Biomedical subjects

P Overath

Publications and source records attributed to P Overath.

At least 37 records · Page 2Linked to original sources

PubMed1995

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei.

In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositol-anchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; KD = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-free ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo- and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.

Animals↗

PubMed1995

The role of macrophage receptors in adhesion and uptake of Leishmania mexicana amastigotes.

Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of mammalian macrophages. Propagation of the infection is considered to occur by host-cell rupture and uptake of released parasites by uninfected macrophages. In this study, the kinetics of binding of L mexicana mexicana amastigotes to COS cells and to COS cells transfected with three different macrophage receptors (FcRII-B2, receptor for the Fc-domain of immunoglobulins; CR3, complement type 3 receptor and the mannose receptor) is compared to the rate of adhesion to peritoneal macrophages. Amastigotes isolated from macrophages cultivated in vitro bind with slow, sigmoid kinetics to COS cells expressing either of the three receptors, or to peritoneal macrophages. In contrast, amastigotes isolated from mouse lesions bind with rapid, hyperbolic kinetics to COS cells expressing the Fc receptor or to peritoneal macrophages but with slow, sigmoid kinetics to COS cells expressing the CR3 or the mannose receptor. As shown by immunofluorescence experiments, lesion-derived amastigotes contain host-derived immunoglobulins (Ig) but no complement component 3 at their surface. It is concluded that amastigotes contain no intrinsic ligand at their surface, which enables high-affinity interactions with macrophages. Opsonization by specific Ig may be of relevance in vivo because firstly, in cryosections of mouse lesions extracellular amastigotes containing surface Ig can be detected and, secondly, B cell-deficient mice reconstituted with parasite-specific Ig show a modest increase in the rate of lesion development. In addition, it is shown that amastigotes are internalized by COS cells and grow in large parasitophorous vacuoles similar to those observed in macrophages.

Adhesiveness↗

PubMed1995

Purification, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexicana amastigotes.

The intracellular amastigote form of the parasitic protozoon Leishmania mexicana expresses a high-molecular weight phosphoglycan, which is antigenically related to the surface glycolipid lipophosphoglycan and the secreted enzyme acid phosphatase of Leishmania promastigotes. This antigen was purified from a cell-free homogenate of infected mouse tissue and from amastigotes. Compositional and immunological analysis of the purified components indicate a proteophosphoglycan structure consisting of serine-rich polypeptide chains and mild acid-labile phosphooligosaccharides capped by mannooligosaccharides. Immunofluorescence and immunoelectron microscopy of parasitized mouse peritoneal macrophages and infected mouse tissue suggest that the proteophosphoglycan is secreted in large amounts by amastigotes via their flagellar pockets into the parasitophorous vacuoles of host cells. In some infected macrophages proteophosphoglycan is also located in vesicles apparently originating from the parasitophorous vacuole, which demonstrates redistribution of a secreted amastigote antigen in parasitized host cells.

Animals↗

PubMed1994

O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.

Acid Phosphatase↗

PubMed1994

Expression of a glycosylphosphatidylinositol-anchored Trypanosoma brucei transferrin-binding protein complex in insect cells.

The expression site-associated gene ESAG 6 was previously implicated in transferrin binding in the protozoan parasite Trypanosoma brucei. ESAG 6 and the closely related ESAG 7 of T. brucei strain AnTat1.3 have now been expressed in insect cells using the baculovirus expression system. Expression of ESAG 6 alone in insect cells gives rise to a glycosylated protein of approximately 52 kDa, which is cell surface-associated through a glycosylphosphatidylinositol anchor at its C terminus. The ESAG 7 product of about 42 kDa is also glycosylated, but lacks the glycosylphosphatidylinositol modification, and is located intracellularly. No transferrin-binding activity is observed when either ESAG is expressed independently. However, their expression results in a cell surface complex of ESAG 6 and 7 products that specifically binds transferrin. This shows that both ESAG 6 and 7 products are necessary and sufficient for binding to transferrin.

Animals↗

PubMed1994

Distribution of parasite cysteine proteinases in lesions of mice infected with Leishmania mexicana amastigotes.

It is well established that Leishmania mexicana amastigotes contain large amounts of cysteine proteinases in their extended lysosomes. In this study it is shown that the cell-free supernatant of homogenized lesion tissue from infected mice contains large amounts of acid proteinases. The majority of this enzymatic activity also corresponds to cysteine proteinases from L. mexicana amastigotes. Immunoelectron microscopy of mouse lesion sections suggests, that frequently amastigotes lyse and release lysosomal cysteine proteinases into the parasitophorous vacuole of infected macrophages. The cysteine proteinases are also found extracellularly in the tissue presumably as a result of macrophage rupture and appear to persist in the lesion tissue, where they may damage host cells and the extracellular matrix.

Amino Acid Sequence↗

PubMed1994

Invariant surface proteins in bloodstream forms of Trypanosoma brucei.

Antigenic variation of the glycoprotein forming the coat of African trypanosomes has been a dominant field of investigation for many years. The extravagant potential of these parasites to change their surface coat has destroyed hopes for a vaccine based on the variant surface glycoprotein. Recently, there has been a rising interest in the characterization of surface proteins that are not subject to antigenic variation. In this review, Peter Overath, Maliha Chaudhri, Dietmar Steverding and Karl Ziegelbauer summarize the present evidence for the occurrence, cellular localization and function of invariant surface proteins in Trypanosoma brucei.

Journal Article↗

PubMed1994

Characterization of phosphoglycan-containing secretory products of Leishmania.

This article presents an overview on phosphoglycan-containing components secreted by the insect and mammalian stages of several species of Leishmania, the causative agents of leishmaniasis in the Old and New World. Firstly, promastigotes of all three species considered, L. mexicana, L. donovani and L. major, shed lipophosphoglycan (LPG) into the culture medium possibly by release of micelles from the cell surface. Like the cell-associated LPG, culture supernatant LPG is amphiphilic and composed of a lysoalkylphosphatidylinositol-phosphosaccharide core connected to species-specific phosphosaccharide repeats and oligosaccharide caps. Secondly, all three species release hydrophilic phosphoglycan. Thirdly, all three species appear to secrete proteins covalently modified by phosphosaccharide repeats and oligosaccharide caps. In the case of promastigotes of L. mexicana, these components are organized as two filamentous polymers released from the flagellar pocket: the secreted acid phosphatase (sAP) composed of a 100 kDa phosphoglycoprotein and a protein-containing high-molecular-weight-phosphoglycan (proteo-HMWPG) and fibrous networks likewise composed of phosphoglycan possibly linked to protein. Structural analyses and gene cloning suggest that the parasites can covalently modify protein regions rich in serine and threonine residues by the attachment of phosphosaccharide repeats capped by oligosaccharides. We propose that the networks formed in vitro correspond to fibrous material previously demonstrated in the digestive tract of infected sandflies. In the case of L. donovani, the sAP is also modified by phosphoglycans but contains neither proteo-HMWPG nor does it aggregate to filaments. Finally, L. mexicana amastigotes release proteo-HMWPG via the flagellar pocket into the parasitophorous vacuole of infected macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗

PubMed1994

Characterization of polymer release from the flagellar pocket of Leishmania mexicana promastigotes.

Trypanosomatids contain a unique compartment, the flagellar pocket, formed by an invagination of the plasma membrane at the base of the flagellum, which is considered to be the sole cellular site for endocytosis and exocytosis of macromolecules. The culture supernatant of Leishmania mexicana promastigotes, the insect stage of this protozoan parasite, contains two types of polymers: a filamentous acid phosphatase (sAP) composed of a 100-kD phosphoglycoprotein with non-covalently associated proteo high molecular weight phosphoglycan (proteo-HMWPG) and fibrous material termed network consisting of complex phosphoglycans. Secretion of both polymers is investigated using mAbs and a combination of light and electron microscopic techniques. Long filaments of sAP are detectable in the lumen of the flagellar pocket. Both sAP filaments and network material emerge from the ostium of the flagellar pocket. While sAP filaments detach from the cells, the fibrous network frequently remains associated with the anterior end of the parasites and can be found in the center of cell aggregates. The related species L. major forms similar networks. Since polymeric structures cannot be detected in intracellular compartments, it is proposed that monomeric or, possibly, oligomeric subunits synthesized in the cells are secreted into the flagellar pocket. Polymer formation from subunits is suggested to occur in the lumen of the pocket before release into the culture medium or, naturally, into the gut of infected sandflies.

Acid Phosphatase↗

PubMed1994

Surface antigens of Leishmania mexicana amastigotes: characterization of glycoinositol phospholipids and a macrophage-derived glycosphingolipid.

Amastigotes of the protozoan parasite Leishmania proliferate in phagolysosomes of macrophages. They abundantly express glycoinositol phospholipids (GIPLs), which are considered necessary for parasite survival by providing a shield at the surface against lysosomal hydrolases and by serving as receptors for the interaction with host cells. The structures of four GIPLs of L. mexicana amastigotes were characterized by a combination of gas-liquid chromatography-mass spectrometry, methylation linkage analysis and enzymatic treatments. They contain the glycan structures Man alpha 1-3Man alpha 1-4GlcN (iM2), Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM3), Man alpha 1-2Man alpha 1-6(Man alpha 1-3)-Man alpha 1-4GlcN (iM4) and (NH2-CH2CH2-PO4)Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (EPiM3), which are linked to alkylacyl-phosphatidylinositol. The predominant amastigote GIPL, EPiM3 (approximately 2 x 10(7) molecules/cell), is located at the parasite cell surface, in the flagellar pocket and in lysosomal membranes, but not on host cell structures as shown by immunofluorescence and immunoelectron microscopy. In addition, amastigotes in infected Balb/c mice contain a glycolipid with similar distribution as EPiM3, which has the same characteristics as the Forssman antigen of mammalian cells. In contrast to EPiM3, there is strong evidence that this glycosphingolipid is not synthesized by amastigotes but by macrophages in the lesion. This suggests a mechanism of lipid transfer from the macrophage to the parasite.

Animals↗

PubMed1994

ESAG 6 and 7 products of Trypanosoma brucei form a transferrin binding protein complex.

In Trypanosoma brucei, the gene for the expressed variant surface glycoprotein (VSG) is preceded by a series of open reading frames designated expression site associated genes (ESAGs), which together with the VSG gene form a polycistronic transcription unit. It is shown that the products derived from two ESAGs (ESAG 6 and 7 in the nomenclature of Pays, E., et al. Cell 57, 835-845 (1989)) form a complex, which binds transferrin with high affinity. Transferrin affinity chromatography yields heterodimers or higher order heteroligomers composed of the products of ESAG 6 and ESAG 7. The former is a heterogeneously glycosylated protein of 50 to 60 kDa modified by a glycosylphosphatidylinositol membrane anchor at the COOH-terminus, while the latter is the previously identified 42 kDa glycoprotein carrying an unmodified COOH-terminus (Schell, D., et al. EMBO J. 10, 1061-1066 (1991) and Schell, D., et al. EMBO J. 12, 2990 (1993)). When isolated from trypanosomes grown in rodents, the complex is in part free and in part associated with transferrin. Also, the complex is present both in the membrane fraction and the soluble fraction of cell lysates. As shown by immunoelectron microscopy, both transferrin and ESAG 6/7-derived proteins can be demonstrated in the lumen of the flagellar pocket, an invagination of the plasma membrane serving as the sole site for endocytotic uptake of macromolecular nutrients. Weak labeling is also obtained on the flagellar pocket membrane and in intracellular vesicles. The possibility that the binding protein complex serves as a receptor for the uptake of transferrin in T. brucei is discussed.

Amino Acid Sequence↗

PubMed1994

Structure and function of GPI-anchored surface proteins of Trypanosoma brucei.

While proteins modified at their COOH-terminal end by a glycosylphosphatidylinositol (GPI) membrane anchor have been found as minor components in many eukaryotic cells, they dominate surface constituents of several parasitic protozoa. In this article, GPI-anchored proteins of Trypanosoma brucei are discussed.

Animals↗

PubMed1993

Monoclonal antibodies directed against Leishmania secreted acid phosphatase and lipophosphoglycan. Partial characterization of private and public epitopes.

Leishmania promastigotes, the stage of the parasite characteristic for the sandfly vector, express an abundant glycoconjugate, called lipophosphoglycan, at their surface. Lipophosphoglycan consists of lysoalkyl-sn-glycerophosphoinositol linked to a phosphosaccharide core conserved in all species, which is connected to PO4-6Gal beta 1,4Man alpha 1 repeats with species-specific substitutions at the Gal residue; the repeats are capped by conserved and species-specific oligosaccharides. Most Leishmania species also secrete an acid phosphatase, which, in Leishmania mexicana, is a filamentous complex composed of a phosphorylated glycoprotein and non-covalently associated proteo-(high-molecular-mass)phosphoglycan. The secreted acid phosphatase complex was used as an antigen to derive a panel of monoclonal antibodies (mAbs). A total of 25 mAbs (17 novel and 8 previously described) were tested by different techniques for their specificity against lipophosphoglycan and secreted acid phosphatase from several Leishmania species. This comparison and the modification of the antigens by chemical or enzymic treatments allowed a classification of the mAbs into several groups. First, from 25 mAbs examined, 22 recognize lipophosphoglycan and the enzyme complex of L. mexicana; only three are specific for secreted acid phosphatase. Two of the latter group are also directed against carbohydrate structures, whereas the third mAb recognizes the 100-kDa polypeptide of the complex. The secreted acid-phosphatase-specific class detects antigen in the flagellar pocket of promastigotes while all anti-lipophosphoglycan mAbs bind to the cell surface. Second, all 15 anti-lipophosphoglycan mAbs investigated in detail appear to be directed against the phosphosaccharide repeats or the cap structure rather than the phosphosaccharide core. Two mAbs recognize terminal cap-structures containing Man alpha 1,2Man residues. Four antibodies are specific for L. mexicana and are probably directed against PO4-6[Glc beta 1,3]Gal beta 1,4Man alpha 1 repeats while six mAbs react with the unmodified repeats. Two antibodies specific for Leishmania major recognize Gal beta 1,3-substituted repeats unique for lipophosphoglycan from this species. Analysis by immunoblotting indicates that the high-molecular-mass proteo-phosphoglycan of L. mexicana secreted acid phosphatase carries epitopes for all anti-lipophosphoglycan mAbs suggesting the presence of capped phosphosaccharide repeats while the enzymically active glycoprotein subunit is modified by caps but probably not by repeats. In the case of Leishmania donovani secreted acid phosphatase, the enzymically active polypeptide may be directly modified by repeats. The mAbs are used to characterize changes in lipophosphoglycan structure, which occur in culture during the transition of promastigotes from the logarithmic to the stationary growth phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗

PubMed1993

The lysosomal gp63-related protein in Leishmania mexicana amastigotes is a soluble metalloproteinase with an acidic pH optimum.

Leishmania mexicana amastigotes express a lysosomal protein, which is antigenically related to the promastigote surface metalloproteinase (gp63). It is shown that the purified gp63-related protein from amastigote is also an active metalloproteinase. The pH-optimum of the enzyme is acidic, similar to lysosomal cysteine proteinases, but distinct from the neutral to basic pH-optimum of the promastigote surface proteinase. This study appears to be the first report on a metalloproteinase with a lysosomal localization.

Acids↗

PubMed1993

Proteolytic release of cell surface proteins during differentiation of Trypanosoma brucei.

The surface of the bloodstream forms of Trypanosoma brucei is covered by the abundant glycosylphosphatidylinositol-anchored variant surface protein (mfVSG). During differentiation of bloodstream forms to the insect-stage or procyclic forms, the mfVSG is replaced by another glycoprotein, designated procyclic acidic repetitive protein (PARP) or procyclin. Shortly after differentiation is triggered in vitro, a cell-associated fragment of mfVSG can be detected which is subsequently released into the culture medium. In the case of the mfVSG of the variant clone MITat 1.4 (470 amino acid residues), fragmentation occurs close to the COOH-terminus (Gln433 or Thr434) as shown by NH2-terminal sequencing, metabolic labeling experiments, and molecular weight determinations by laser desorption/ionization mass spectrometry. Two invariant surface glycoproteins, which are anchored in the membrane by hydrophobic sequences close to their COOH-termini, are lost from the surface with similar kinetics as mfVSG. The data suggest that trypanosomes synthesize or activate a developmentally-regulated proteinase which degrades the glycoproteins at the surface, at the membrane lining the flagellar pocket, and/or in an early endocytic compartment.

Amino Acid Sequence↗

PubMed1993

Leishmania mexicana promastigotes induce cytotoxic T lymphocytes in vivo that do not recognize infected macrophages.

The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli beta-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicana-gal induces beta-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a beta-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of beta-galactosidase in the context of H-2Kd; however, they do not recognize L. mexicana-gal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming.

Animals↗

PubMed1993

Course of Leishmania infection in beta 2-microglobulin-deficient mice.

Mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption lack the beta 2-m protein and are deficient in functional major histocompatibility complex class I (MHC I) molecules. The mutant mice have normal numbers of CD4+8- T helper cells but lack MHC I-directed CD4-8+ alpha/beta T cells. The beta 2-m mutant and wild-type mice were infected with Leishmania major or L. mexicana, which cause cutaneous leishmaniasis in the Old and New World, respectively. In both mutant and wild-type mice, the infection with L. major was controlled at a low level of parasitization, while L. mexicana caused a progressive disease. Assuming the absence of compensatory mechanisms, it is concluded that MHC I-directed CD8+ T cells are not important for the course of a Leishmania infection, supporting the prevailing view that control or exacerbation of the disease is modulated by type 1 (TH1) or type 2 (TH2) CD4+8- T cells, respectively.

Animals↗

PubMed1993

Expression of lipophosphoglycan, high-molecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana.

The abundant surface glycoconjugate of Leishmania promastigotes, lipophosphoglycan (LPG), forms a blue-colored complex (lambda max = 649 nm) with the cationic dye Stains-all, which can be quantitated densitometrically on polyacrylamide gels of cell lysates. Promastigotes of Leishmania mexicana, Leishmania major and Leishmania donovani yield values of 1-3 x 10(6) LPG molecules cell-1. In amastigotes the LPG content is down-regulated below the detection limit (< 10(3) molecules cell-1) in L. mexicana and L. donovani, but remains significant in L. major (2 x 10(3) molecules cell-1). In the case of L. mexicana, these results are supported by immunological studies. Using several monoclonal and polyclonal antibodies, LPG is undetectable by immunoblotting in lysates of either amastigotes or infected macrophages and the amastigote surface is devoid of LPG as judged by immunofluorescence and immunoelectron microscopy. Immunoblotting experiments demonstrate that amastigotes synthesize hydrophilic high-molecular weight compounds which stain blue with Stains-all and cross-react with the monoclonal and polyvalent antibodies suggesting the presence of similar phosphoglycan structures as in LPG. The high-molecular weight phosphoglycan appears to be located in the lumen of the flagellar pocket of mouse lesion amastigotes and may be secreted from there into the lumen of the parasitophorous vacuole of parasitized macrophages. In L. mexicana promastigotes the surface protease gp63 is amphiphilic and comprises about 1% of the cellular proteins. In contrast, in amastigotes gp63-related proteins are predominantly hydrophilic; they amount to only about 0.1% of the cellular proteins and are mainly located in the lumen of the extended lysosomes (megasomes) characteristic for this species.

Animals↗