STAGE-SPECIFIC PROTEOPHOSPHOGLYCAN FROM LEISHMANIA-MEXICANA AMASTIGOTES - STRUCTURAL CHARACTERIZATION OF NOVEL MONOPHOSPHORYLATED, DIPHOSPHORYLATED, AND TRIPHOSPHORYLATED PHOSPHODIESTER-LINKED OLIGOSACCHARIDES

Intracellular amastigotes of the protozoan parasite Leishmania mexican a secrete a macromolecular proteophosphoglycan (aPPG) into the phagoly sosome 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 spectrom etry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosacc harides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glyca ns represent amastigote stage-specific and novel structures. These inc lude neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1- 3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several mo nophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modi fications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monop hosphorylated aPPG tri- and tetrasaccharides that are uniquely phospho rylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4 -6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3G lc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4 -6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-G al]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1 -4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-G al]beta 1-4Man. These glycans are linked together by the conserved pho sphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Ma n alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backb one most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransfera ses which may be potential anti-parasite drug targets.