Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids

In addition to utilizing glycosylated phosphatidylinositols (GPIs) as anchors for surface proteins, protozoan parasites of the genus Leishmania synthesize two novel classes of GPI: the polydisperse lipophosphoglycans (LPGs) and a family of low molecular weight glycoinositol phospholipids (GIPLs). We now show that LPG is expressed in high copy number (6 X 10(6) molecules/cell) in the promastigote (insect) stage of L. donovani but not in the amastigote stage, which infects mammalian macrophages. Detection of these molecules was by gas chromatography-mass spectrometric analyses and by a sensitive radiolabeling procedure. In contrast, a novel family of GIPLs was present in high copy number (approximately 10(7) molecules/cell) in both promastigote and amastigote stages of L. donovani. These glycolipids were purified and analyzed by gas chromatography-mass spectrometry, methylation analysis, and by chemical and enzymatic sequencing after deamination and NaB3H4 reduction. Promastigotes contained three major GIPLs species with the following generalized structure ... where R=H for isoM2, Man alpha1- for isoM3 or Man alpha1-2Man alpha1- for isoM4. Amastigotes contained two major GIPL species that lacked the alpha1-3-linked mannose branch and had the linear structures Man alpha1-6Man alpha1-4GlcN (M2) and Man alpha1-2Man alpha1-6Man alpha1-4GlcN (M3) linked to alkylacyl-PI. The 1-O-alkyl-2-acyl-PI moieties of all of these species contained predominantly C18:0 alkyl chains and C16:0 or C18:0 fatty acids. Amastigotes contained, in addition, a GalNAc beta1-3 terminating glycosphingolipid with homology to the mammalian para Forssman glycolipid. This glycolipid appeared to be a constituent of the parasite membrane but was not metabolically labeled with [3H]glucose, suggesting that it was acquired from host cells. These results suggest that LPG may not be required for amastigote survival in the mammalian host and that the GIPLs are likely to be major components on the surface membrane in both stages.