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 ) up to a density of 2 
   10 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 .
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 .
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-m filter , and loaded onto a precharged 
  ni 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 ni 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-m syringe filter .
the sample was passed 
  through a gstrap hp column ( ge healthcare ) connected to an 
  ni 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-m syringe 
  filter and further purified using anion exchange chromatography on a 
  hitrap q hp - sepharose column ( ge healthcare ) .
the hplc 
  assay used 0.05 g of tbuap - his6 incubated in 100 l of the 
  hplc assay buffer ( 50 mm tris - hcl , ph 7.5 , 250 m 
  utp , 10 mm mgcl2 , 1 mm dithiothreitol , 20% 
  glycerol , 250 m glcnac-1-p ) for 10 min , terminated by boiling 
  for 5 min .
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 
  m . 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 g of 
  tbuap - his6 in a 96-well plate format ( nunc ) in 90 l of 
  50 mm tris - hcl , ph 7.5 , 250 m utp , 250 
  m 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 l 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 ) .
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 ) .
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 g / ml 
  tetracycline .
the parasites were then introduced into groups of five mice 
  ( dosed with and without doxycycline , respectively ) by intraperitoneal 
  injection of 3  10 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 
  g / ml tetracycline for the conditional null mutant ) to a density of 1 
   10 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 mgso4 , 0.1 
  m glucose adjusted to ph 7.45 with hcl ) to a density of 4  
  10 cells / ml . aliquots ( 15 l ) 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 l 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 .
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 ) .
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 ) .
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 ) and analyzed using 
  multiple reaction - monitoring liquid chromatography - tandem mass spectrometry 
  ( 19 ) .
brucei cells washed 
  with trypanosome dilution buffer and hypotonically lysed in 300 l of water 
  containing 0.1 mm 1-chloro-3-tosylamido-7-amino-2-heptone ( tlck ) 
  and 1 g / 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  10 
  or 5  10 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 g / 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 ) .
t. brucei 
  cultures ( 100 ml ) were washed in trypanosome dilution buffer and resuspended 
  in 300 l of lysis buffer ( 10 mm 
  nah2po4-na2hpo4 , ph 8.0 , 0.1 
  mm tlck , 1 g / ml leupeptin , and 1 g / 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 l of de52 ( whatman ) preequilibrated in 10 mm 
  nah2po4-na2hpo4 , ph 8.0 , buffer 
  and eluted with 4  200 l of fresh lysis buffer .
the eluates were 
  pooled and concentrated to 100 l 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 g/l in 0.5% 
  sds , 0.1 m dithiothreitol and boiled for 10 min . for 
  endoglycosidase h ( endo h )
digests , 5 l of the svsg was added to 20 l 
  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 l 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 g/l 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 ) .
identification , cloning , and expression of tbuap  a blastp 
  search of the t. brucei predicted protein data base with homo 
  sapiens and saccharomyces cerevisiae uap amino acid sequences , 
  accession numbers np_003106 and np _ 010180 , revealed a putative tbuap 
  gene ( gene number tb11.02.0120 ) .
the tbuap open reading frame was 
  amplified by pcr using pfu polymerase from genomic dna prepared from 
  t. brucei strain 427 .
two different consensus sequences emerged 
  ( accession numbers am909685 and am909686 ) , suggesting that there are two 
  slightly different tbuap alleles , with eight base pair differences 
  and one amino acid difference between them ( ser or gly at position 507 ) . 
 
southern blot analysis using a tbuap orf probe confirmed that tbuap 
  is present as a single copy per haploid genome ( supplemental fig .
the tbuap amino 
  acid sequence contains the expected pyrophosphorylase 
  lx2gxgtx6pk motif , and 13 of 
  15 residues shown to be involved in substrate binding in the human uap crystal 
  structures ( 36 ) are conserved 
  in the tbuap sequence ( supplemental fig .
the tbuap orf was cloned into a pet15b expression vector , with a 
  his6 tag at the n terminus and a modified pet15b vector with a 
  prescission protease site between the protein and the his6 tag . 
 
both were expressed in e. coli and purified as described under 
   experimental procedures 
analysis 
  of the recombinant tbuap - his6 protein by analytical 
  ultracentrifugation ( at 1 mg / ml ) indicated that the majority ( > 80% ) of 
  tbuap is monomeric at this concentration ( data not shown ) .
enzymatic activity of tbuap  the activity and substrate 
  specificity of tbuap was assessed by incubating recombinant 
  tbuap - his6 with utp and glcnac-1-p , galnac-1-p , glc 1-phosphate , or 
  gal 1-phosphate and analyzing the products by hplc .
using glcnac-1-p as the 
  substrate , a single uv - absorbing peak that co - eluted with authentic udp - glcnac 
  was observed ( fig . 
 
no sugar nucleotide product was observed in the 
  absence of tbuap - his6 ( fig . 
 
1b ) or when galnac-1-p , glc 1-phosphate , or gal 
  1-phosphate was used as a substrate ( fig . 
  1 , c - e ) .
these data show that glcnac-1-p is the preferred 
  substrate of tbuap under these conditions . to test the metal ion dependence of the tbuap - his6 activity ,
the 
  enzyme was preincubated with 5 mm edta and then incubated with utp , 
  glcnac-1-p , and 10 mm mgcl2 , mncl2 , 
  cacl2 , cucl2 , zncl2 , or no divalent cation . 
 
the products of triplicate experiments were analyzed by hplc , and relative 
  yields of udp - glcnac were determined . in the presence of edta alone , no 
  detectable udp - glcnac was formed , suggesting that tbuap is divalent metal 
  ion - dependent .
similar levels of activity were restored with mg 
  and mn , whereas ca , cu , and 
  zn failed to restore detectable activity ( supplemental fig . 
 
the ph dependence of the activity was studied over the ph range 5.0 - 9.5 , 
  using the same hplc - based assay .
the enzyme displayed a broad ph optimum 
  between ph 6.0 and 9.0 ( supplemental fig . 6 ) . based on the aforementioned 
  experiments ,
the enzyme was assayed at ph 7.5 in the presence of 10 
  mm mgcl2 . in order to measure the apparent km values for the two 
  substrates of tbuap , a discontinuous , colorimetric ,
the assay relies on pyrophosphatase to convert the ppi 
  component of the utp + glcnac-1-p  udp - glcnac + ppi reaction 
  to inorganic phosphate , which is subsequently measured using malachite green . 
 
the enzyme was assayed with a fixed concentration of utp and varying 
  concentrations of glcnac-1-p and with a fixed concentration of glcnac-1-p and 
  varying concentrations of utp ( supplemental figs . 7 and 8) .
the apparent 
  km values for utp and glcnac-1-p are 26  6 and 39 
   13 m , respectively , with a vmax of 
  0.4  0.1 nmol / min and a specific activity of 24  6 
  mol / min / mg . these values are compared with those of other recombinant 
  eukaryotic uaps 
  ( 36 - 38 ) 
  in table 1 .
table 1comparison of recombinant eukaryotic uap propertiesspeciesmolecular massglcnac-1-p kmutp kmspecific activityalternative substratesreference / sourcekda m m molminmg 
  1 t .
brucei ( tbuap ) 
   60.1 
   39  13 
   26  6 
   26  6 
   none 
   this study 
  g. intestinalis ( giuap ) 
   49.6 
   300  120 
   38  17 
   9.2  1.9 , 55.4  11.1 
   glc 1-phosphate , galnac-1-p 
   ref .
38 
  s. cerevisiae ( scuap ) 
   53.5 
   14 
   21 
   17 
   glc 1-phosphate 
   ref .
37 
  h. sapiens ( agx1 ) 
   57.0 
   5.3 
   53 
   69 
   galnac-1-p 
   ref .
36 
  h. sapiens ( agx2 ) 
 
  58.8 
 
  6.0 
 
  49 
 
  68 
 
  galnac-1-p 
 
  ref
. 36 
   comparison of recombinant eukaryotic uap properties construction of a tbuap conditional null mutant  in order to 
  test the hypothesis that udp - glcnac biosynthesis is essential for bloodstream 
  form t. brucei and that there is no alternate route to this 
  metabolite , a conditional null mutant of the final step of udp - glcnac 
  biosynthesis was created by the replacement of both endogenous copies of the 
  tbuap gene and the introduction of an ectopic inducible copy of 
  tbuap under tetracycline control .
a genetically modified strain 427 
  t. brucei cell line was used to generate the conditional null mutant . 
 
this cell line is engineered to constitutively express t7 polymerase and 
  tetracycline repressor protein under the control of a t7 promoter .
this cell line will be 
  referred to as  wild type  from hereon .
the first endogenous allele of tbuap was replaced 
  with the pac gene , creating tbuap : pac . the 
  ectopic copy ( plew100-tbuap ) was then inserted into the rdna region , 
  creating tbuap : pac / tbuap .
the 
  second allele of tbuap was then replaced with the hyg gene , 
  creating 
  tbuap : pac / tbuap/tbuap : hyg . 
 
genomic dna from the wild type and three mutant cell lines 
  produced during the creation of the tbuap conditional null mutant was 
  digested with bglii and nhei and probed with tbuap open reading 
  frame .
genomic dna was from wild type ( lane 1 ) , 
  tbuap : pac ( lane 2 ) , 
  tbuap : pac / tbuap ( lane 
  3 ) , and the final 
  tbuap : pac / tbuap/tbuap : hyg 
  conditional null mutant ( lane 4 ) .
the first endogenous allele of tbuap was replaced 
  with the pac gene , creating tbuap : pac . the 
  ectopic copy ( plew100-tbuap ) was then inserted into the rdna region , 
  creating tbuap : pac / tbuap .
the 
  second allele of tbuap was then replaced with the hyg gene , 
  creating 
  tbuap : pac / tbuap/tbuap : hyg . 
 
genomic dna from the wild type and three mutant cell lines 
  produced during the creation of the tbuap conditional null mutant was 
  digested with bglii and nhei and probed with tbuap open reading 
  frame .
genomic dna was from wild type ( lane 1 ) , 
  tbuap : pac ( lane 2 ) , 
  tbuap : pac / tbuap ( lane 
  3 ) , and the final 
  tbuap : pac / tbuap/tbuap : hyg 
  conditional null mutant ( lane 4 ) .
the first tbuap allele was replaced by homologous recombination 
  following electroporation of the parasites in the presence of linear dna 
  containing a pac gene flanked by about 500 bp of tbuap 
  5- and 3-utr . following selection with puromycin ,
a 
  tbuap : pac clone was selected and transformed with an 
  ectopic , tetracycline - inducible , copy of tbuap , introduced into the 
  ribosomal dna locus under phleomycin selection 
  ( 33 ) . after tetracycline 
  induction , the second endogenous allele was replaced by a hyg gene to 
  yield the desired 
  tbuap : pac / tbuap/tbuap : 
  hyg clone ( fig . 
 
after each round of transformation , genomic dna was 
  extracted for southern blot analysis using a tbuap orf probe and 
  genomic dna digested with bglii and nhei .
under these conditions , the 
  endogenous tbuap gene produces a fragment of 5.6 kb , whereas the 
  ectopic copy produces a fragment of 2.5 kb . the blot 
  ( fig .
2b ) shows the 
  successful introduction of the ectopic copy and replacement of both endogenous 
  alleles in the 
  tbuap : pac / tbua - p/tbuap : hyg 
  clone used for further studies .
this cell line will be referred to from hereon 
  as the tbuap conditional null mutant .
the tbuap gene is essential to bloodstream form t. brucei in vitro and 
  in vivo 
triplicate cultures of wild type and tbuap 
  conditional null mutant cells , under permissive and nonpermissive conditions 
  ( i.e. with and without tetracycline , respectively ) , were inoculated 
  at 1  10 cells / ml and subcultured every 2 days . the 
  tbuap conditional null mutant cultures under permissive conditions 
  had a reduced growth rate and grew to approximately half the cell density of 
  the wild type but were otherwise healthy 
  ( fig .
3 , a and 
  b ) . under nonpermissive conditions the cells grew for 2 
  days but , upon subculturing , failed to grow for more than 5 days 
  ( fig .
the 
  eventual resumption of growth is typical of bloodstream form t. 
  brucei conditional null mutants for essential genes 
  ( 6 , 
  14 , 
  18 , 
  39 - 42 ) , 
  whereby the essential gene becomes constitutively active through loss of 
  tetracycline control due to deletion of the tetracycline repressor protein 
  gene ( 14 ) .
analysis by reverse 
  transcription - pcr confirmed that tbuap mrna was undetectable by 12 h 
  of tetracycline removal but that the cells that resumed growth , 10 days after 
  tetracycline removal , were reexpressing tbuap mrna 
  ( fig .
3d ) . figure 3.tbuap expression is essential for the growth of bloodstream 
  form t. brucei in culture .
b , growth curves for 
  tbuap conditional null mutant cells , subcultured every 2 days , grown 
  under permissive ( plus tetracycline ) conditions .
c , growth curves for 
  tbuap conditional null mutant cells grown under nonpermissive ( minus 
  tetracycline ) conditions .
d , ethidium bromide - stained agarose gel of 
  reverse transcription - pcr products from rna extracted from tbuap 
  conditional null mutant cells after 0 , 2 , 6 , 12 , and 48 h and 10 days without 
  tetracycline , as indicated .
the upper panel shows reverse 
  transcription - pcr products using tbuap primers , the middle 
  panel is a control without reverse transcriptase , and the lower 
  panel is a control using dol - p - man synthetase ( dpms ) primers to 
  show equal rna input .
tbuap expression is essential for the growth of bloodstream 
  form t. brucei in culture .
b , growth curves for 
  tbuap conditional null mutant cells , subcultured every 2 days , grown 
  under permissive ( plus tetracycline ) conditions .
c , growth curves for 
  tbuap conditional null mutant cells grown under nonpermissive ( minus 
  tetracycline ) conditions .
d , ethidium bromide - stained agarose gel of 
  reverse transcription - pcr products from rna extracted from tbuap 
  conditional null mutant cells after 0 , 2 , 6 , 12 , and 48 h and 10 days without 
  tetracycline , as indicated .
the upper panel shows reverse 
  transcription - pcr products using tbuap primers , the middle 
  panel is a control without reverse transcriptase , and the lower 
  panel is a control using dol - p - man synthetase ( dpms ) primers to 
  show equal rna input .
the tbuap conditional null mutant cells were subcultured and grown 
  for 24 h with and without tetracycline and then introduced into groups of five 
  mice that were dosed with or without doxycycline in the drinking water .
the 
  animals dosed with the drug showed high blood parasitemias ( > 3  
  10/ml ) within 4 days and were sacrificed , whereas the 
  doxycycline - free animals did not show any signs of infection for up to 14 
  days , when the experiment was terminated .
these data show that the 
  tbuap gene is essential to bloodstream form t. brucei both 
  in vitro and in vivo .
subcellular localization of tbuap  to obtain antibodies , 
  tbuap - his6 was used to inoculate mice and the resulting anti - serum 
  was affinity - purified on a column of recombinant prescission protease - treated 
  ( tag - free ) tbuap coupled to cnbr - sepharose beads .
the mouse anti - tbuap 
  affinity - purified antibody was used together with rabbit anti - gapdh antibody 
  as a glycosomal marker .
the secondary antibodies were anti - mouse alexa 594 
  ( red ) and anti - rabbit alexa 488 ( green ) .
the fluorescence 
  micrographs of wild type cells show that the anti - gapdh co - localized with the 
  anti - tbuap , indicating that tbuap is located in glycosome microbodies in 
  bloodstream form t. brucei ( fig . 
  4 , a - c ) .
a similar experiment was performed with the 
  tbuap conditional null mutant grown with and without tetracycline for 
  48 h. the results show that the tbuap signal co - localizes with the glyosomal 
  gapdh marker ( as before ) ( fig . 4 , 
  d - f ) but that the intensity of the tbuap signal is 
  greatly reduced after 48 h without tretracycline 
  ( fig .
4 , g - i ) , 
  demonstrating the strict specificity of the antibody for tbuap in 
  immunofluorescence microscopy .
sugar nucleotide levels in the tbuap conditional null 
  mutant  to analyze the effect of the selective removal of 
  tbuap gene expression on parasite udp - glcnac levels , sugar 
  nucleotides were extracted from the tbuap conditional null mutant 
  under permissive and nonpermissive conditions , chromatographed as described in 
  ( 35 ) , and quantitated by the 
  method described in ref .
briefly , sugar nucleotides were extracted from t. brucei , separated 
  by reverse phase hplc and quantitated by multiple reaction monitoring tandem 
  mass spectrometry using an internal standard ( gdp - glucose ) , a sugar nucleotide 
  that is not found in trypanosomes .
the multiple reaction monitoring approach 
  exploits characteristic transitions between precursor and product ions to 
  identify specific metabolites in complex mixtures .
for example udp - glcnac 
  gives rise to an [ m - h ] precursor ion at m / z 606 
  that fragments to produce a major product ion of 
  [ udp - h2o ] at m / z 385 .
thus , a 
  chromatogram of the mass transition 606  385 is highly selective for 
  udp - glcnac .
similarly , gdp - man and gdp - glc can be monitored using the 
  [ gdp - hex ] to [ gdp - h2 o ] transition of 
  m / z 604  424 .
5 ) illustrate the 
  dramatic reduction in udp - glcnac levels , relative to the gdp - glc internal 
  standard , in the tbuap conditional null mutant after 48 h in the 
  absence of tetracycline .
these data and the effects on other sugar nucleotides 
  are summarized in ( table 2 ) . 
 
the levels of udp - glc , udp - gal , and gdp - man in the tbuap conditional 
  null cell line under permissive conditions agree reasonably well with the wild 
  type levels determined previously 
  ( 19 ) .
however , even under 
  permissive conditions , the level of udp - glcnac is significantly lower in the 
  conditional null mutant ( 16 pmol/1  10 cells ) than in the 
  wild type ( 80 pmol/1  10 cells ) .
the reduced level may be 
  because tbuap expression is no longer under the control of its 
  endogenous promoter but rather under the control of the procyclin promoter in 
  the plew100-tbuap ectopic copy .
thus , the lower growth rate of the 
  mutant under permissive conditions may be a result of the reduced level of 
  udp - glcnac . under nonpermissive conditions
, the cells stopped dividing between 
  48 and 60 h and died , by cell lysis , by around 72 h. at 48 h , the level of 
  udp - glcnac was 2.9 pmol/1  10 , < 5% of wild type 
  levels .
table 2sugar nucleotide levels in the tbuap conditional null mutant 
  under permissive and nonpermissive conditionssugar nucleotidewild 
  typeatbuap cko ( 0 
  h)btbuap cko ( 48 
  h)bpmol/10 cellspmol/10 cellspmol/10 cells udp - glcnac 
   80  20 
   16 
   2.9 
   gdp - man 
   5.6  3.9 
   9.5 
   19 
   udp - gal 
   55  3 
   44 
   35 
 
  udp - glc 
 
  123  7 
 
  141 
 
  70 
  asugar nucleotide levels for wild type t. brucei taken from ref . 
 
sugar nucleotide levels in the tbuap conditional null mutant 
  under permissive and nonpermissive conditions sugar nucleotide levels for wild type t. brucei taken from ref . 
  19 .
wild type ( a - c ) and 
  tbuap conditional null mutant cells grown with tetracycline 
  ( d - f ) and without tetracycline for 48 h ( g - i ) were stained 
  with rabbit anti - gapdh ( a glycosomal marker ) and alexa 488 anti - rabbit 
  ( green channel ; a , d , and g ) and affinity - purified 
  mouse anti - tbuap and alexa 594 anti - mouse ( red channel ; b , 
  e , and h ) . merged images are shown in c , f , 
  and i. the merged image in i also includes a 
  4,6-diamidino-2-phenylindole stain ( blue ) for dna .
no 
  significant signals were obtained when primary antibodies were omitted ( data 
  not shown ) .
wild type ( a - c ) and 
  tbuap conditional null mutant cells grown with tetracycline 
  ( d - f ) and without tetracycline for 48 h ( g - i ) were stained 
  with rabbit anti - gapdh ( a glycosomal marker ) and alexa 488 anti - rabbit 
  ( green channel ; a , d , and g ) and affinity - purified 
  mouse anti - tbuap and alexa 594 anti - mouse ( red channel ; b , 
  e , and h ) .
merged images are shown in c , f , 
  and i. the merged image in i also includes a 
  4,6-diamidino-2-phenylindole stain ( blue ) for dna .
no 
  significant signals were obtained when primary antibodies were omitted ( data 
  not shown ) .
changes in the levels of other sugar nucleotides in the tbuap 
  conditional null mutant under nonpermissive conditions were less profound 
  ( table 2 ) .
however , when the 
  level of udp - glcnac falls , there is an accumulation of gdp - man .
since the 
  consumption of gdp - man for protein n - glycosylation and gpi anchor 
  synthesis depends on the availability of udp - glcnac , an accumulation of 
  gdp - mannose is not unexpected .
the reason for the reduction in udp - glc and 
  udp - gal levels , to about 60% of wild type , is less clear .
however , a 
  comparable phenomenon was seen in the tbgale ( udp - glc 
  4-epimerase ) conditional null mutant , where rapid loss of udp - gal under 
  nonpermissive conditions was followed by a reduction in udp - glc and udp - glcnac 
  levels ( 16 ) .
effects of udp - glcnac starvation on protein 
  glycosylation  ricin , which binds nonreducing terminal galactose 
  residues , has been shown to bind to the flagellar pocket of t. brucei 
  ( 43 ) and to the 
  endosomal / lysosomal system of the parasite 
  ( 27 ) .
the 
  poly - n - acetyllactosamine - specific ( gal1 - 4glcnac ) , lectin from 
  lycopersicon esculentum ( tomato ) has also been shown to bind 
  exclusively to glycoproteins in the flagellar pocket and endosomal / lysosomal 
  system in t. brucei 
  ( 26 ) .
the ricin and tomato 
  lectin binding oligosaccharide structures were characterized and were shown to 
  include a family of unusually large n - linked 
  poly - n - acetyllactosamine - containing glycans with an average of 54 
  n - acetyllactosamine repeats / glycan 
  ( 27 ) .
the effect of udp - glcnac starvation on these structures was assessed using 
  western blots of whole cell lysates from wild type and the tbuap 
  conditional null mutant cell lines probed with tomato lectin .
a large smear 
  was detected in the wild type cell lysate , which decreased progressively from 
  0 to 48 h in the absence of tetracycline in the tbuap conditional 
  null mutant ( fig .
the decrease in intensity , and a downward shift in apparent molecular weight , 
  of the tomato lectin binding high molecular weight glycoproteins indicated a 
  reduction in total poly - n - acetyllactosamine synthesis as the cellular 
  levels of udp - glcnac fall .
the specificity of the tomato lectin blot for 
  carbohydrate was confirmed by including chitin hydrolysate , a tomato lectin 
  inhibitor ( fig .
6b ) . 
  to distinguish whether this reduction in tomato lectin binding was 
  specifically caused by udp - glcnac starvation and not just a general phenomenon 
  in dying cells ,
the same experiment was performed with a tbgpi12 
  conditional null mutant ( 6 ) . 
 
this is another ( lethal ) glycosylation conditional null mutant but , this time , 
  in the gpi biosynthetic pathway . under nonpermissive ( without tetracycline ) 
  conditions
, this cell line also ceases cell division and dies , but , in this 
  case , the cell lysate blots showed relatively little change in tomato lectin 
  binding ( supplemental fig .
9 ) . to observe the effects of udp - glcnac starvation on a specific glycoprotein , 
 
wild type and tbuap conditional null mutant cells , grown in the 
  presence and absence of tetracycline for 24 and 48 h , were harvested , lysed , 
  and analyzed by sds - page and western blotting with the p67-specific monoclonal 
  antibody mab139 ( supplemental fig .
the intensity of the staining was 
  slightly decreased , compared with wild type , in the tbuap conditional 
  null mutant under permissive conditions ( 0 h ) , which may reflect the overall 
  difference in udp - glcnac levels between the mutant and wild type cell line 
  described earlier .
after 48 h in the absence of tetracycline , both the 
  intensity and the apparent molecular weight of the p67 smear had decreased . 
 
figure 5.measurement of sugar nucleotides in the tbuap conditional null 
  mutant under permissive and nonpermissive conditions .
representative 
  liquid chromatography - tandem mass spectrometry chromatograms of sugar 
  nucleotides extracted from the tbuap conditional null mutant before 
  ( a ) and after ( b ) withdrawal of tetracycline for 48 h. the 
  upper chromatogram in each panel shows the peaks 
  corresponding to gdp - man and the gdp - glc internal standard , and the lower 
  chromatogram shows the peak corresponding to udp - glcnac .
measurement of sugar nucleotides in the tbuap conditional null 
  mutant under permissive and nonpermissive conditions .
representative 
  liquid chromatography - tandem mass spectrometry chromatograms of sugar 
  nucleotides extracted from the tbuap conditional null mutant before 
  ( a ) and after ( b ) withdrawal of tetracycline for 48 h. the 
  upper chromatogram in each panel shows the peaks 
  corresponding to gdp - man and the gdp - glc internal standard , and the lower 
  chromatogram shows the peak corresponding to udp - glcnac . to further demonstrate that the poly - n - acetyllactosamine 
  structures were being affected by udp - glcnac starvation , wild type , and the 
  tbuap conditional null mutant cells , after 0 , 24 , and 48 h without 
  tetracycline , were analyzed using fluorescence microscopy 
  ( fig .
cells 
  were fixed onto coverslips and incubated with biotinylated tomato lectin and 
  fluorescein - conjugated streptavidin . using this approach , 
  poly - n - acetyllactosamine structures specific to the flagellar pocket 
  and endosomal / lysosomal region
the 
  specificity of this reaction was confirmed by including chitin hydrolysate as 
  a tomato lectin inhibitor ( fig . 
 
little difference in tomato lectin staining was seen 
  in the tbuap conditional null mutant cells after 24 h in the absence 
  of tetracycline ( fig .
7 , b and 
  c ) , but after 48 h , almost no signal was seen within the 
  flagellar pocket or endosomal / lysosomal region 
  ( fig .
these 
  results correlate well with the tomato lectin blot data in 
  ( fig .
results using wild type cells and 
  including 4,6-diamidino-2-phenylindole staining to highlight the 
  positions of the nuclear and kinetoplast dna , are included 
  ( fig .
7 , f - j ) . to assess the effects of udp - glcnac starvation on gross cellular morphology 
  and flagellar pocket morphology , including the appearance of the flagellar 
  pocket lumenal contents , wild type cells and tbuap conditional null 
  mutant cells , grown in the absence of tetracycline for 48 h , were subjected to 
  scanning and transmission electron microscopy ( supplemental figs . 11 and 12 , 
  respectively ) .
however , no significant differences in the images were 
  recorded . to observe the effects udp - glcnac starvation had on vsg glycosylation , svsg 
  was purified from wild type and tbuap conditional null mutant cells , 
  grown in the presence and absence of tetracycline for 48 h. analysis by 
  sds - page and coomassie blue staining revealed that svsg from wild type and 
  tbuap conditional null cells grown under permissive conditions appear 
  as single bands , whereas svsg from the tbuap conditional null grown 
  for 48 h in the absence of tetracycline appeared as a doublet 
  ( fig .
the formal 
  possibility that the altered svsg profile may have been caused by the cells 
  switching vsg expression to a new vsg variant was eliminated by tryptic mass 
  fingerprinting , by which both bands were positively identified as vsg221 ( data 
  not shown ) .
a , extracts of wild type and tbuap 
  conditional null mutant ( tbuap cko ) cells 0 , 24 , and 48 h after 
  tetracycline removal ( as indicated ) were subjected to sds - page , transferred to 
  nitrocellulose , and probed with biotinylated tomato lectin followed by 
  horseradish peroxidase - streptavidin .
b , as in a , except that 
  the tomato lectin inhibitor , chitin hydrolysate , was included with the 
  lectin . tomato lectin blotting of wild type the tbuap conditional null 
  mutant extracts .
a , extracts of wild type and tbuap 
  conditional null mutant ( tbuap cko ) cells 0 , 24 , and 48 h after 
  tetracycline removal ( as indicated ) were subjected to sds - page , transferred to 
  nitrocellulose , and probed with biotinylated tomato lectin followed by 
  horseradish peroxidase - streptavidin .
b , as in a , except that 
  the tomato lectin inhibitor , chitin hydrolysate , was included with the 
  lectin . aliquots of svsg from wild type cells and the tbuap conditional 
  null mutant grown for 48 h in the absence of tetracycline were digested with 
  the enzymes endo h and pngase f and analyzed by sds - page and coomassie blue 
  staining . as previously described 
  ( 44 ) , wild type svsg showed a 
  small shift in molecular weight when digested with endo h , due to the removal 
  of the c - terminal oligomannose n - linked glycan , and a greater shift 
  when digested with pngase f , due to the removal of both n - linked 
  glycans ( fig .
when the svsg from the tbuap conditional null mutant was digested 
  with endo h , the upper band collapsed into the lower band , and when digested 
  with pngase f , both bands collapsed into a fully deglycosylated form 
  ( fig .
these data 
  suggest that the vsg doublet present in the mutant represents vsg molecules 
  that are similar to those found in wild type ( i.e. the upper band ) 
  and vsg molecules that have selectively lost an endo h - sensitive 
  n - linked glycan ( i.e. the lower band ) . to analyze this further ,
wild type and tbuap conditional null mutant cells grown under 
  permissive conditions gave the expected glycoform mass ranges for svsg221 
  ( 16 , 
  44 ) 
  ( table 3 ) .
however , svsg from 
  the tbuap conditional null mutant grown under nonpermissive 
  conditions for 48 h displayed two discrete sets of svsg glycoforms , 
  corresponding to the two bands seen by sds - page .
one set was similar to that 
  of wild type svsg , except that it lacks the higher molecular weight glycoforms 
  that contain five glcnac residues ( table 
  3 ) .
the other set of glycoforms have masses consistent with the 
  absence of oligomannose structures at the c - terminal ( asn-428 ) n - glycosylation 
  site ( consistent with the endo h resistance of the lower vsg band on 
  sds - page ) .
analysis of the glycopeptide fraction of a pronase digest of the 
  mutant vsg sample , prepared and analyzed by electrospray tandem mass 
  spectrometry according to ref . 
 
54 , revealed a range of 
  asn-263 and asn-428 n - linked glycopeptide and ser-433 gpi 
  glycopeptide species4 
  similar to those of wild type vsg ( supplemental fig .
these results rule 
  out the possibility that lower molecular weight vsg glycoforms arise from 
  changes at the n - linked and gpi glycosylation sites that happen to be 
  equivalent in mass to the loss of the oligomannose structures from 
  asn-428 .
9 ) for svsg221 samples from wild type ( wt ) and tbuap 
  conditional null cells grown with ( cko + tet ) or without tetracycline ( cko - 
  tet ) for 48 h are tabulated in that order , followed by ( in parentheses ) the 
  theoretical mass of the assigned vsg composition ( theo . ) .
the abundance of each isobaric group of vsg molecules is indicated as follows : 
  + + + , mass peaks > 70% of the biggest species ; + + , mass peaks > 40% of the 
  biggest species ; + , mass peaks < 40% of the biggest species.measured ( and theoretical ) molecular mass of wt/+tet/tet ( theo.)proteinaglcn - ino - cpbetnpbhexnachexosewtcko + tetcko  tetda 51,542/51,546/na ( 51,531 ) 
   1 
   1 
   1 
   5 
   23 
   + 
   + 
    
   51,500/51,505/na ( 51,490 ) 
   1 
   1 
   1 
   4 
   24 
   + 
   + 
    
   51,379/51,384/na ( 51,369 ) 
   1 
   1 
   1 
   5 
   22 
   + 
   + 
    
   51,338/51,343/na ( 51,328 ) 
   1 
   1 
   1 
   4 
   23 
   + 
   + 
    
   51,215/51,219/na ( 51,207 ) 
   1 
   1 
   1 
   5 
   21 
   + 
   + 
    
   51,174/51,179/na ( 51,166 ) 
   1 
   1 
   1 
   4 
   22 
   + 
   + 
    
   51,053/51,058/na ( 51,045 ) 
   1 
   1 
   1 
   5 
   20 
   + 
   + 
    
   51,012/51,017/51,016 ( 51,004 ) 
   1 
   1 
   1 
   4 
   21 
   + 
   + 
   + 
   50,891/50,897/50,895 ( 50,883 ) 
   1 
   1 
   1 
   5 
   19 
   + 
   + 
    
   50,850/50,856/50,854 ( 50,842 ) 
   1 
   1 
   1 
   4 
   20 
   + + + 
   + + + 
   + + + 
   50,688/50,694/50,691 ( 50,680 ) 
   1 
   1 
   1 
   4 
   19 
   + + 
   + + 
   + + 
   50,526/50,531/50,530 ( 50,518 ) 
   1 
   1 
   1 
   4 
   18 
   + 
   + 
   + 
   50,364/50,369/50,368 ( 50,356 ) 
   1 
   1 
   1 
   4 
   17 
   + 
   + 
   + 
   na / na/49,311 ( 49,302 ) 
   1 
   1 
   1 
   2 
   13 
    
    
   + 
   na / na/49,149 ( 49,140 ) 
   1 
   1 
   1 
   2 
   12 
    
    
   + + + 
   na / na/48,987 ( 48,978 ) 
   1 
   1 
   1 
   2 
   11 
    
    
   + + + 
   na / na/48,826 ( 48,816 ) 
   1 
   1 
   1 
   2 
   10 
    
    
   + 
   na / na/48,663 ( 48,654 ) 
   1 
   1 
   1 
   2 
   9 
    
    
   + 
 
  na / na/48,501 ( 48,492 ) 
 
  1 
 
  1 
 
  1 
 
  2 
 
  8 
 
   
 
   
 
  + 
  athe average molecular weight of svsg221 polypeptide ( 46,284 da ) minus amino 
  acids 1 - 27 ( signal peptide ) and residues 460 - 476 ( gpi attachment signal 
  sequence ) with four disulphide bonds 
  ( 44).bcomponents of gpi common to all glycoforms of svsg221 : glcn - ino - cp , 
  glucosamine-1 - 6-myo - inositol-1,2-cyclic phosphate ; etnp , 
  ethanolamine phosphate
9 ) for svsg221 samples from wild type ( wt ) and tbuap 
  conditional null cells grown with ( cko + tet ) or without tetracycline ( cko - 
  tet ) for 48 h are tabulated in that order , followed by ( in parentheses ) the 
  theoretical mass of the assigned vsg composition ( theo . ) .
the abundance of each isobaric group of vsg molecules is indicated as follows : 
  + + + , mass peaks > 70% of the biggest species ; + + , mass peaks > 40% of the 
  biggest species ; + , mass peaks < 40% of the biggest species . the average molecular weight of svsg221 polypeptide ( 46,284 da ) minus amino 
  acids 1 - 27 ( signal peptide ) and residues 460 - 476 ( gpi attachment signal 
  sequence ) with four disulphide bonds 
  ( 44 ) . components of gpi common to all glycoforms of svsg221 : glcn - ino - cp , 
  glucosamine-1 - 6-myo - inositol-1,2-cyclic phosphate ; etnp , 
  ethanolamine phosphate . taken together ,
these data show that under udp - glcnac starvation , the 
  elaboration of the man3glcnac2 and 
  man4glcnac2 glycans at the fully occupied asn-263 
  n - glycosylation site glycans with glcnac and gal1 - 4glcnac is 
  significantly reduced and that the occupancy of the c - terminal asn-428 site is 
  selectively and dramatically reduced .
whereas some enzymes of carbohydrate metabolism in t. brucei are 
  closer to their prokaryote counterparts 
  ( 45 , 
  46 ) , this is not the case for 
  tbuap , which has 31% sequence identity and 50% sequence similarity to its 
  human counterpart and , unlike its bacterial counterpart , glmu , is not fused to 
  a glucosamine-1-phosphate n - acetyltransferase domain 
  ( 47 ) .
analysis of the recombinant uap from t. brucei demonstrated that 
  tbuap is a conventional divalent cation - dependent pyrophosphorylase with 
  kinetic parameters similar to those reported for other eukaryotic uaps . 
 
however , tbuap differs from most other uaps in that it is highly selective for 
  its sugar phosphate substrate , accepting only glcnac 1-phosphate , and that it 
  is compartmentalized in a microbody , the glycosome . with respect to the former ,
the inability of tbuap to accept galnac-1-p 
  reflects the fact that t. brucei , like all of the trypanosomatids , 
  does not incorporate galnac into its glycoconjugates ( reviewed in ref . 
 
thus , the organism has no 
  need to make udp - galnac either from galn or galnac or by epimerization of 
  udp - glcnac .
accordingly , both tbuap ( this study ) and tbgale 
  ( 15 ) are exclusively 
  glcnac-1-phosphate- and udp - glc / gal - specific , respectively .
this is unlike 
  their mammalian counterparts that can also utilize galnac-1-p 
  ( 36 ) and udp - glcnac / galnac 
  ( 48 ) , respectively .
comparison 
  of the tbuap sequence with those of the human agx1 and agx2 uap splice 
  variants , for which there are crystal structures 
  ( 36 ) , indicates that tbuap has 
  a conserved active site , containing only a 2-amino acid difference among the 
  15 identified substrate - interacting residues ( i.e. arg-313 in place 
  of pro-288 and ala-489 in place of lys-455 ) .
( 36 ) reported that the 
  equatorial glcnac c4 hydroxyl of udp - glcnac forms one hydrogen bond with 
  gly-290 and one with asn-327 of agx1 and that the axial galnac c4 hydroxyl of 
  udp - galnac forms two hydrogen bonds with asn-327 .
however , the context of the key asn residue 
  ( underlined ) in tbuap is kfncanissnlc 
  whereas in the agx enzymes it is 
  lfnagnianhff .
nonequivalent adjacent 
  residues ( in italic type ) , particularly the bulkier ca versus ag 
  sequence immediately before the asn residue , may affect the latter hydrogen 
  bond network and account for the observed selectivity of tbuap for glcnac-1-p 
  over galnac-1-p .
figure 7.fluorescence microscopy of tomato lectin binding to wild type and 
  tbuap conditional null mutant cells .
wild type ( wt ) and 
  conditional null mutant ( cko ) cells after 0 , 24 , and 48 h without 
  tetracycline were visualized by phase - contrast and tomato lectin fluorescence 
  microscopy , as indicated ( a - e ) . also shown
are wild type cells 
  stained with 4,6-diamidino-2-phenylindole ( dapi ) and tomato 
  lectin ( tl ) ( f - j ) .
fluorescence microscopy of tomato lectin binding to wild type and 
  tbuap conditional null mutant cells
. wild type ( wt ) and 
  conditional null mutant ( cko ) cells after 0 , 24 , and 48 h without 
  tetracycline were visualized by phase - contrast and tomato lectin fluorescence 
  microscopy , as indicated ( a - e ) . also shown
are wild type cells 
  stained with 4,6-diamidino-2-phenylindole ( dapi ) and tomato 
  lectin ( tl ) ( f - j ) . with respect to the glycosomal , rather than cytosolic , location of the 
  native enzyme , a recent computational search of the t. brucei , t. 
  cruzi , and leishmania major genomes did not identify their uaps 
  as glycosomal enzymes ( 45 ) . 
 
however , that study used the pts1 prosite pattern ps00342 of 
  ( sta - gcn)-(rkh)-(livmafy)$ ( where $ denotes the end of the protein ) 
  supplemented with the pattern s - s-(lif)$. thus , the tbuap c - terminal 
  tripeptide sequence , snm$ , did not pass this filter . on the other hand
, the 
  same authors noted that the most abundant residues , in order of frequency , in 
  t. brucei pts1 sequences are ( sagpyn)-(khrnq)-(lm - avy)$ , indicating 
  that although the snm$ permutation has not been identified previously as a 
  t. brucei pts1 sequence , it appears to be one .
significantly , the 
  c - terminal tripeptide sequences of the t. cruzi and l. major 
  uaps are gnm$ and anm$ , respectively , which also fit the aforementioned 
  potential pts1 permutations .
it remains to be determined experimentally 
  whether a c - terminal ( sga)-nm$ sequence is sufficient for glycosomal import . 
 
the alternative is that tbuap is targeted to the glycosome through association 
  with another pts1 or pts2 targeted protein , as has been described previously 
  for peroxisomal import 
  ( 49 ) . in all trypanosomatids , the two - step conversion of glucose to fructose 
  6-phosphate , via hexokinase and glucose-6-phosphate isomerase , occurs in the 
  glycosome , and it is conceivable that the entire udp - glcnac biosynthetic 
  pathway is located in this organelle . although 
  glucosamine - fructose-6-phosphate transaminase , glucosamine - phosphate 
  n - acetyltransferase , and phosphoacetylglucosamine mutase lack obvious 
  pts1 or pts2 sequences , the t. cruzi and l. major 
  glucosamine-6-phosphate deaminase sequences do contain pts1 sequences 
  ( 46 ) .
our 
  current hypothesis , which we are currently testing , is that 
  glucosamine - fructose-6-phosphate transaminase , glucosamine - phosphate 
  n - acetyltransferase , and phosphoacetylglucosamine mutase are 
  piggybacked into the glycosome via oligomerization with glucose-6-phosphate 
  isomerase and/or glucosamine-6-phosphate deaminase and/or tbuap to provide 
  functional udp - glcnac synthesis machinery in a single location .
indeed , it may 
  be that all sugar nucleotide biosynthesis occurs in this location in 
  trypanosomes , since both udp - glc 4-epimerase and gdp - man 
  4,6-dehydratase , required for in udp - gal and gdp - fuc synthesis , respectively , 
  have also been shown to be glycosomal in t. brucei 
  ( 15 , 
  18 ) .
a glycosomal location for 
  the synthesis of sugar nucleotides further suggests that there may be specific 
  transporters or antiporters in the glycosome membrane to , for example , 
  exchange utp for udp - sugars and gtp for gdp - sugars or ndp - sugars for 
  ppi . in this regard ,
it is worth noting that , whereas most sugar 
  nucleotide antiporters exchange ndp - sugars for nmps 
  ( 50 ) ( requiring nucleoside 
  diphosphatases to convert ndps to nmps ) , ndp - sugar / ndp and ndp - sugar / ndp - sugar 
  antiporter activities have also been recently reported 
  ( 51 ) .
figure 8.endoglycosidase digestions of svsg221 from wild type and tbuap 
  conditional null mutant cells . a , svsg221 purified from wild type 
  and tbuap conditional null mutant ( tbuap cko ) cells before 
  ( 0 h ) and after 48 h without tetracycline ( 48 h )
b , aliquots of wild type svsg221 were 
  digested with endo h ( that removes only oligomannose n - linked 
  glycans ) or pngase f ( that removes all n - linked glycans ) , and the 
  products were subjected to sds - page and coomassie blue staining .
species 
  containing two , one , and no n - linked glycans were resolved , as 
  indicated .
c , aliquots of svsg221 from tbuap conditional 
  null cells grown for 48 h without tetracycline were digested with endo h or 
  pngase f , and the products were subjected to sds - page and coomassie blue 
  staining .
species containing two , one , and no n - linked glycans were 
  resolved , as indicated .
a , svsg221 purified from wild type 
  and tbuap conditional null mutant ( tbuap cko ) cells before 
  ( 0 h ) and after 48 h without tetracycline ( 48 h ) were subjected to sds - page 
  and coomassie blue staining .
b , aliquots of wild type svsg221 were 
  digested with endo h ( that removes only oligomannose n - linked 
  glycans ) or pngase f ( that removes all n - linked glycans ) , and the 
  products were subjected to sds - page and coomassie blue staining .
species 
  containing two , one , and no n - linked glycans were resolved , as 
  indicated .
c , aliquots of svsg221 from tbuap conditional 
  null cells grown for 48 h without tetracycline were digested with endo h or 
  pngase f , and the products were subjected to sds - page and coomassie blue 
  staining .
species containing two , one , and no n - linked glycans were 
  resolved , as indicated .
figure 9.electrospray mass spectrometry of svsg221 from wild type and 
  tbuap conditional null mutant cells .
aliquots of svsg221 from 
  wild type cells ( a ) and tbuap conditional null mutant cells 
  grown in the presence of tetracycline ( b ) and the absence of 
  tetracycline for 48 h ( c ) were analyzed by positive ion electrospray 
  mass spectrometry , and the data were processed by baysian protein 
  reconstruction to produce mass graphs of isobaric glycoforms .
models of some 
  of the principal glycoforms are indicated , and the compositions of the 
  detected glycoforms are shown in table 
  3 .
electrospray mass spectrometry of svsg221 from wild type and 
  tbuap conditional null mutant cells .
aliquots of svsg221 from 
  wild type cells ( a ) and tbuap conditional null mutant cells 
  grown in the presence of tetracycline ( b ) and the absence of 
  tetracycline for 48 h ( c ) were analyzed by positive ion electrospray 
  mass spectrometry , and the data were processed by baysian protein 
  reconstruction to produce mass graphs of isobaric glycoforms .
models of some 
  of the principal glycoforms are indicated , and the compositions of the 
  detected glycoforms are shown in table 
  3 .
the phenotype generated through udp - glcnac starvation with regard to tomato 
  lectin binding is similar to that caused by udp - gal starvation 
  ( 16 ) ( i.e. a 
  reduction in lectin binding in western blots and fluorescence microscopy ) , 
  indicating a reduction in both size and quantity of the giant poly - lacnac 
  structures normally found throughout the flagellar pocket and 
  endosomal / lysosomal system 
  ( 26 , 
  27 ) .
for example , tomato lectin blots remain 
  unchanged for a tbgpi12 conditional null mutant 
  ( 6 ) under nonpermissive 
  conditions ( supplemental fig .
transmission electron microscopy images of 
  gold - conjugated ricin binding in the flagellar pocket had suggested that the 
  bloodstream form - specific fibrous material in the flagellar pocket might 
  correspond to ricin - binding glycoconjugates 
  ( 43 ) .
however , although a 
  significant reduction in tomato lectin binding was observed in this study , 
  transmission electron microscopy images did not show a significant reduction 
  in the fibrous material when compared with wild type cells .
this suggests that 
  the fibrous material is either unrelated to the poly - lacnac - containing 
  glycoproteins of the flagellar pocket or that the fibrous appearance of the 
  glycoproteins in transmission electron microscopy is unaffected by reducing 
  their poly - lacnac content .
hopefully , future studies on the composition of the 
  lumenal contents of the flagellar pocket will resolve this issue .
another way of assessing the glycosylation phenotype of bloodstream form 
  t. brucei mutants is to use the abundant vsg as a reporter and to 
  assess the status of its two n - glycosylation sites and gpi anchor 
  ( 16 , 
  44 ) . in the case of udp - gal 
  starvation ,
the copy number and integrity of the vsg coat is not affected , 
  although the gpi anchors , which normally sport an average of five gal side 
  chain residues , are free of galactose 
  ( 16 ) . the effect on vsg 
  glycosylation was very different under udp - glcnac starvation .
mass 
  spectrometry and endoglycosidase digestion of vsg221 isolated from the 
  tbuap conditional null mutants under nonpermissive conditions 
  revealed the presence of two major species of vsg221 in approximately equal 
  amounts .
one form was almost indistinguishable from wild - type vsg221 , whereas 
  the other , lower molecular mass form , specifically lacked the c - terminal 
  ( asn-428 ) n - linked glycan . from a hierarchical point of view , it is 
  not surprising that gpi anchor synthesis is maintained , even when protein 
  n - glycosylation and n - glycan elaboration are affected , since 
  gpi synthesis and transfer to protein are clearly essential to bloodstream 
  form t. brucei 
  ( 5 - 8 ) . 
 
furthermore , from a structural point of view , the asn-263 n - linked 
  glycan may be more important to the correct folding of the vsg .
( 52 ) analyzed the 
  crystal structures of two vsg variants , vsg iltat.1.24 and vsg221 , and found 
  that a short -helix in iltat.1.2 is absent in vsg221 but replaced by 
  the protein - proximal sugars of the asn-263 n - linked glycan .
however , 
  this does not provide a mechanistic explanation for the hierarchy of asn-263 
  n - glycosylation in strict preference to the n - glycosylation 
  of asn-428 under udp - glcnac starvation .
radiolabeling studies suggest that 
  bloodstream form t. brucei contain very low steady - state levels of 
  man9glcnac2-pp - dolichol and a relatively large pool of 
  man5glcnac2-pp - dolichol 
  ( 53 ) . despite their 
  differences in abundance
, the organism appears to use both of these 
  oligosaccharyl - pp - dolichol species for transfer to protein , the former to 
  sites destined to contain oligomannose glycans and the latter to sites 
  destined to become complex type structures 
  ( 44 ) . in most eukaryotes ,
a 
  single type of dolichol - pp - linked oligosaccharide precursor 
  ( glc3man9glcnac2-pp - dolichol ) is transferred 
  to the nascent peptide in the er through the action of 
  oligosaccharyltransferase ( ost ) , a complex of typically eight different 
  proteins ( 54 ) .
trypanosomatids 
  lack candidate genes for all but the catalytic subunit , stt3 , of this complex . 
  however , there are three copies of the tbstt3 gene in the t. 
  brucei genome .
two of the tbstt3 genes encode almost identical 
  proteins , and we speculate that the unique tbstt3 utilizes either exclusively 
  man5glcnac2-pp - dolichol or 
  man9glcnac2-pp - dolichol and that one or both of the two 
  similar tbstt3s utilizes the other donor . under udp - glcnac starvation ,
gdp - man 
  levels rise slightly , and presumably dol - p - man is also present at greater than 
  normal levels . from this point of view
, one might expect the ratio of 
  man9glcnac2-pp - dolichol / man5glcnac2-pp - dolichol 
  to increase slightly .
however , the absolute levels of both precursors are 
  presumably significantly reduced as udp - glcnac becomes limiting for the 
  synthesis of the common glcnac2-pp - dolichol core .
thus , the 
  simplest explanation for the selective loss of the c - terminal oligomannose 
  n - linked glycans from vsg221 under udp - glcnac starvation is that the 
  effective concentration of man9glcnac2-pp - dolichol falls 
  so far below the effective km for the relevant tbstt3 that 
  most vsg molecules appearing in the er fail to receive a c - terminal 
  n - glycan . comparative studies on the substrate specificities and 
  kinetic parameters of the two classes of tbstt3 should resolve this issue . 
 
meanwhile , additional support for such a dual oligosaccharyltransferase model 
  comes from the analysis of a t. brucei alg3 mutant that can only make 
  man5glcnac2 and that also underglycosylates the 
  c - terminal asn-428 site of vsg221 
  ( 55 ) and from pulse - chase 
  studies that show , in at least two different vsgs , that endo h - sensitive 
  oligomannose sites ( but not endo h - resistant sites ) can be 
  n - glycosylated post - translationaly 
  ( 30 , 
  56 ) .
finally , through the creation of the conditional null mutant , 
  tbuap was shown to be essential for the growth and survival of 
  bloodstream t. brucei in culture , suggesting that this enzyme might 
  be considered as a potential therapeutic target for human african sleeping 
  sickness .
further in vivo support for this notion was obtained when 
  the tbuap conditional null was shown to be infectious to mice dosed 
  with doxycycline in their drinking water but not to doxycycline - free mice . 
 
although the essentiality of udp - glcnac biosynthesis via tbuap was expected , 
  experimental validation in vitro and in vivo is , 
  nevertheless , important , since it rules out any possible metabolic and/or 
  nutritional bypasses .
compared with other conditional null mutants generated 
  in this laboratory , the ablation of udp - glcnac synthesis led to more rapid 
  killing of the parasite ( < 72 h ) than ablation of either udp - gal synthesis 
  ( 14 , 
  16 ) or gpi biosynthesis 
  ( 6 ) , which both take 4 - 5 days . 
 
the rapidity of cell death presumably reflects the fact that udp - glcnac is 
  simultaneously required for gpi anchor biosynthesis , core protein 
  n - glycosylation , and the subsequent decoration of complex 
  n - glycans with lactosamine units .
of course , udp - glcnac is also 
  essential to mammalian cells 
  ( 57 ) , and selective inhibition 
  of the parasite uap would be a therapeutic requirement .
 a gene encoding trypanosoma brucei 
  udp - n - acetylglucosamine pyrophosphorylase was identified , and the 
  recombinant protein was shown to have enzymatic activity . the parasite enzyme 
  is unusual in having a strict substrate specificity for 
  n - acetylglucosamine 1-phosphate and in being located inside a 
  peroxisome - like microbody , the glycosome . 
 a bloodstream form t. 
  brucei conditional null mutant was constructed and shown to be unable to 
  sustain growth in vitro or in vivo under nonpermissive 
  conditions , demonstrating that there are no alternative metabolic or 
  nutritional routes to udp - n - acetylglucosamine and providing a genetic 
  validation for the enzyme as a potential drug target . 
 the conditional null 
  mutant was also used to investigate the effects of 
  n - acetylglucosamine starvation in the parasite . 
 after 48 h under 
  nonpermissive conditions , about 24 h before cell lysis , the status of parasite 
  glycoprotein glycosylation was assessed . under these conditions , 
  
 udp - n - acetylglucosamine levels were less than 5% of wild type . 
 lectin 
  blotting and fluorescence microscopy with tomato lectin revealed that 
  poly - n - acetyllactosamine structures were greatly reduced in the 
  parasite . 
 the principal parasite surface coat component , the variant surface 
  glycoprotein , was also analyzed . 
 endoglycosidase digestions and mass 
  spectrometry showed that , under udp - n - acetylglucosamine starvation , 
  the variant surface glycoprotein was specifically underglycosylated at its 
  c - terminal asn-428 n - glycosylation site . 
 the significance of this 
  finding , with respect to the hierarchy of site - specific 
  n - glycosylation in t. brucei , is discussed . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material