Cloning, Expression, and Characterization of T. brucei UDP-N-acetylglucosamine Pyrophosphorylase

Parasite Culture—T. brucei bloodstream form
parasites strain 427, variant MITat1.2 (also known as variant 221), that
express T7 polymerase and tetracycline repressor protein under G418 selection
were cultured in HMI-9 medium
(33 (link)) up to a density of ∼2
× 10⁶ cells/ml at 37 °C with 5% CO₂.
Cloning and Sequencing of TbUAP—The TbUAP open
reading frame identified in the T. brucei genome data base was
amplified by PCR from genomic DNA with Pfu polymerase using forward and
reverse primers containing BamHI sites (underlined) of
5′-cgcggatccaatgagtgacagggacgtgtg-3′ and
5′-cgcggatccttacatgttcgatgattcgg-3′, respectively. The
products of six separate PCRs were cloned into pCR-BluntII-Topo®, and a
representative clone from each PCR was sequenced. The primer
5′-cgcagcggttcttcgaggagaattcctac-3′ was also used to obtain
complete sequence coverage of the ORF.
Reverse Transcription-PCR—RNA was extracted using the RNeasy
extraction kits with on-column DNase digestion (RNase-free DNase; Qiagen). RNA
samples (50 ng) were treated with Ominiscript reverse transcriptase (Qiagen)
to generate cDNA. The cDNAs were then amplified by PCR using Taq polymerase
and TbUAP ORF primers (forward,
5′-aatgagtgacagggacgtgtg-3′; reverse,
5′-ttacatgttcgatgattcgg-3′) and DPMS (Dol-P-Man
synthetase) primers (forward, 5′-aatggatgcggaccttcagcacccac-3′;
reverse, 5′-tagaaccgtgagcgcggtgccatac-3′) to show equal RNA
addition.
Southern Blotting—Genomic DNA (5 μg) was digested with
appropriate restriction endonucleases. A DNA probe was made using the
TbUAP ORF and the random primer labeling kit (GE Healthcare). The
probe was then detected using the CDP-Star™ detection kit (GE
Healthcare).
TbUAP Protein Expression and Purification—The TbUAP ORF was cloned into the BamHI site of the expression vector pET15b (Novagen)
to create pET15b-TbUAP, which incorporated a His₆ tag when
expressed. Expression was performed using BL21 (DE3) Escherichia
coli. The cells were grown overnight at room temperature with 0.05
mm isopropyl β-d-1-thiogalactopyranoside. Cells
were harvested and washed in 50 mm Tris-HCl, pH 8.0, 0.3
m NaCl, 1 mg/ml lysozyme, and Roche complete protease inhibitor
mixture tablets (Roche Applied Science) and then lysed in a French press. The
lysate was cleared by centrifugation (40,000 × g, 60 min, 4
°C), passed through a 0.2-μm filter, and loaded onto a precharged
Ni²⁺ HiTrap™ chelating HP column (GE Healthcare).
TbUAP-His₆ was eluted with 50 mm Tris-HCl (pH 8.0), 0.3
m NaCl with 0.1-0.2 m imidazole. The protein was then
dialyzed overnight using a Slide-A-Lyser® dialysis cassette (Pierce) with
10 kDa molecular mass cut-off at 4 °C in 25 mm Na₂HPO₄-NaH₂PO₄ buffer, pH 8.0.
The sample was then filtered as above before being loaded onto a HiTrap™
Q HP-Sepharose column (Amersham Biosciences), preequilibrated with 25
mm Na₂HPO₄-NaH₂PO₄, pH
8.0. The column was washed with 25 mm Na₂HPO₄-NaH₂PO₄, pH 8.0, followed
by a gradient to 25 mm Na₂HPO₄-NaH₂PO₄, pH 8.0, 0.5
m NaCl over 30 min. Fractions (3 ml) were collected and checked by
SDS-PAGE. TbUAP-His₆-containing fractions were pooled and
concentrated, and the buffer was exchanged to 50 mm Tris-HCl, pH
7.5, 10 mm MgCl₂, and 20% glycerol using a Vivaspin
concentrator (Vivascience) 10 kDa molecular mass cut off at 4 °C. The
protein was then stored at -80%.
To obtain a PreScission protease cleavable His₆-tagged TbUAP
protein, the TbUAP open reading frame was amplified by PCR from the
aforementioned pET15b-TbUAP plasmid using the forward primer,
5′-ctccatgggcagcagccatcatcatcatcatcacagcagcggcctggaagttctgttccaggggcccggatccATGAGTGACAGGGACGTGTGCATTCAG-3′,
containing an NcoI restriction site (underlined), the coding sequences for
MGSSHHHHHHSSG (italic type), and a PreScission protease cleavage site of
LEVLFQGP (boldface type), followed by a BamHI restriction site (underlined)
and a TbUAP gene-specific sequence (uppercase) and the reverse
primer,
5′-gctcgagatctggatccTTACATGTTCGATGATTCGGAGACCACC-3′,
containing restriction sites for BglII and BamHI (italic type) and a
TbUAP gene-specific sequence (uppercase). The PCR product was cloned
into the pGEM-T Easy PCR cloning vector (Promega) and subsequently digested
with NcoI and BglII and inserted between the NcoI and BamHI sites of the
pET15b protein expression vector (Novagen). The resulting construct,
pET15b-His₆-PP-TbUAP, that encodes the full TbUAP coding
sequence preceded by the sequence MGSSHHHHHHSSGLEVLFQGPGS (where PreScission
protease cleaves between the Q and the G) was expressed in E. coli and purified on an Ni²⁺ HiTrap™ chelating HP column, as
described above. The sample was then digested with ∼2 mg of
GST-PreScission protease (a kind gift of Bill Hunter; University of Dundee) in
50 mm Tris, pH 8.0, 100 mm NaCl, 10 mm EDTA,
and 1 mm dithiothreitol at room temperature for 4-16 h at 4 °C.
The sample was dialyzed for 2 h using a Slide-A-Lyser® dialysis cassette
(10 kDa molecular mass cut-off) at 4 °C in 2 liters of 50 mm Tris-HCl, pH 8.0, and 50 mm NaCl to remove the EDTA, and then the
sample was passed though a 0.2-μm syringe filter. The sample was passed
through a GSTrap™ HP column (GE Healthcare) connected to an
Ni²⁺ HiTrap™ chelating HP column. The flow-through was then
dialyzed overnight using a Slide-A-Lyser® dialysis cassette at 4 °C in
1 liter of 25 mm Na₂HPO₄-NaH₂PO₄, pH 8.0, with two
changes of buffer. The sample was then passed though a 0.2-μm syringe
filter and further purified using anion exchange chromatography on a
HiTrap™ Q HP-Sepharose column (GE Healthcare).
TbUAP Assays—Two methods were used to assay TbUAP. The HPLC
assay used 0.05 μg of TbUAP-His₆ incubated in 100 μl of the
HPLC assay buffer (50 mm Tris-HCl, pH 7.5, 250 μm UTP, 10 mm MgCl₂, 1 mm dithiothreitol, 20%
glycerol, 250 μm GlcNAc-1-P) for 10 min, terminated by boiling
for 5 min. The samples were analyzed using conditions based on Ref.
34 (link). The HPLC assay buffer was
altered to study substrate specificity, metal ion dependence, and pH
dependence. For substrate specificity, GlcNAc-1-P was changed to glucose
1-phosphate, galactose 1-phosphate, or GalNAc-1-P, all at 250
μm. For metal ion dependence, MgCl₂ was replaced with
CaCl₂, CuCl₂, ZnCl₂, or MnCl₂. For
pH dependence, the Tris-HCl buffer was replaced with a dual buffer of 50
mm Tris, 50 mm sodium acetate with the pH adjusted with
HCl.
The TbUAP colorimetric assay was performed with 0.05 μg of
TbUAP-His₆ in a 96-well plate format (Nunc™) in 90 μl of
50 mm Tris-HCl, pH 7.5, 250 μm UTP, 250
μm GlcNAc-1-P, 10 mm MgCl₂, 1
mm dithiothreitol, 20% glycerol, 0.04 units/ml pyrophosphatase
(Sigma). The reaction was left for 10 min and terminated by the addition of
100 μl of the color reagent (0.2% ammonium molybdate, 0.5% Triton X-100,
0.7 n HCl, 0.03% malachite green). Absorbance at 655 nm was
measured after 5 min using a SpectraMax 340 PC (Molecular Devices).
Construction of a TbUAP Conditional Null Mutant—The gene
replacement cassettes were generated by PCR amplification of 500 bp of UTR
immediately flanking the 5′- and 3′-ends of the TbUAP ORF
with Taq polymerase using the forward and reverse primers
5′-aaggaaaaaaGCGGCCGCagatgcgtgcacaacaaaaa-3′ and
5′-gtttaaacttacggaccgtcaagctttatctataacacacggagcc-3′
and 5′-gacggtccgtaagtttaaacggatccgtggacgttgcagcgcccgg-3′
and 5′-aaggaaaaaaGCGGCCGCcaccacagttcaccatccag-3, respectively. The two
PCR products were then used in a separate PCR to produce a construct
containing the 5′-UTR linked to the 3′-UTR by a short HindIII,
PmeI, and BamHI cloning site (italic type). The resulting PCR product was then
ligated into pGEM-5Zf(+) vector (Promega) using the NotI site (uppercase).
Antibiotic resistance markers were cloned into the HindIII/BamHI restriction
sites between the two UTRs to produce two constructs, one containing the
PAC (puromycin acetyltransferase) drug resistance gene and one
containing the HYG (hygromycin phosphotransferase) drug resistance
gene. To generate the tetracycline-inducible ectopic copy of the
TbUAP ORF, the Nde1 site in the ORF was silenced using the primers
5′-aagcttgggatagcatacgtgcagattggaa-3′ and
5′-attccaatctgcacgtatgctatcccaagct-3′. The primers
5′-catatgatgagtgacagggacgtgtg-3′ and
5′-ttaattaattacatgttcgatgattcgg-3′ were then used to
PCR-amplify the ORF, which was cloned into the vector pLew100 using the NdeI
and PacI sites (italic type)
(33 (link)).
These constructs were purified using the Qiagen Maxiprep kit, digested with
NotI to linearize, precipitated, washed twice with 70% ethanol, and
redissolved in sterile water. The linearized DNA was electroporated into
T. brucei bloodstream cells (strain 427, variant 221) that were
stably transformed to express T7 RNA polymerase and the tetracycline repressor
protein under G418 selection. Cell culture, transformation, and selection were
carried out as previously described
(33 (link)).
Mouse Infection Studies—The TbUAP conditional null
mutant cells were subcultured and grown without selection drugs (hygromycin,
puromycin, phleomycin, and G418) for 24 h with and without 1 μg/ml
tetracycline. The parasites were then introduced into groups of five mice
(dosed with and without doxycycline, respectively) by intraperitoneal
injection of 3 × 10⁵ parasites in 0.2 ml of HMI-9 medium. The
plus doxycycline group of animals were dosed with doxycycline in the drinking
water (0.2 mg/ml in a 5% sucrose solution) for 1 week prior to infection and
until the experiment was terminated. Infections were assessed by tail
bleeding, diluting the blood 1:200 in HMI-9 medium and counting on a Neubauer
hemocytometer.
TbUAP Localization—Two BALB/c adult mice were used to raise
polyclonal antibodies against His₆-tagged TbUAP protein with
Freund's complete adjuvant. Each mouse received two further immunizations with
Freund's incomplete adjuvant over 2 months. Antibodies were then
affinity-purified on CNBr-Sepharose-immobilized TbUAP that had had its
His₆ tag removed with PreScission protease.
Wild type and TbUAP conditional null mutant bloodstream form
T. brucei cells were grown in HMI-9 medium (with or without 1
μg/ml tetracycline for the conditional null mutant) to a density of 1
× 10⁶ cells/ml over 48 h, harvested by centrifugation, and
resuspended in trypanosome dilution buffer (0.1 m Na₂HPO4, 0.01 m NaH₂PO₄, 0.025
m KCl, 0.4 m NaCl, 5 mm MgSO_4, 0.1
m glucose adjusted to pH 7.45 with HCl) to a density of 4 ×
10⁷ cells/ml. Aliquots (15 μl) were added to 13-mm coverslips
(VWR), left at room temperature for 15 min, fixed in 1 ml of 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 1 h followed by three
5 min washes in 2 ml of PBS. Cells were permeabilized with 0.05% Triton X-100
in PBS containing 0.5 mg/ml bovine serum albumin for 10 min at room
temperature. Samples were then blocked in 2 ml of PBS, 0.5% bovine serum
albumin, for 1 h at room temperature. The coverslips were incubated with mouse
anti-TbUAP (1:5,000 dilution) and rabbit anti-glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antiserum (1:10,000; a kind gift of Paul Michels,
Catholic University of Louvain) in PBS, 0.5% bovine serum albumin. Samples
were then washed, as above, in PBS, 0.5% bovine serum albumin and incubated
with 50 μl of Alexa 594-conjugated anti-mouse IgG and Alexa 488-conjugated
anti-rabbit IgG (containing 4′,6-diamidino-2-phenylindole in the case of
the wild type cells) for 1 h. Coverslips were mounted on glass slides (VWR),
sealed with Hydromount containing 2.5% 1,4-diazabicyclo[2.2.2]octane and left
to dry in the dark for 30 min. Microscopy was performed on a Zeiss Axiovert
200 M fluorescence microscope for wild type cells and on a Zeiss LSM 510 META
confocal microscope for the TbUAP conditional null mutant cells.
FIGURE 1.

Substrate specificity of TbUAP. Recombinant TbUAP-His₆ was incubated with UTP and different sugar-1-phosphate substrates, as
indicated, and the products were analyzed by HPLC. A sugar nucleotide product
(UDP-GlcNAc) was observed using GlcNAc-1-P (A) but not without
GlcNAc-1-P (B) or with GalNAc-1-P, Glc 1-phosphate, or Gal
1-phosphate (C-E, respectively).

Sugar Nucleotide Analysis—Sugar nucleotide analysis was
performed as described elsewhere
(19 (link)). Briefly, cells were
pelleted by centrifugation, washed in ice-cold PBS, and lysed in 70% ethanol
in the presence of 10 pmol of GDP-glucose internal standard. Sugar nucleotides
were extracted using EnviCarb columns
(35 (link)) and analyzed using
multiple reaction-monitoring liquid chromatography-tandem mass spectrometry
(19 (link)).
Lectin and Antibody Blotting—T. brucei cells washed
with trypanosome dilution buffer and hypotonically lysed in 300 μl of water
containing 0.1 mm 1-chloro-3-tosylamido-7-amino-2-heptone (TLCK)
and 1 μg/ml leupeptin. Cell ghosts were harvested by centrifugation (13,000
× g for 10 min), and the pellet was resuspended in SDS-sample
buffer containing 8 m urea. The lysed extracts were then subjected
to electrophoresis under reducing conditions, with 1.5 × 10⁷ or 5 × 10⁷ cell equivalents/lane, on a NuPAGE® 4-12%
BisTris gradient (Invitrogen) using MOPS SDS running buffer. Proteins were
then transferred to a nitrocellulose membrane under normal Western blotting
conditions. Membranes were stained with Ponceau S solution to demonstrate
equal loading, blocked with 0.25% bovine serum albumin, 0.05% Igepal detergent
(Sigma), 0.15 m NaCl in 50 mm Tris-HCl, pH 7.4, and then
incubated with 0.33 μg/ml biotinylated tomato lectin (Vector Laboratories),
with or without 3 mg/ml chitin hydrolysate (Vector Laboratories), and then
with 1:10,000 diluted ExtraAvidin-horseradish peroxidase conjugate (Sigma).
All membranes were then developed by chemiluminescent detection (ECL-plus; GE
Healthcare).
To probe for p67, T. brucei was lysed with SDS-sample buffer and
loaded onto a 10% SDS-polyacrylamide gel before being transferred to
nitrocellulose membrane. The membrane was then probed with MAb139 (a kind gift
from Jay Bangs, Madison) at a dilution of 1:2,000 as the primary antibody and
then with 1:10,000 diluted anti-mouse IgG conjugated with horseradish
peroxidase, followed by ECL reagent as described above.
Purification and Endoglycosidase Digestion of Soluble Form Variant
Surface Glycoprotein (sVSG)—The VSG coat of trypanosomes can be
conveniently released in a soluble form through osmotic cell lysis at 37
°C. This causes cleavage of the dimyristoylglycerol component of the GPI
membrane anchors by the action of an endogenous GPI-specific phospholipase C
(30 (link)). T. brucei cultures (100 ml) were washed in trypanosome dilution buffer and resuspended
in 300 μl of lysis buffer (10 mm NaH₂PO₄-Na₂HPO₄, pH 8.0, 0.1
mm TLCK, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) and
incubated at 37 °C for 10 min. This was then cooled on ice for 2 min and
centrifuged for 5 min at 16,000 × g, and the supernatant was
applied to 200 μl of DE52 (Whatman) preequilibrated in 10 mm NaH₂PO₄-Na₂HPO₄, pH 8.0, buffer
and eluted with 4 × 200 μl of fresh lysis buffer. The eluates were
pooled and concentrated to ∼100 μl using a YM-10 spin concentrator
(Microcon). The majority of the buffer salts were removed by diafiltration
with three additions of 0.5 ml of water.
For each enzyme digestion, sVSG was dissolved at 0.2 μg/μl in 0.5%
SDS, 0.1 m dithiothreitol and boiled for 10 min. For
endoglycosidase H (Endo H) digests, 5 μl of the sVSG was added to 20 μl
of 50 mm sodium citrate, pH 5.5, 10 mm phenylmethylsulfonyl fluoride, and 0.025 units of Endo H. For
N-glycosidase F (PNGase F) digests, 5 μl of sVSG was added to 50
mm sodium phosphate, pH 7.5, 0.1% Triton X-100, 10 mm phenylmethylsulfonyl fluoride, and 0.025 units of PNGase F. The digests were
then left overnight at 37 °C.
Electrospray Mass Spectrometry of sVSG—Intact sVSG was
diluted to 0.05 μg/μl in 50% methanol, 1% formic acid and loaded into
Micromass type-F nanotips. The sVSG was analyzed by positive ion electrospray
tandem mass spectrometry using an Applied Biosystems Q-StarXL instrument, and
the masses were calculated using the Bayesian protein reconstruction algorithm
(ABI Analyst Software).