic. Thus, UDP-Glc made in the cytoplasm most likely enters the glycosome, via a specific sugar nucleotide transporter or pore, to be epimerized into UDP-Gal. The UDP-Gal must then be transported back to the cytoplasm and, from there, into the lumen of the endoplasmic reticulum and the Golgi apparatus for use by a range of UDP-Gal-dependent {alpha}- and {beta}-galactosidase transferases. Why the UDP-Glc/UDP-Gal epimerization process would need to be compartmentalized in this way is not clear.



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  		FIG. 8.
Comparison of procyclic form T. brucei mutant phenotypes. Wild-type procyclic form T. brucei cells express a total of about 4 x 106 GPI molecules (32), of which about 3 x 106 (6) are attached to procyclins and the balance are presumably mostly cell surface GIPLs (7–9). The procyclins have GPI side chains of about 10 N-acetyl-lactosamine (Gal{beta}1-4GlcNAc) units with about 5 terminal sialic acid residues (6, 20), whereas the GIPL side chains are substantially smaller with about 4 N-acetyl-lactosamine units (see footnote 2). Deletion of all procyclin genes (7) or RNA interference of TbGPI8 (8) or gene deletion of TbGPI10 or TbGPI8 (9) leads to the up-regulation of GIPL expression and cell growth in culture. On the other hand, galactose starvation in TbGALE conditional null mutants under non-permissive conditions (this study) leads to 10-fold up-regulation of procyclin protein expression, but the cells stop growing once GPI N-acetyl-lactosamine side chains are depleted.

 
The UDP-Glc 4'-epimerase, and therefore, galactose metabolism, has been shown to be essential in bloodstream form T. brucei (24). Similarly, we were unable to create a TbGALE null mutant in procyclic form T. brucei, and the cessation of growth under non-permissive conditions in a procyclic form conditional null mutant (Fig. 5) supports the conclusion that UDP-Glc 4'-epimerase activity is also essential for cell growth in this life cycle stage of the parasite. The long delay (7–8 days) between the suppression of TbGALE mRNA transcription and the cessation of cell growth could be due to high TbGALE mRNA levels in the tetracycline-induced conditional null mutant, leading to increased epimerase protein levels, combined with a relatively long protein half-life. Thus, it could take several cell divisions to reduce epimerase levels to unsustainable levels. The inability of free Gal in the medium to rescue the conditional null mutants shows that, like bloodstream form T. brucei, procyclic form T. brucei is unable to take up (43) and metabolize Gal by the Leloir pathway (44).

The sugar nucleotide UDP-Gal is the ubiquitous donor for eukaryote galactosyltransferases. Therefore, the most likely explanation for the essentiality of the TbGALE gene is an absolute requirement for one or more Gal-containing oligosaccharides on one or more parasite glycoproteins. In bloodstream form parasites, there are several known Gal-containing glycoproteins. All variant surface glycoprotein variants contain Gal in their N-linked oligosaccharides and/or GPI membrane anchor side chains (38, 45–47) and, according to ricin binding (48), tomato lectin binding (49), and structural characterization (50), several other glycoproteins in the flagellar pocket and endosomal/lysosomal system contain poly-N-acetyl-lactosamine glycans. Some of these N-linked poly-N-acetyl-lactosamine glycans are extremely large and of unusual structure (50). In procyclic form T. brucei, these large poly-N-acetyl-lactosamine glycans are absent, and the N-linked oligosaccharide repertoire is limited to oligomannose (Man9-5GlcNAc2) structures (6, 47, 51, 52). Thus, in this life cycle stage, Gal appears to be largely restricted to the poly-N-acetyl-lactosamine-containing side chains of the procyclin GPI anchors (6, 47) and of the free GIPLs (7–9). An obvious hypothesis is that these GPI side chain poly-N-acetyl-lactosamine structures play an important (possibly anti-adhesive) protective role on the cell surface. A way to test this would be to make null, conditional null, or inducible RNA interference mutants for the {beta}-Gal- and {beta}-GlcNAc-transferases involved in poly-N-acetyl-lactosamine synthesis. However, this is not yet experimentally amenable because bioinformatic analysis has not provided obvious candidates for these genes.

Thus far, the normal molecular architecture of procyclic form T. brucei has been perturbed by gene knockouts of procyclin genes and by gene knockouts and RNA interference of GPI biosynthesis genes. The TbGPI10 and TbGPI8 genes, which encode the third {alpha}-mannosyltransferase and the catalytic subunit of the GPI:protein transamidase, respectively, are essential for bloodstream form T. brucei but non-essential for the procyclic form in culture, provided that non-adherent tissue culture ware is used (8, 9). GPI-minus TbGPI10 and TbGPI8 knock-out cells do not express procyclin on their surface and are significantly impaired (particularly the TbGPI8 knockout) in their ability to colonize the tsetse fly midgut. Analysis of these cells and total procyclin gene (GPEET, EP1, EP2, and EP3) knock-out cells (7) revealed that the absence of cell surface procyclin is compensated for by an increase in the copy number of GIPLs that are, in essence, GPI anchors not attached to protein and with fewer (about four) N-acetyl-lactosamine repeats.2 In this study, we have analyzed the results of a different kind of cell surface perturbation, brought about by galactose-starvation using procyclic form T. brucei TbGALE single allele knock-out and TbGALE conditional null mutant cells.

The TbGALE single allele knock-out clones exhibited a reduction in the apparent molecular weights of their EP procyclins (Fig. 6) that was traced to a reduction in their Gal content of about 30% (Table I). This clearly had little or no impact on cell viability but serves to demonstrate that procyclic form T. brucei does not express an excess of epimerase activity to supply its galactosylation needs. Indeed, the barely detectable levels of TbGALE mRNA and undetectable levels of epimerase protein in wild-type procyclic cells is consistent with this notion. A similar example of haploid insufficiency with respect to a biochemical phenotype (rather than viability) was reported for the ConA 1-1 procyclic form mutant that lacks one functional allele of polyprenol reductase, an enzyme involved in synthesis of dolichol. This partial defect affected procyclin N-glycosylation and rendered the parasites resistant to killing by the lectin concanavalin A (52).

A more dramatic phenotype than that of the TbGALE single-allele knockout was observed with the epimerase conditional null mutant following the withdrawal of tetracycline (Fig. 7A). In this case, after 9 days, when growth essentially ceased, the cells still expressed the same EP procyclins as the tetracycline-induced control cells but with dramatically lower apparent molecular weights. The entire reduction in molecular weight was mapped to the GPI anchor side chains that were almost entirely free of galactose (Table I). This reduction in size correlated with a 10-fold up-regulation in procyclin protein expression, presumably an attempt to compensate for the inability of the parasite to cover the cell surface with bulky trans-sialylated poly-N-acetyl-lactosamine units. Thus, although procyclic form T. brucei appears to be able to compensate for a lack of procyclin protein by up-regulation of poly-N-acetyl-lactosamine-containing GIPLs (7–9), the converse is not the case (Fig. 8). We suggest, therefore, that the primary role of procyclins (at least in the early stages of parasite differentiation and tsetse fly colonization) is to act as a platform for the efficient expression of anti-adhesive, membrane-protecting trans-sialylated poly-N-acetyl-lactosamine oligosaccharides.


   FOOTNOTES
 
* This work was supported by Wellcome Trust grants 71463 and 60669. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Astra-Zeneca, Charnwood, LE11 5RH. Back

§ Supported by a Ph.D. studentship from The States of Guernsey. Back

Supported by a Wellcome Trust Travelling Research Fellowship. Present address: Wellcome Centre for Molecular Parasitology, University of Glasgow, G11 6NU. Back

|| To whom correspondence should be addressed. E-mail: m.a.j.ferguson{at}dundee.ac.uk.

1 The abbreviations used are: GIPLs, glycoinositol phospholipids; GPI, glycosylphosphatidylinositol; PAC, puromycin acetyl transferase; BSR, blasticidin resistance; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; GC-MS, gas chromatography-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; aq.HF, aqueous hydrogen fluoride; Gal{beta}1-4Glc-NAc, N-acetyl-lactosamine. Back

2 A. Acosta-Serrano and M. A. J. Ferguson, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Fred Opperdoes and Paul Michels for the anti-glyceraldehyde-3-phosphate dehydrogenase antibody and for advice on subcellular fractionation. We thank Mick Urbaniak for providing recombinant T. brucei epimerase and Alan Fairlamb for helpful suggestions.



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 DISCUSSION
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