RESUMO
The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.
Assuntos
Glicosilfosfatidilinositóis/química , Leishmania guyanensis/química , Lipídeos/química , Polissacarídeos/química , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicoesfingolipídeos/química , Leishmania guyanensis/crescimento & desenvolvimento , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
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 ([Glcbeta1-3]1-2Galbeta1-4Man, Galbeta1-3Galbeta1-4Man, Galbeta1-3Glcbeta1-3Galbeta1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta1-4Man) but carrying stage-specific modifications (R = Galbeta1-, [Glcbeta1-3]1-2Glcbeta1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glcbeta1-3Galbeta1-4Man, PO4-6-Glcbeta1-3Glcbeta1-3Galbeta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3Galbeta1-4Man). In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glcbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-+ ++4Man, PO4-6-Glcbeta1-3[PO4-6-Glc]beta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3[PO4-6-Glc]beta1-3Glcbeta1-3[PO4-6-Gal]beta1 -4Man, and PO4-6-Glcbeta1-3[PO4-6-Glc]beta1-3Glcbeta1-3[PO4-6-Gal]beta1 -4Man. These glycans are linked together by the conserved phosphodiester R-Manalpha1-PO4-6-Gal-R or the novel phosphodiester R-Manalpha1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Manalpha1-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.