Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

Abstract

The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. The trypanosomatids are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Like most eukaryotes, the cell surfaces and endosomal/lysosomal systems of these organisms are rich in glycoconjugates, some of which play essential roles in their survival, infectivity, or virulence. The glycoconjugate repertoires of the three trypanosomatids studied here, Trypanosoma brucei, T. cruzi, and Leishmania major, are fundamentally different, reflecting their disparate life cycles, modes of infection, and disease pathologies (see references 5,27,28,38,47,65, and 69 and references therein). The monosaccharides that make up these glycoconjugates vary between the three trypanosomatid species. For example, all three contain D-mannose (Man), D-Nacetylglucosamine (GlcNAc), D-glucosamine (GlcN), D-glucose (Glc), and D-galactopyranose (Galp), while only T. cruzi and L. major contain D-galactofuranose (Galf), only T. cruzi contains D-xylose (Xyl), L-rhamnopyranose (Rha), and L-fucose (Fuc), and only L. major contains D-arabinopyranose (Ara) ( Table 1). However, a unifying theme of their glycobiology is an abundance of cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and/or non-proteinlinked GPI structures.The protein-linked GPI glycans of the leishmania are the simplest in structure, consisting of the conserved Man␣1-2Man␣1-6Man␣1-4GlcN core. Those of T. cruzi are principally Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN, and those of T. brucei are the most complex, with ␣-D-Galp and ␤-D-Galp side chains in the bloodstream form and sialylated poly-N-acetyllactosamine (poly-LacNAc) side chains in the procyclic form (27).The non-protein-linked GPI structures include the so-called glycoinositolphospholipids, or GIPLs, that are most abundant in the leishmania and T. cruzi species (58, 64, 90) but are also found in procyclic-form T. brucei (60,73,122) and bloodstream form T. brucei (39,63). Non-protein-linked GPIs also include the more exotic leishmania-specific lipophosphoglycan (LPG) structures (38,47,64,65). The leishmania LPGs contain characteristic phosphosaccharide repeats of Galp␤1-4Man␣1-P, which in L. major can have ␤-D-Galp and ␣-D-Arap side chains (66,67). These repeats are also found in the secr...

Leishmania UDP-sugar Pyrophosphorylase

“…In Leishmania, however, where D-Ara is present, the monosaccharide is exclusively activated by GDP, and a putative GDP-Ara pyrophosphorylase has been identified in the genome (8). In addition to GDP-␣-D-Ara, L. major promastigote synthesizes UDP-Glc, UDP-Gal (in the pyranosic and furanosic form), UDP-GlcNAc, GDP-Man, and low amounts of GDP-Fuc, but neither UDP-Xyl nor its precursor UDP-GlcA are produced (8).…”

“…Consequently, mutants deficient in the formation of UDP-Galf or in the transport of UDP-Gal into the secretory pathway organelles present an altered glycocalyx associated with parasite attenuation (4 -7). A route to UDP-Gal formation is via epimerization of the abundant nucleotide sugar UDP-glucose (UDP-Glc) by the UDP-Glc 4-epimerase (8). The biosynthesis of UDP-Gal is thus intimately linked to glucose metabolism (Fig.…”

Metabolically programmed quality control system for dolichol-linked oligosaccharides

“…The amounts of GDP-Man, UDP-Glc, and UDP-GlcNAc were simultaneously measured in samples by HPLC coupled with UV detection, as described previously (27). Three nucleotide sugars were also analyzed by ion-pair, reversed-phase liquid chromatography electrospray ionization-MS/MS, as described previously (39).…”

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

“…Lectin affinity chromatography showed significant improvements in discrimination between epimers, but has not been widely adopted [9]. Liquid chromatography-tandem mass spectrometry operated in multiple reaction monitoring mode was used to determine sugar nucleotides in three trypanosomatids during 45 min analyses [10]. Capillary electrophoresis showed excellent separation of UDP-GlcNAc and UDPGalNAc in a method that required at least 18 min [11].…”