Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmaniaspecies. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Galβ1-4Manα1-) backbone and capped with a neutral oligosaccharide. In Leishmania majorthe backbone is substituted at the C(O)3 of the Galpresidue with side chains containing Galp, Glcpand Arapresidues whereas in Leishmania donovanithe backbone is unsubstituted. We report the solubilization of a (β1-3)galactosyltranferase [(β1-3)GalT] from a L. majormicrosomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (β1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovaniLPG 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]Galβ1-3-4Manα1-) found in L. majorLPG. 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 (β1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (β1-3)GalT that adds the first βGal residue to the acceptor. The (β1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the Km values for UDP-Gal (445 μM) and L. donovaniacceptor (280 μM 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 (β1-3)GalT. The detergent-solubilized (β1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (β1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to β(1-3)GalT and is probably involved in the correct folding of the enzyme. The (β1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (β1-3)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.

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