Abstract
A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Crystallography
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Escherichia coli / genetics
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Gene Knockout Techniques
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Glucosamine 6-Phosphate N-Acetyltransferase / analysis
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Glucosamine 6-Phosphate N-Acetyltransferase / chemistry*
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Glucosamine 6-Phosphate N-Acetyltransferase / genetics
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Glucosamine 6-Phosphate N-Acetyltransferase / metabolism
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Mass Spectrometry
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Microbodies / metabolism
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Molecular Sequence Data
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Mutation
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Phylogeny
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Polysaccharides / analysis
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Protozoan Proteins / chemistry
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Protozoan Proteins / genetics
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Protozoan Proteins / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Sequence Alignment
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Trypanosoma brucei brucei / enzymology*
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Trypanosoma brucei brucei / genetics
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Trypanosomiasis, African
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Variant Surface Glycoproteins, Trypanosoma / chemistry
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Variant Surface Glycoproteins, Trypanosoma / genetics
Substances
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Polysaccharides
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Protozoan Proteins
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Recombinant Proteins
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Variant Surface Glycoproteins, Trypanosoma
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poly-N-acetyllactosamine
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Glucosamine 6-Phosphate N-Acetyltransferase
Associated data
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GENBANK/FR715998
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PDB/FR715998