J Biol Chem, Vol. 273, Issue 22, 13509-13523, May 29, 1998

Stage-specific Proteophosphoglycan from Leishmania mexicana Amastigotes
STRUCTURAL CHARACTERIZATION OF NOVEL MONO-, DI-, AND TRIPHOSPHORYLATED PHOSPHODIESTER-LINKED OLIGOSACCHARIDES

Thomas Ilg^§, David Craik, Graeme Currie, Gerd Multhaup^**, and Antony Bacic

From the  Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3052, Australia, the ^§ Walter and Eliza Hall Institute of Medical Research, P. O. Royal Melbourne Hospital, Victoria 3050, Australia, the  Centre for Drug Design and Development, University of Queensland, Brisbane, Queensland 4072, Australia, and the ^** Centre for Molecular Biology, Heidelberg, Federal Republic of Germany

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as ¹H and ³¹P NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase. However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc1-3]_1-2Gal1-4Man, Gal1-3Gal1-4Man, Gal1-3Glc1-3Gal1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO₄-6-Gal]1-4Man) but carrying stage-specific modifications (R = Gal1-, [Glc1-3]_1-2Glc1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO₄-6-Glc1-3Gal1-4Man, PO₄-6-Glc1-3Glc1-3Gal1-4Man, PO₄-6-Gal1-3Glc1-3Gal1-4Man). In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO₄-6-Glc1-3Glc1-3[PO₄-6-Gal]1-4Man, PO₄-6-Gal1-3Glc1-3[PO₄-6-Gal]1-4Man, PO₄-6-Gal1-3Glc1-3Glc1-3[PO₄-6-Gal]1-4Man, PO₄-6-Glc1-3[PO₄-6-Glc]1-3[PO₄-6-Gal]1-4Man, PO₄-6-Gal1-3[PO₄-6-Glc]1-3Glc1-3[PO₄-6-Gal]1-4Man, and PO₄-6-Glc1-3[PO₄-6-Glc]1-3Glc1-3[PO₄-6-Gal]1-4Man. These glycans are linked together by the conserved phosphodiester R-Man1-PO₄-6-Gal-R or the novel phosphodiester R-Man1-PO₄-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man1-PO₄-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.