Biosynthesis of lipophosphoglycan from Leishmania major: solubilization and characterization of a (beta 1-3)-galactosyltransferase.
Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.
Ng
K
K
Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
Handman
E
E
Bacic
A
A
D000818:Animals; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002474:Cell-Free System; D003902:Detergents / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D018320:Leishmania major / Q000201:enzymology*; D008566:Membranes / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology; D012995:Solubility; D014530:Uridine Diphosphate / Q000494:pharmacology
D002413:Cations, Divalent; D003902:Detergents; D006028:Glycosphingolipids; D011135:Polysaccharides, Bacterial; D012492:Salts; C008290:lipophosphonoglycan; D014530:Uridine Diphosphate; D005700:Galactosyltransferases; D004047:Diethyl Pyrocarbonate