Probing Elongating and Branching β-d-Galactosyltransferase Activities in Leishmania Parasites by Making Use of Synthetic Phosphoglycans

Abstract

Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [−6)Gal(β1-4)Man(α1-OPO 3 H−]. The repeat structures are assembled by sequential addition of Manα1-OPO 3 H and β-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-3 H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [ 3 H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with β-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating β-D-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating β-Dgalactosyltransferase and a branching β-D-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior. Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsThe trypanosomatid protozoan parasite Leishmania infects over 12 million people worldwide, causing a variety of diseases that range from mild cutaneous lesions to fatal visceral infections. General aspects of leishmaniasis and overall control strategies have been reviewed. 1,2 The parasite exists in two forms: the flagellated promastigote in the female sand fly vector and the amastigote in the mammalian host. Amastigotes are obligate intracellular parasites of macrophages, where they survive and multiply. A key step of the infectious cycle is the ability of the parasite to be transmitted to new hosts by the insect vector. In the recent years, visceral leishmaniasis is reported to be rapidly emerging as an opportunistic infection in HIV patients, 3,4 in pregnant females, and in organ transplant patients. 5 Globalization and consequent travel of people across the world has increased the chances of spreading the infection. 6 Pentavalent antimonials were brought into use against leishmaniasis more than 50 years ago. Along with Pentostam and Glucantime, non-antimonial polyene antibiotic Amphotericin B, as well as Paromomicin and Miltefosine have also proved to be successful for the treatment of L. donovani induced visceral leishmaniasis. However, these have the disadvantage of high toxicity. [1][2][3][4] Since the available treatment for leishmaniasis poses ma...

TcNST2encodes a Golgi-localized UDP-galactose transporter inTrypanosoma cruzi

“…Galactose residues inside the Golgi lumen are then added to GlcNAc by specific galactosyl transferases. These enzymes have not yet been studied in T. cruzi, but their activities have already been described in T. brucei (Pingel and Duszenko, 1992; Pingel et al ., 1999; Ramasamy and Field, 2012) and Leishmania (Sizova et al ., 2011).…”

Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates

“…2,[83][84][85][86][87][88] The authors have shown that substrate recognition strongly depends on several functional parameters: the negative charge of the phosphodiester bridge, the β-anomeric configuration of the Galp entity, and the H-bonding ability of the primary hydroxyls of both Manp and Galp residues. 89 Once again, synthetic substrates underlined the requirement for phosphodiester linkages. 89 Once again, synthetic substrates underlined the requirement for phosphodiester linkages.…”

Synthetic neoglycoconjugates of cell-surface phosphoglycans of Leishmania as potential anti-parasite carbohydrate vaccines

“…All the prepared phosphoglycans contain a dec-9-enyl aglycone moiety as they were purposely designed [55] for (a) studying Leishmania biosynthetic enzymes and (b) further preparation of neoglycoconjugates, which could be tested as potential anti-leishmaniasis vaccines. The hydrophobicity of the dec-9-enyl chain has been exploited for assaying, first, the mannosylphosphate transferases [57, 58] and then the galactosyltransferases [59, 60] involved in the LPG biosynthesis in Leishmania . As for the bioconjugation, the high lability of the anomeric phosphodiester bridges in phosphoglycans was given the top priority while selecting techniques for the neoglycoconjugate preparation.…”