Molecular and Biochemical Parasitology 94 (1998) 27 – 40
Leishmania donoani has distinct
mannosylphosphoryltransferases for the initiation and
elongation phases of lipophosphoglycan repeating unit
biosynthesis
Albert Descoteaux
1,a
, Brenda Jo Mengeling
a
, Stephen M. Beverley
b
,
Salvatore J. Turco
a,
*
a
Department of Biochemistry, Uniersity of Kentucky Medical Center, Lexington, 40536 KY, USA
b
Department of Molecular Microbiology, Washington Uniersity School of Medicine, St. Louis, 63110 MO, USA
Received 12 January 1998; received in revised form 16 March 1998; accepted 18 March 1998
Abstract
Lipophosphoglycan (LPG) is the predominant surface glycoconjugate of Leishmania promastigotes and plays
several roles in the infectious cycle of this protozoan parasite. The salient feature of LPG is the presence of 15–30
copies of a disaccharide-phosphate repeating unit Gal( 1,4)Man( 1-PO
4
), which is also found on many other secreted
molecules (secretory acid phosphatase, phosphoglycan, proteophosphoglycan). This structural diversity suggests that
a multiplicity of enzymes mediating repeating unit addition may exist, especially for the mannosylphosphoryltrans-
ferases (MPTs), which initiate repeating unit synthesis. This work has taken a combined biochemical-genetic approach
to resolve this issue. An lpg
-
mutant of Leishmania donoani, JEDI, was obtained by antibody selection against cells
expressing a repeating unit epitope of LPG. Metabolic and surface labeling experiments revealed that JEDI cells
accumulated a truncated form of LPG bearing only a single repeating unit: [Gal( 1,4)Man( 1-PO
4
)]-
[Gal( 1,6)Gal( 1,3)Gal
f
( 1,3)[Glc( 1-PO
4
)]Man( 1,3)Man( 1,4)GlcN( 1,6)]-PI. Enzymatic assays of microsomal
preparations showed that JEDI lacked MPT activity when tested with a repeating unit acceptor but retained wild-type
Abbreiations: AnhMan, 2,5-anhydromannose; CHAPS, 3-(3-chloramidopropyldimethyl-ammonio)-1-propanesulfonic acid;
eMPT, elongation LPG mannosylphosphoryltransferase; GIPL, glycosylinositolphospholipid; HBSS, Hank’s balanced salt solution;
HPLC, high pressure liquid chromatography; iMPT, initiating LPG mannosylphosphoryltransferase; LPG, lipophosphoglycan;
MPT, mannosylphosphoryltransferase; PI-PLC, phosphatidylinositol-specific phospholipase C; TLCK, 1-chloro-3-tosylamido-
7amino-2-heptone; WT, wild-type.
* Corresponding author. Tel.: +1 606 3236693; fax: +1 606 3231037; e-mail: turco@pop.uky.edu
1
Present address: Centre de recherche ´ en immunologie, Institut Armand-Frappier, CP 100, 531 boul. des Prairies Laval, H7N 4Z3
Quebec, Canada.
0166-6851/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.
PII S0166-6851(98)00047-4