Purification and Structural Characterization of a Filamentous, Mucin-like Proteophosphoglycan Secreted by Leishmania Parasites

Abstract

Parasitic protozoa of the genus Leishmania secrete a filamentous macromolecule that forms networks and appears to be associated with cell aggregation. We report here the purification of this parasite antigen from Leishmania major culture supernatant and its compositional (75.6% carbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ultrastructural characterization as a highly unusual proteophosphoglycan (PPG). Mild acid hydrolysis, which cleaves preferentially hexose 1-phosphate bonds, releases the PPG glycans. Their structures are Gal␤1-4Man, Man␣1-2Man, Gal␤1-3Gal␤1-4Man, PO 4 -6(Gal␤1-3) 0 -2 Gal␤1-4Man, and PO 4 -6(Ara␤1-2Gal␤1-3)Gal␤1-4Man. These glycans are also components of the parasite glycolipid lipophosphoglycan, but their relative abundance and structural organization in PPG are different. Some of them represent novel forms of protein glycosylation. 31 P NMR on native PPG demonstrates that phosphate is exclusively in phosphodiester bonds and that the basic structure R-Man␣1-PO 4 -6-Gal-R connects the glycans. A phosphodiester linkage to phosphoserine (most likely R-Man␣1-PO 4 -Ser) anchors the PPG oligosaccharides to the polypeptide. PPG has a unique amino acid composition; glycosylated phosphoserine (>43 mol %), serine, alanine, and proline account for more than 87 mol % and appear to be clustered in large proteinase-resistant domains. Electron microscopy of purified PPG reveals cable-like, flexible, long (to 6 m), and unbranched filaments. The overall structure of PPG shows many similarities to mammalian mucins. Potential functions of this novel mucin-like molecule for the parasites are discussed.Parasitic protozoa of the genus Leishmania are the causative agent of a wide range of human and animal diseases, which are transmitted by an insect vector, the sandfly. The digenetic life cycle of the parasite encompasses the infective metacyclic and several forms of procyclic, extracellular promastigotes in the digestive tract of the insect and the intracellular amastigotes residing in parasitophorous vacuoles of the mammalian host cells, the macrophages. Expression of complex and unique glycoconjugates appears to be crucial for the survival and development of the parasites in the sandfly vector and the mammalian host (reviewed in Refs. 1-3). The dominant cell surface glycolipid of Leishmania promastigotes is lipophosphoglycan (LPG). 1 In the sandfly, LPG serves as a ligand for the attachment of procyclic promastigotes to the midgut wall lining and may protect the parasites against the hydrolytic environment of the insect's digestive tract. After transmission of metacyclic promastigotes to the mammalian host, LPG confers complement resistance; it has been shown to act as a receptor for invasion of macrophages and may protect the parasites against the microbicidal response of the host cell (reviewed in Refs. 1, 2).The structure of LPG from five different Leishmania species has been described (4 -10), including life stage-and strainspecific features (8,11,12). Conserved structural elements are...

Transcriptional Inhibition of Interleukin-12 Promoter Activity in Leishmania Spp.–Infected Macrophages

“…The repeating PG domain is a characteristic feature of other molecules of the leishmania glycocalyx, including proteo-PG Ilg, Stierhof et al, 1994;Ilg et al, 1996), secreted PG, and acid phosphatase (Shakarian and Dwyer, 2000). The PG domain contains species and strain-specific substitutions (Tolson et al, 1989;McConville et al, 1990;Ilg et al, 1992) and developmentally regulated polymorphisms (Glaser et al, 1991;Sacks, 1992;Moody et al, 1993).…”

Olfaction in vector-host interactions

“…This promastigote secretory gel (PSG) is formed by the parasites and has been shown to enhance cutaneous infections when deposited onto the skin with sand fly saliva (Rogers et al 2004, Titus 1998. A major component of PGS is a filamentous glycoprotein, proteophosphoglycan (fPPG) (Ilg et al 1996), which forms the 3D matrix of the plug that blocks the gut (Stierhof et al 1999) and keeps the valve open. The production of PSG has been shown to occur is several Leishmania-sand fly combinations (Stierhof et al 1999).…”

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

“…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.…”