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Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Galactose starvation in a bloodstream form Trypanosoma brucei UDP-glucose 4'-epimerase conditional null mutant
AU - Urbaniak, Michael D.
AU - Turnock, Daniel C.
AU - Ferguson, Michael A. J.
PY - 2006/11
Y1 - 2006/11
N2 - Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4'-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal beta-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4'-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.
AB - Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4'-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal beta-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4'-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.
KW - Animals
KW - Animals, Genetically Modified
KW - Cell Shape
KW - Galactose
KW - Glycoproteins
KW - Glycosylphosphatidylinositols
KW - Humans
KW - Lectins
KW - Phenotype
KW - Protein Binding
KW - Starvation
KW - Trypanosoma brucei brucei
KW - UDPglucose 4-Epimerase
U2 - 10.1128/EC.00156-06
DO - 10.1128/EC.00156-06
M3 - Journal article
C2 - 17093269
VL - 5
SP - 1906
EP - 1913
JO - Eukaryotic Cell
JF - Eukaryotic Cell
SN - 1535-9778
IS - 11
ER -