Abstract
Nucleotide sugar transporters (NSTs) are indispensible for the biosynthesis of glycoproteins by providing the nucleotide sugars needed for glycosylation in the lumen of the Golgi apparatus. Mutations in NST genes cause human and cattle diseases and impaired cell walls of yeast and fungi. Information regarding their function in the protozoan parasite, Trypanosoma brucei, a causative agent of African trypanosomiasis, is unknown. Here, we characterized the substrate specificities of four NSTs, TbNST1-4, which are expressed in both the insect procyclic form (PCF) and mammalian bloodstream form (BSF) stages. TbNST1/2 transports UDP-Gal/UDP-GlcNAc, TbNST3 transports GDP-Man, and TbNST4 transports UDP-GlcNAc, UDP-GalNAc, and GDP-Man. TbNST4 is the first NST shown to transport both pyrimidine and purine nucleotide sugars and is demonstrated here to be localized at the Golgi apparatus. RNAi-mediated silencing of TbNST4 in the procyclic form caused underglycosylated surface glycoprotein EP-procyclin. Similarly, defective glycosylation of the variant surface glycoprotein (VSG221) as well as the lysosomal membrane protein p67 was observed in Δtbnst4 BSF T. brucei. Relative infectivity analysis showed that defects in glycosylation of the surface coat resulting from tbnst4 deletion were insufficient to impact the ability of this parasite to infect mice. Notably, the fact that inactivation of a single NST gene results in measurable defects in surface glycoproteins in different life cycle stages of the parasite highlights the essential role of NST(s) in glycosylation of T. brucei. Thus, results presented in this study provide a framework for conducting functional analyses of other NSTs identified in T. brucei.