THE SUPPRESSION OF GALACTOSE METABOLISM IN PROCYLIC FORM TRYPANOSOMA BRUCEI CAUSES CESSATION OF CELL GROWTH AND ALTERS PROCYCLIN GLYCOPROTEIN STRUCTURE AND COPY NUMBER*

Galactose metabolism is essential in bloodstream form T.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


Galactose metabolism is essential in bloodstream form T.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 f r a c t i o n a t i o n ,
s u g g e s t i n g a n o v e l compartmentalization of galactose metabolism in this organism. The epimerase is encoded by the TbGALE gene and procyclic form T.brucei singleallele knockouts and conditional (tetracyclineinducible) 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 procylic form glycoproteins, the procyclins, were analysed in TbGALE single-allele knockouts and in the conditional null mutant after removal of t e t r a c y c l i n e .
T h e 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 lifecycle 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 ten-fold more procyclin. The implications of these data with respect to the relative roles of procyclin polypeptide and carbohydrate are discussed.
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 host's innate immune system through the expression of a dense monolayer of 10 7 variant surface glycoprotein (VSG) molecules and it avoids specific immune responses through antigenic variation (1,2). Thus, each parasite expresses only one of a repertoire of several hundred VSG genes at a time. The bloodstream form parasites exist as dividing 'slender' forms and non-dividing 'stumpy' forms that are pre-adapted for survival in the tsetse fly. Following ingestion in a blood meal, the stumpy trypomastigote form differentiates into the dividing procyclic form that colonises the tsetse midgut. The procyclic trypanosomes express a radically different cell surface coat made up about 3 x 10 6 procyclin glycoproteins (3)(4)(5)(6) and a smaller number of free glycoinositolphospholipids (GIPLs) (7)(8)(9). The procyclins are polyanionic, rod-like (6,10), proteins encoded by four procyclin genes: GPEET, that encodes a protein with 5 or 6 Gly-Pro-Glu-Glu-Thr repeats and EP1, EP2 and E P 3 that 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 6 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 Man 5 GlcNAc 2 oligosaccharide, at the N-terminal side of the Glu-Pro repeat domain (6,11). Whereas neither EP2 nor GPEET procyclin is N-glycosylated, GPEET1 procyclin is phosphorylated on six out of seven Thr residues (13)(14)(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 characterised by the presence of large poly-disperse branched poly-N-acetyllactosamine (Galb1-4GlcNAc) containing side-chains (with an average of about 8 to 12 repeats, depending on the preparation) that can terminate with a2-3 linked sialic acid residues (6,20). Sialic acid is transferred from serum sialoglycoconjugates to terminal bGal residues by the action of a cell-surface trans-sialidase enzyme (21)(22)(23) and trans-sialylation of surface components plays a role in the successful colonisation of the tsetse fly (9). In vivo, the Ntermini 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 paper, 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
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 mg/ml hygromycin and 15 mg/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 o C and 5% CO 2 as described in (24).
G e l F i l t r a t i o n a n d A n a l y t i c a l Ultracentrifugation of Recombinant T. brucei UDP-Glc 4'-epimerase -Bacterial expression and purification of recombinant epimerase have been described previously (24). Recombinant protein (100 mg at 1 mg/ml in phosphate-buffered saline (PBS)) was loaded onto a Superdex 200 HR30 HPLC column (Pharmacia Biotech). 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 weight standards (cytochrome C 12.4 kDa, carbonic anhydrase 29 kDa, ovalbumin 43 kDa, bovine serum albumin 66 kDa, alcohol dehydrogenase 150 kDa, b -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 analysed 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 analysed using the SEDFIT programme (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 b-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 SAX column (25 cm x 0.46 cm; HiChrom) and analysed under conditions that separate UDP-hexoses (28). The column was eluted at 1.5 ml/min with 5 mM Na 2 B 4 O 7 for 2 min then with a linear gradient to 200 mM Na 2 B 4 O 7 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, 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 MgSO 4 , 100 mM Na 2 HPO 4 , 10 mM NaH 2 PO 4 and 100 mM glucose) at a final concentration of 2 x 10 7 cells/ml. The parasites were air-dried on cover slips (13 mm) at room temperature, fixed in 4% paraformaldehyde in trypanosome dilution buffer for 30 min at 4 o C and permeabilized with 1% NP40 in phosphate-buffered saline (PBS). After washing four times with (PBS), fixed parasites were treated with 0.1% bovine serum albumen in PBS for 1 h to block non-specific binding and incubated for 1 h with pre-immune rabbit serum or rabbit antiserum (diluted 1/500 with 0.1% bovine serum albumen in PBS) raised against T.brucei glyceraldehyde phosphate dehydrogenase (GAPDH) or against recombinant T.brucei UDP-Glc 4'epimerase (24). The coverslips were washed six times with PBS and incubated for 1 h with Alexa488 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 and purified over DEAE-cellulose (29) and fractionated into large granular, small granular, microsomal and cytosolic fractions according to (30). Aliquots of these fractions (equivalent to 2.9 x 10 8 cells) were subjected to SDS-PAGE on 10% NuPage (Invitrogen) gels and transferred to nitrocellulose, 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 (pH7.4), 0.05% NP40, 2.5 mg/ml bovine serum albumen, washed three times with the same buffer, incubated 1 h with anti-rabbit conjugated to horse radish peroxidase (Scottish Antibody production Unit) diluted 1/5000 with the same buffer, washed three times and developed with ECL (Amersham) according to the manufacturers instructions.

Generation of TbGALE Single-Allele Knockout and Conditional Null Mutant Procyclic form T.brucei Clones -Puromycin (PAC) and
blasticidin (BSR) antibiotic resistance genes were cloned into TbGALE-targeted gene replacement plasmids as described in (24). The tetracyclineinducible expression plasmid (pLew100) containing the TbGALE ORF has been described previously (24). Linearised plasmids (10 mg) were introduced into mid-log (4-8 ¥ 10 6 cells/ml) procyclic cells, that had been washed and resuspended in cytomix (31) at 4 ¥ 10 7 cells/ml, by electroporation at 1.7 kV (3 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 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 were 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 addition of 1 mg/ml tetracycline. To test if 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 ¥ 10 6 cells/ml. Cells were counted daily and cultures were diluted to 1 ¥ 10 6 cells/ml when densities were around 1 x 10 7 cells/ml.
Total RNA for Northern blots was prepared using Qiagen RNAeasy Midi kits. Samples of RNA (5 mg) were run on formaldehyde agarose gels and transferred to Hybond N nylon membrane (Amersham Pharmacia) for hybridisation with [a-32 P]dCTP-labelled T. brucei T b G A L E probe (Stratagene, Prime-It RmT Random Primer labelling kit). The control b-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 approximately 5 ¥ 10 7 cells, as described in (12). Aliquots of 9% butanolextracted procyclins (from the equivalent of 3 ¥ 10 7 cells) were freeze dried and treated with 50 µl icecold 50% aqueous hydrogen fluoride (aq. HF) for 24 h at 0 o C to cleave the GPI anchor ethanolamine-phosphate bond. After freeze drying, aliquots equivalent to 3 ¥ 10 6 cells were further treated with 50 µl 40 mM trifluoroacetic acid (TFA), 100 o C for 20 min, to cleave Asp-Pro bonds and remove N-glycosylated N-termini. The samples were dried and redissolved in 5 µl 0.1% TFA. Aliquots (0.5 µl) of each sample were mixed with 0.5 ml 20 mg/ml sinapinic acid in 70 % acetonitrile, 0.1 % TFA and analysed by negative-ion MALDI-Tof. Data collection was in linear mode on a Voyager-DE STR instrument. The accelerating voltage was 2500 V, grid voltage was set at 94 %, with an extraction time delay of 700 nsec. Data were collected manually at 100 shots per spectrum, with 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 2 x concentrated SDS sample buffer. Aliquots, from either total parasite lysates or butanol extracts, equivalent to 5 x 10 6 cells, were subjected to SDS-PAGE on 4-12% Nu-PAGE (Invitrogen) gels and transferred to PVDF Hybond-P membranes (Amersham Pharmacia) in a semi-dry transfer apparatus at 40 mA for 1h. In the case of the data in Fig. 7A, butanol extracts (2 x 10 7 cell equivalents) were submitted to mild TFA 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 (mAb 247) (5) diluted 1:1000 in PBS, washed three times with PBS and incubated for 1 h with alkaline phosphatase-conjugated goat antimouse IgG (Sigma 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 NBT/BCIP detection tablet (Roche) until bands developed. The membranes were rinsed with water and dried.
GC-MS Composition Analyses of Procyclins -Samples of extracted procyclins (from 2 ¥ 10 7 cells) were mixed with 200 pmol scyllo-inositol internal standard and subjected (in triplicate) to total GPI quantification according to protocol A of (32) and/or to monosaccharide analysis according to (33).

T. brucei UDP-Glc 4'-Epimerase is a Dimer
in Aqueous Solution -The purification and kinetic properties of recombinant T. brucei epimerase has 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 either UDP-Glc, UDP-Gal, UDP-GlcNAc or UDP-GalNAc and the products were analysed by SAX-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 inter-conversion of UDP-Glc and UDP-Gal ( Fig.  2A, B). However, the T. brucei enzyme was unable to inter-convert UDP-GlcNAc and UDP-GalNAc (Fig.  2C, 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. 3A, B). The punctate staining pattern throughout the cell body is similar to that observed using polyclonal rabbit antibodies to the glycosome marker enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) (Fig. 3C, D). Pre-immune sera did not show immunoreactivity against fixed parasites (Fig. 3E, 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 (mitochondriaenriched), small granular (glycosome-enriched), microsomal (endoplasmic reticulum and Golgi apparatus-enriched) and cytosolic fractions by differential centrifugation according to (30). Aliquots of these fractions were analysed 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.
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 drugresistance 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 (TbGALE Ti D TbGALE::PAC/DTbGALE::BSR) were obtained and Southern blots confirmed the replacement of both chromosomal TbGALE alleles with antibiotic resistance genes (Fig. 4).
To test whether the T b G A L E 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 10 7 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 expressed 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).
T. brucei UDP-Glc 4'-epimerase Single-Allele Knockout Clones Exhibit Haploid-Insufficiency with Respect to Procyclin Galactosylation -We analysed whole cell lysates from wild-type cells and from two independent single-allele knockout clones (DTbGALE::PAC) and (DTbGALE::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 knockout cells (Fig. 6A). We also extracted procylins from the same cells and analysed them by negativeion 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 DTbGALE::BSR cells would account for a slight decrease in procyclin average molecular weight (about 0.3 kDa), it seemed likely that the majority of the observed reduction in procyclin apparent molecular weight 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 TMSderivatisation (33) and normalized the figures to Man = 7.0 (Table I). EP1-1, EP1-2 and EP3 Procyclins contain 7 measurable Man residues per molecule because all three contain the same single Man 5 GlcNAc 2 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 knockout 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.

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 re-analysed following mild acid treatment, a procedure that cleaves EPprocyclins 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 Cterminal portion of the molecule. Negative ion M A L D I -T o f a n a l y s i s o f t h e 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 Man 5 GlcNAc 2 N-linked oligosaccharide on all procyclin species (data not shown). The mass spectrometric data, therefore, confirmed that changes in apparent molecular weight observed in (Fig. 7A) were indeed due to changes in the C-terminal portion of the EP procyclins.
The anti-EP procyclin Western blot also suggested a substantial increase in the amount of (lower-molecular weight) EP procyclin at day-9 (compare Fig. 7A, lane 1 with lane 3). To analyse 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 approximately 10-fold higher in the day-9 cells compared to the day-0 cells (Table I). We further anlaysed the Man: Gal ratio in these samples by GC-MS and found that the day-9 procyclins were hypo-galactosylted and contained, on average, 27.5-fold less Gal than day-0 procyclins; i.e., an average of 0.3 Gal residues per 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).
The epimerase appears to be located in glycosomes in the bloodstream and procyclic form of T . b r u c e i according to immunofluorescence microscopy ( Fig. 3A-F) and in bloodstream forms by subcellular fractionation and Western blotting (Fig.  3G). Subcellular fractionation and Western blotting was 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 b-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-1phosphate, is presumably transported out of the glycosome and into the cytosoplasm to react with U T P v i a UTP:glucose-1-phosphate uridylyltransferase to form UDP-Glc. The putative T . b r u c e i UTP:glucose-1-phosphate uridylyltransferase gene sequence (Tb10.389.0330) predicts neither a PTS1-nor 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 epimerised into UDP-Gal. The UDP-Gal must then be transported back to the cytoplasm and, from there, into the lumen of the ER and the Golgi apparatus for use by a range of UDP-Gal-dependent aand b-Gal transferases. Why the UDP-Glc/UDP-Gal epimerization process would need to be compartmentalized in this way is not clear.
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 celldivisions 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 u b i q u i t o u s d o n o r f o r e u k a r y o t e galactosyltransferases. Therefore, the most likely explanation for the essentiality of the TbGALE gene is an absolute requirement for one or more Galcontaining oligosaccharides on one or more parasite glycoproteins. In bloodstream form parasites, there are several known Gal-containing glycoproteins. All VSG variants contain Gal in their N-linked oligosaccharides and/or GPI membrane anchor sidechains (38,(45)(46)(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-acetyllactosamine glycans are absent and the N-linked oligosaccharide repertoire is limited to oligomannose (Man 9-5 GlcNAc 2 ) 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)(8)(9). An obvious hypothesis is that these GPI side-chain poly-N-acetyllactosamine structures may 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 RNAi mutants for the bGaland bGlcNAc-transferases involved in poly-Nacetyl-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 RNAi of GPI biosynthesis genes. The TbGPI10 and T b G P I 8 genes, that encode the third amannosyltransferase 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 non-adherent tissue culture ware is used (8,9). GPI-minus TbGPI10 and TbGPI8 knockout 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) knockout 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 4) N-acetyl-lactosamine repeats (Acosta-Serrano and Ferguson, unpublished data). In this paper, we have analyzed the results of a different kind of cell surface perturbation, brought about by galactose-starvation using procyclic form T.brucei T b G A L E single allele knockout and T b G A L E conditional null mutant cells.
The TbGALE single allele knockout 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 procylin 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 upregulation in procyclin protein expression, presumably an attempt to compensate for the parasite's inability to cover the cell surface with bulky trans-sialylated poly-N-acetyllactosamine units. Thus, while procyclic form T.brucei appears to be able to compensate for a lack of procyclin protein by upregulation of poly-N-acetyllactosamine-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. 1 The abbreviations used are: VSG, variant surface glycoprotein; GIPLs, glycoinositol phospholipids; GPI, glycosylphosphatidylinositol; PAC, puromycin acetyl transferase; BSR, blasticidin resistance; PBS, phosphatebuffered saline; GAPDH, glyceraldeyde phosphate dehydrogenase; TFA, trifluoroacetic acid.       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 (lanes 1-3) and following the resumption of cell growth after 15 days (lane 4). Panel B: MALDI-Tof mass spectra of aq. HF dephosphorylated and mild acid-treated procyclins from the UDP-Glc 4'-epimerase conditional (tetracyclineinducible) 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. Fig. 8. Comparison of procyclic form T.brucei mutant phenotypes. Wild-type procyclic form T.brucei cells express a total of about 4 x 10 6 GPI molecules (32) of which about 3 x 10 6 (6) are attached to procyclins and the balance are presumably mostly cell-surface GIPLs (7)(8)(9). The procyclins have GPI side-chains of about 10 Nacetyl-lactosamine (Galb1-4GlcNAc) units with about 5 terminal sialic acid residues (6,20) while the GIPL side-chains are substantially smaller with about 4 N-acetyl-lactosamine units (Acosta Serrano and Ferguson, unpublished data). Deletion of all procyclin genes (7) or RNAi of TbGPI8 (8) or gene deletion of TbGPI10 or TbGPI8 (9) leads to the upregulation of GIPL expression and cell survival in culture. On the other hand, galactose starvation in TbGALE conditional null mutants under non-permissive conditions (this study) leads to 10-fold upregulation of procyclin protein expression but the cells stop growing once GPI N-acetyl-lactosamine side-chains are depleted. TbGALE conditional null cells, day 0 2.8 ± 0.6 x 10 6 7.0 7.8 ± 1.9 TbGALE conditional null cells, day 9 2.6 ± 0.2 x 10 7 7.0 0.3 ± 0.05

Notes:
All figures are the means of triplicate analyses ± 1 standard deviation. nd = not determined. a Quantified by GC-MS according to (32). b Quantified by GC-MS according to (33). Ratio fixed to Man = 7.0 to reflect the 7 Man residues per mol procyclin (5 from the Man 5 GlcNAc 2 N-linked oligosaccharide and 2 from the GPI anchor) that are detected by this method.