Showing papers by "Thomas Ilg published in 1999"

Proteophosphoglycans of Leishmania mexicana. Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes.

Abstract: Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Gopfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressed ppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

Proteophosphoglycan, a major secreted product of intracellular Leishmania mexicana amastigotes, is a poor B-cell antigen and does not elicit a specific conventional CD4+ T-cell response.

Abstract: Leishmania spp. are the etiologic agents of a spectrum of human diseases. These protozoans have a digenetic life cycle and are transmitted by insect vectors as flagellated, extracellular promastigotes to their mammalian host, where they replicate as nonmotile amastigotes in parasitophorous vacuoles derived from the phagolysosomal compartment of parasitized macrophages (2). Healing of the disease and killing of the parasites is dependent on activation of potent microbicidal mechanisms in the host cells. This process is initiated by CD4+ T lymphocytes that secrete lymphokines such as gamma interferon (IFN-γ) and tumor necrosis factors which act via specific receptors on macrophages. The secretion of these activating cytokines by Leishmania antigen-specific CD4+ T lymphocytes is triggered by the interaction of their T-cell receptors with their cognate major histocompatibility complex (MHC) class II peptide complexes (15). The question of whether macrophages harboring an established infection can provide parasite-derived peptides bound to MHC class II molecules on their surface for the interaction with CD4+ T lymphocytes has been studied by using genetically engineered parasites (20, 21). The results of these investigations indicate that the parasitophorous vacuole membrane communicates with the macrophage plasma membrane and may be the compartment where peptides derived from parasite proteins are produced and then loaded onto MHC class II molecules. Provided that MHC class II expression is induced by IFN-γ in infected macrophages, T-cell activation by the host cell is possible. The data further suggest that only the subset of parasite antigens which are secreted or exposed on the parasite surface is efficiently presented by macrophages harboring live parasites, while intracellular proteins are presented only if the parasites are killed (20, 21). The search for cell surface proteins in amastigotes has proven to be difficult, and, at least for L. mexicana, a major amastigote surface protein has not been identified (19), although such proteins are typical for several parasitic protozoa. Instead, the surface appears to be covered with glycoinositolphospholipids and glycosphingolipids (19). However, L. mexicana amastigotes secrete large amounts of a stage-specific proteophosphoglycan (aPPG) (8). This is so far the only secreted amastigote product that has been identified in infected tissue and has been analyzed in some detail: aPPG belongs to a novel class of serine- and threonine-rich Leishmania proteins that are extensively modified by phosphodiester-linked phosphooligosaccharides and terminal mannooligosaccharides (9). Lesions of L. mexicana-infected mice yield 40 to 100 μg of aPPG per g of tissue, and in the parasite-containing phagolysosome its concentration may reach several milligrams per milliliter (8, 17). Macrophages infected in vitro contain 0.1 to 0.2 pg of aPPG per cell, which is consistent with the in vivo estimate (1, 17). Since purified aPPG has an apparent molecular mass ranging from 4 × 105 to 2 × 106 Da, the number of aPPG molecules per infected macrophage (assuming an average molecular mass of 106 Da) is calculated to be about 105. The continuous secretion of aPPG into the phagolysosome may contribute to the enlargement of this compartment, which is typical for macrophages infected with L. mexicana (17). Occasionally, aPPG is detected by immunocytochemistry in macrophage vesicles, suggesting that aPPG is also exported from the parasitophorous vacuole and, possibly, secreted by viable infected host cells (8). Upon rupture of infected macrophages, aPPG is released and can be taken up by other phagocytes, most probably by receptor-mediated endocytosis. As a highly abundant and secreted parasite product, aPPG could provide an ideal target for the cellular immune response of the host in L. mexicana infections. In the present study, we investigated the immune response to this secreted parasite product in mice infected with L. mexicana as well as in those immunized with the purified molecule. We demonstrated that in spite of the very high local concentration and the large amounts present in infected tissue, aPPG elicited no B-cell response in most infected mice and was not recognized by conventional CD4+ T cells. Likewise, in immunized animals, the purified native compound was a very poor B-cell antigen and was not CD4+ T-cell immunogenic, in contrast to the Escherichia coli-expressed recombinant form. These results suggest that L. mexicana amastigotes avoid the stimulation of the immune system of their mammalian host by heavy glycosylation of their major secretory product, aPPG, which appears to minimize its immunogenicity.

Mucin-Like Proteophosphoglycans from the Protozoan Parasite Leishmania

Abstract: ヒトではリーシュマニア(住血鞭毛虫)は偏性細胞内寄生生物(偏性-obligatory-というのは、「ある特定の環境にのみ生活しうる」という意味)であり、単核の食細胞系細胞の中で増殖する。この病原性の原虫はサシチョウバエの消化管に住んでいてそこから感染する。リーシュマニアの特製の表面やリーシュマニアが分泌する分子こそが、この寄生虫がサシチョウバエに定着し、かつマクロファージに進入して生き延びるために欠かせない重要な因子であることが分かってきた。私たちは前鞭毛型リーシュマニア、および無鞭毛型リーシュマニアにより分泌されて表面に存在する非常にユニークなムチン様プロテオホスフォグリカン(PPG)を研究してきた。この総説ではこの新しいプロテオグリカンの構造と機能の最新の知見について述べたい。一次構造が調べられて、タンパク質や、ホスフォグリカン構造、タンパク質-炭水化物結合ががきわめてユニークであることが明らかとなったので、予想される機能と合わせ考えることで、ワクチンや抗寄生虫薬剤の開発の良い対象となるであろう。

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

Abstract: Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manalpha1-PO(4)-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50. 5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of >60 mol% neutral glycans, most likely (Manalpha1-2)(0-5)Man and Galbeta1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO(4)-6Galbeta1-4Man and PO(4)-6(Glcbeta1-3)Galbeta1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone.

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

Abstract: Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manalpha1-PO(4)-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50. 5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of >60 mol% neutral glycans, most likely (Manalpha1-2)(0-5)Man and Galbeta1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO(4)-6Galbeta1-4Man and PO(4)-6(Glcbeta1-3)Galbeta1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone.