Mariño Karina, Güther M Lucia Sampaio, Wernimont Amy K, Qiu Wei, Hui Raymond, Ferguson Michael A J
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
Eukaryot Cell. 2011 Jul;10(7):985-97. doi: 10.1128/EC.05025-11. Epub 2011 Apr 29.
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.
一个预测编码布氏锥虫葡糖胺6-磷酸N-乙酰转移酶（TbGNA1；EC 2.3.1.4）的基因被克隆并在大肠杆菌中表达。重组蛋白具有酶活性，其高分辨率晶体结构在1.86 Å下获得。内源性TbGNA1蛋白定位于过氧化物酶体样微体即糖体。构建了布氏锥虫血流形式的GNA1条件性敲除突变体，结果表明其在非允许条件下无法在体外维持生长，这表明除了通过6-磷酸葡糖胺外，不存在其他合成UDP-GlcNAc的代谢或营养途径。对非允许条件下TbGNA1突变体的蛋白质糖基化表型分析表明，寄生虫中的多聚N-乙酰乳糖胺结构大大减少，并且主要寄生虫表面被膜成分即变异表面糖蛋白（VSG）的糖基化谱发生了改变。讨论了这些结果的意义以及TbGNA1作为非洲昏睡病新型药物靶点的潜力。
