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

摘要

Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [−6)Gal(β1-4)Man(α1-OPO3H−]. The repeat structures are assembled by sequential addition of Manα1-OPO3H 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-[6-³H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [³H]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-d-galactosyltransferase 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.