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.
PIGF is a protein involved in the ethanolamine phosphate (EtNP) transfer steps of glycosylphosphatidylinositol (GPI) biosynthesis. PIGF forms a heterodimer with either PIGG or PIGO, two enzymes that transfer an EtNP to the second or third mannoses of GPI respectively. Heterodimer formation is essential for stable and regulated expression of PIGO and PIGG, but the functional significance of PIGF remains obscure. In the present study, we show that PIGF binds to PIGO and PIGG through distinct molecular domains. Strikingly, C-terminal half of PIGF was sufficient for its binding to PIGO and PIGG and yet this truncation mutant could not complement the PIGF defective mutant cells, suggesting that heterodimer formation is not sufficient for PIGF function. Furthermore, we identified a highly conserved motif in PIGF and demonstrated that the motif is not involved in binding to PIGO or PIGG, but critical for its function. Finally, we identified a PIGF homologue from Trypanosoma brucei and showed that it binds specifically to the T. brucei PIGO homologue. These data together support the notion that PIGF plays a critical and evolutionary conserved role in the ethanolamine-phosphate transfer-step, which cannot be explained by its previously ascribed binding/stabilizing function. Potential roles of PIGF in GPI biosynthesis are discussed.
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