Stage-specific Proteophosphoglycan from Leishmania mexicana Amastigotes

Abstract

Intracellular amastigotes of the protozoan parasiteLeishmania 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 1 H and 31 P 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 ([Glc␤1-3] 1-2 Gal␤1-4Man, Gal␤1-3Gal␤1-4Man, Gal␤1-3Glc␤1-3Gal␤1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO 4 -6-Gal]␤1-4Man) but carrying stage-specific modifications (R ‫؍‬ Gal␤1-, [Glc␤1-3] 1-2 Glc␤1-), and monophosphorylated aPPG tri-and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO 4 -6-Glc␤1-3Gal␤1-4Man, PO 4 -6-Glc␤1-3Glc␤1-3Gal␤1-4Man, PO 4 -6-Gal␤1-3Glc␤1-3Gal␤1-4Man). In addition aPPG contains highly unusual di-and triphosphorylated glycans whose major species are PO 4 -6-Glc␤1-3Glc␤1- Protozoan parasites of the genus Leishmania are the causative agent of a spectrum of human diseases. Leishmania have a digenetic life cycle that encompasses the extracellular promastigotes in the digestive tract of the parasite-transmitting insect vector, the sandfly, and the disease-causing intracellular amastigotes living in parasitophorous vacuoles of mammalian macrophages.The parasites produce unusual glycoconjugates, which are thought to play crucial roles for survival, development, and virulence in both developmental stages of the parasite. The best characterized Leishmania glycoconjugate is the promastigote cell-surface glycolipid, lipophosphoglycan (LPG).1 The structure of LPG from five different Leishmania species has been determined. LPG contains a conserved lyso-alkylphosphatidylinositol membrane anchor linked to a phosphohexasaccharide core structure, a conserved backbone of up to 40 phosphodiester-linked disaccharide repeats (PO 4 -6-Gal␤1-4Man␣1-), and species-, strain-, and stage-specific components linked to the core and repeats as well as terminating neutral (cap) glycans at the non-reducing end of the molecule (reviewed in Refs. 1 and 2). In the sandfly, LPG serves as a ligand for the attachment of non-infectious procyclic promastigotes to the midgut wall lining and may protect the parasites against the hydrolytic environment of the insect's digestive tract. A stagespecific form of LPG confers complement resistance to the highly infectious metacyclic promastigotes, which are injected by the sandfly into the skin of the mammalian host. LPG also acts as a receptor for the invasion of macrophages by metacyclic promastigotes and may protect this transient mammalian parasite stage against the initial microbici...

Molecular Cloning and Characterization of a Novel Repeat-containing Leishmania major Gene,ppg1, That Encodes a Membrane-associated Form of Proteophosphoglycan with a Putative Glycosylphosphatidylinositol Anchor

“…Using affinity-purified antibodies isolated from this serum, SDS-PAGE/immunoblots of total cell lysates from PBS-washed L. major promastigotes showed a strong signal in the region corresponding to the stacking gel and the top of the separating gel (Fig. 11, lane 1), a position typical for PPGs (25,28,29). This cell-bound PPG1 protein remained associated with the membrane pellet after disruption of the cells and ultracentrifugation (Fig.…”

“…This group of molecules includes secreted acid phosphatase synthesized by promastigotes of all Leishmania species except Leishmania major (23,24), non-filamentous proteophosphoglycan of Leishmania mexicana amastigotes (25), and a filamentous proteophosphoglycan (fPPG) secreted by promastigotes of all species investigated so far (5,26). Capped phosphoglycan chains are linked to the polypeptide backbone of these proteins via phosphodiester linkages to serine, an unusual type of protein modification called phosphoglycosylation (27)(28)(29). Among the PPGs, fPPG exhibits some unique features; the contour length of filaments observed by electron microscopy in purified preparations may exceed 6 m. The fPPG protein backbone is largely formed by Ser, Ala, and Pro (together Ͼ87 mol%), and more than 40 mol% of its amino acids are phosphoglycosylated Ser residues.…”

Proteophosphoglycans of Leishmania mexicana: Identification, purification, structural and ultrastructural characterization of the secreted promastigote proteophosphoglycan pPPG2, a stage-specific glycoisoform of amastigote aPPG

“…Eight C57\Bl6 mice were immunized with native aPPG (50 µg) and with aPPG that had been deglycosylated by mild acid hydrolysis (200 µg) [27]. For primary immunizations and booster immunizations, the compounds were resuspended in 0n5 ml 50 % complete or 0n5 ml 50% incomplete Freund's adjuvant in phosphate-buffered saline, pH 7n3 (PBS), respectively.…”

“…In the case of PPGs, phosphosaccharide repeats and cap glycans are linked to a protein backbone via phosphodiester linkages to serine, an unusual form of protein glycosylation termed phosphoglycosylation [25][26][27][28]. The group of PPGs consists primarily of secreted parasite products such as acid phosphatase (SAP [25,[29][30][31][32]), filamentous proteophosphoglycan from promastigotes (fPPG [26,33]) and a non-filamentous proteophosphoglycan from amastigotes (aPPG [27,34]).…”

“…The group of PPGs consists primarily of secreted parasite products such as acid phosphatase (SAP [25,[29][30][31][32]), filamentous proteophosphoglycan from promastigotes (fPPG [26,33]) and a non-filamentous proteophosphoglycan from amastigotes (aPPG [27,34]). Recently, however, a cell surface membrane-bound PPG with a putative GPI anchor has been identified on L. major promastigotes [35] and ultrastructural studies suggest that PG-modified molecules may also be abundant in the extended lysosomes of L. mexicana amastigotes [36,37].…”