DEFECTIVE GALACTOFURANOSE ADDITION IN LIPOPHOSPHOGLYCAN BIOSYNTHESIS IN A MUTANT OF LEISHMANIA-DONOVANI

A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphogly can, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 paras ites with either [H-3]galactose or [H-3]mannose, the main glycosylphos phatidylinositide product that accumulated was O4-Man-Man-GlcN-lyso-1- O-alkylphosphatidylinositol (PI). The metabolic defect was further def ined using a cell-free glycosylation system. When membrane preparation s from wild-type cells were incubated with UDP-[H-3]galactose and unla beled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-G lcN-PI or Gal(f)-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the abs ence of exogenous acceptor, the truncated glycosylphosphatidylinositid e Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized . However, when exogenous Gal(f)-Man-Man-GN-PI was added to the R2D2 i n vitro system, radioactive lipophosphoglycan was synthesized. Collect ively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect i n the synthesis of the ''activated'' galactofuranosyl donor or the lac k of a functional galactofuranosyltransferase.