Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan.
The life cycle of Leishmania parasites within its sand fly vector involves the development of extracellular promastigotes from a noninfective, "procyclic" stage into an infective, "metacyclic" stage that is adapted for transmission in the fly and survival in the mammalian host. Lipophosphoglycan (LPG), the predominant surface glycoconjugate in both procyclic and metacyclic stages, is a critical virulence determinant. LPG is a multidomain molecule; the structural polymorphisms among species lie in branching from the backbone 6Galbeta1,4Man(alpha1)-PO(4) repeat units and in the composition of the small oligosaccharide caps. We have recently demonstrated that the LPG from an Indian isolate of Leishmania donovani differs from a Sudanese strain by possessing one or two side chain beta(1,3)-linked glucose residues. We now have characterized the glucosyltransferase activities responsible for glucosylating the LPG. When incubated with UDP-[(3)H]glucose and Mn(2+), microsomal membranes from the Indian isolate transferred [(3)H]glucose to the repeat units of the exogenous acceptor Sudanese L. donovani LPG, which does not contain any side chain branching. Glucose addition was maximal at 28 degrees C, the optimal growth temperature of procyclic L. donovani. Consistent with the lack of side chain branching in its LPG, Sudanese L. donovani showed minimal glucosyltransferase activity. Indian metacyclic promastigotes, in contrast to procyclic promastigotes, express no glucose side chains off the repeat units. Therefore, we compared the relative activity of the glucosyltransferases in microsomes from procyclic and metacyclic promastigotes and observed approximately 80% less activity in the latter. These results provide evidence that the glucose side chain addition to LPG is developmentally regulated during the parasite's life cycle and that the glucosyltransferases of L. donovani are strain specific.
Mahoney
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Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky, 40536, USA.
Turco
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SJ
D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002855:Chromatography, Thin Layer; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007194:India; D007700:Kinetics; D007893:Leishmania donovani / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D008018:Life Cycle Stages; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism; D013397:Sudan; D013696:Temperature; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D002413:Cations, Divalent; D006028:Glycosphingolipids; D011134:Polysaccharides; D015800:Protozoan Proteins; C008290:lipophosphonoglycan; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose