Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana

Menz, Beatrice; Winter, Gerhard; Ilg, Thomas; Lottspeich, Friedrich; Overath, Peter

doi:10.1016/0166-6851(91)90152-v

Cited by 53 publications

(44 citation statements)

References 24 publications

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“…Two-site ELISA of culture supernatants from L. major promastigotes transfected with pXmodMAP constructs were performed using mAb AP4-coated (50 l/well 20 g/ml, 50 mM NaHCO 3 , pH 9.6, 1 h 25°C) flexible polvinylchloride ELISA plates (Becton-Dickinson, Heidelberg, Germany). mAb AP4 recognizes a conformational peptide epitope of L. mexicana modMAP (41,45). The plates were washed twice with TBS/T (200 l/well), and nonspecific binding sites were blocked with TBS/MT (200 l/well, 1 h, 25°C).…”

Section: Methodsmentioning

confidence: 99%

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

Ilg¹,

Montgomery²,

Stierhof³

et al. 1999

Journal of Biological Chemistry

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Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/ gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(؎S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions.Leishmania are kinetoplastid protozoan parasites with a digenetic life cycle and are responsible for several important human diseases, ranging from mild skin ulcers to fatal visceral disease. Several forms of extracellular flagellated Leishmania promastigotes colonize the digestive tract of vector sandflies. After transmission to the mammalian host by the bite of the insect, the parasites transform to the obligatory intracellular, nonflagellated amastigotes, which reside in the phagolysosomes of macrophages (1). Both Leishmania life stages synthesize complex and unique glycoconjugates, which are either displayed on their surface (2) or secreted (3). These glycoconjugates, which are thought to have crucial functions for parasite survival, development, and virulence in the sandfly vector and the mammalian macrophage, include a family of phosphoglycan-modified molecules, which comprises lipid-linked, protein-linked, and unlinked forms (4 -6). Lipophosphoglycan (LPG) 1 is the best studied representative of this family: it consists of 1-alkyl-2-lyso-phosphatidylinositol that is linked via a diphosphoheptasaccharide core to a linear polymer of up to 40 phosphodiester-linked disaccharides that are modified by species-, strain-, and stage-specific side chains. The polymer chain terminates with variable neutral cap oligosaccharides (2).

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Section: Methodsmentioning

confidence: 99%

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

Ilg¹,

Montgomery²,

Stierhof³

et al. 1999

Journal of Biological Chemistry

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“…order to prove this hypothesis, these regions and their common C-termini were linked to a lysosomal acid phosphatase (LAP) (Menz et al, 1991;Wiese, 1991) lacking its putative C-terminal transmembrane anchor by the expression of suitable gene fusions. These proteins were designated FP2 and FP3, respectively ( Figure 7A).…”

Section: Sssst (E-mentioning

confidence: 99%

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

et al. 1995

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The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, Imsapl and lmsap2, were cloned and sequenced. Imsapl predicts a protein with features characteristic of acid phosphatases and a remarkable serine-and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsapl encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase.

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“…Promastigotes of L. mexicana MNYC\BZ\62\M379 (wild type, WT) were grown in itro in semi-defined medium 79 supplemented with 5 % heat-inactivated fetal bovine serum (iFCS) as previously described [41]. For some experiments a L. mexicana mutant in which both genes encoding secreted acid phosphatase have been deleted (L. mexicana ∆lmsap1\2, [42]) were used.…”

Section: Parasitesmentioning

confidence: 99%

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

Klein

Göpfert

Goehring

et al. 1999

Biochemical Journal

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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 Manα1-PO % -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 polypeptidespecific 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 (50n5 mol % vs. 44n6 mol %), but the structure of their phosphoglycan chains is developmentally regulated : in

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Purification and characterization of a membrane-bound acid phosphatase of Leishmania mexicana

Cited by 53 publications

References 24 publications

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

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

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

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

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