LEISHMANIA-DONOVANI HAS DISTINCT MANNOSYLPHOSPHORYLTRANSFERASES FOR THE INITIATION AND ELONGATION PHASES OF LIPOPHOSPHOGLYCAN REPEATING UNIT BIOSYNTHESIS

Lipophosphoglycan (LPG) is the predominant surface glycoconjugate of L eishmania promastigotes and plays several roles in the infectious cycl e of this protozoan parasite. The salient feature of LPG is the presen ce of 15-30 copies of a disaccharide-phosphate repeating unit Gal(beta 1,4)Man(alpha 1-PO4), which is also found on many other secreted mole cules (secretory acid phosphatase, phosphoglycan, proteophosphoglycan) . This structural diversity suggests that a multiplicity of enzymes me diating repeating unit addition may exist, especially for the mannosyl phosphoryltransferases (MPTs), which initiate repeating unit synthesis . This work has taken a combined biochemical-genetic approach to resol ve this issue. An lpg(-) mutant of Leishmania donovani, JEDI, was obta ined by antibody selection against cells expressing a repeating unit e pitope of LPG. Metabolic and surface labeling experiments revealed tha t JEDI cells accumulated a truncated form of LPG bearing only single r epeating unit: [Gal(beta 1,4)Man(alpha 1-PO4)]-[Gal(alpha 1,6)Gal(alph a 1,3)Gal(f)(beta 1,3 )[Gic(alpha 1-PO4)]Man(alpha 1,3)Man(alpha 1,4)G lcN(alpha 1,6)]-PI. Enzymatic assays of microsomal preparations showed that JEDI lacked MPT activity when tested with a repeating unit accep tor but retained wild-type levels of the MPT activity with an LPG glyc an core acceptor. These data indicate that at least two distinct MPT a ctivities are required for LPG repeating unit synthesis: one involved in the 'initiation' of repeating unit synthesis on the LPG core (iMPT) , and a second (lacking in JEDI) participating in the 'elongation' pha se of repeating unit addition (eMPT), leading to the mature full-lengt h LPG. (C) 1998 Elsevier Science B.V. All rights reserved.