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Elisabeth Jamet - One of the best experts on this subject based on the ideXlab platform.
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The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A toolbox for sequential plant n-glycan processing
Journal of Biological Chemistry, 2015Co-Authors: Stéphanie Dupoiron, Claudine Zischek, Laetitia Ligat, Julien Carbonne, Alice Boulanger, Thomas Duge De Bernonville, Martine Lautier, Pauline Rival, Matthieu Arlat, Elisabeth JametAbstract:N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three Mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-Mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-Mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.
Dingeman C. Rijken - One of the best experts on this subject based on the ideXlab platform.
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The Mannose receptor, localization and role in the clearance of tissue-type plasminogen activator
Fibrinolysis and Proteolysis, 1998Co-Authors: Femke Noorman, M.m. Barrett-bergshoeff, Eric Barbe, Jef J. Emeis, Erik A L Biessen, Dingeman C. RijkenAbstract:Summary The thrombolytic and antithrombotic effects of tissue-type plasminogen activator (t-PA) depend on its concentration in the blood. The Mannose receptor is one of the receptors that mediate the rapid clearance of t-PA from the blood by the liver (Noorman and Rijken, Fibrinolysis & Proteolysis 1997; 11: 173–186). We hypothesized that by blocking the binding of t-PA to this receptor it might be possible to decrease the clearance of t-PA and thereby increase the plasma concentration of endogenous t-PA and exogenous t-PA. Inhibitors of the t-PA-Mannose receptor interaction may thus be useful drugs in thrombolytic and antithrombotic therapy. We developed monoclonal antibodies against the human Mannose receptor. These antibodies are able to inhibit binding of t-PA to the Mannose receptor. By use of the antibodies it was shown that the Mannose receptor is expressed by few human cell types. The expression of the Mannose receptor on macrophages is highly regulated and depends on the type of macrophage activation. The expression of the Mannose receptor in the liver is limited to endothelial cells and Kupffer cells. We developed high affinity ligands of the Mannose receptor that are able to inhibit the plasma clearance of therapeutic t-PA doses by maximally 60% in the rat. Therapeutical concentrations of the low affinity Mannose receptor ligand and antithrombotic agent dextran partially inhibit t-PA plasma clearance (33%) and thereby increase endogenous t-PA plasma concentrations by 20–60% in the rat. Thus Mannose receptor inhibitors can be considered as a new tool to increase the t-PA concentration in blood and thereby increase its thrombolytic and antithrombotic effects.
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Role of carbohydrate and protein in the binding of tissue‐type plasminogen activator to the human Mannose receptor
FEBS Journal, 1998Co-Authors: Femke Noorman, M.m. Barrett-bergshoeff, Dingeman C. RijkenAbstract:The 175-kDa Mannose receptor is one of the receptors that mediates the clearance of tissue-type plasminogen activator (t-PA). The affinity of t-PA for the Mannose receptor is much higher than the affinity of other high-Mannose-type oligosaccharide-containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t-PA with the isolated human Mannose receptor in several in vitro binding assays. t-PA showed a high affinity (Ki = 0.2 nM) for the Mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the Mannose receptor. The interaction was not affected by non-glycosylated t-PA. The high affinity differed slightly between t-PAs synthesized by various cell types (range Ki 0.2−0.7 nM) and between various glycoforms of t-PA. No statistically significant difference in affinity between t-PA and t-PA complexed to inhibitors was observed. In contrast to intact t-PA, a trypsin digest of t-PA had a low affinity (Ki = 0.5 μM) for the Mannose receptor. Both intact and trypsin digests of the high-Mannose-type oligosaccharide-containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5−1.5 μM) for the Mannose receptor. We conclude that neither protein−protein interactions, nor the complex-type oligosaccharides and the fucose residue on t-PA contribute significantly to the high-affinity binding of t-PA. We suggest that the conformation of the high-Mannose-type oligosaccharide on t-PA is influenced by the protein moiety of t-PA in such a way that the oligosaccharide has a high affinity for the Mannose receptor.
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Monoclonal antibodies against the human Mannose receptor that inhibit the binding of tissue-type plasminogen activator.
Thrombosis and Haemostasis, 1997Co-Authors: M.m. Barrett-bergshoeff, Femke Noorman, Dingeman C. RijkenAbstract:To study the role of the Mannose receptor in cellular uptake and degradation of tissue-type plasminogen activator (t-PA), a set of five monoclonal antibodies (Moab) was generated against the Mannose receptor isolated from human placental tissue. All Moab specifically recognised the 175 kDa Mannose receptor in a crude placenta extract, as shown in Western blot analysis. By use of immunohistochemistry, we showed that in human placenta only the Hofbauer cells (fetal macrophages) express the Mannose receptor. Epitope competition experiments indicated that the Moab bound to at least two different epitopes on the receptor molecule. Moab 14-3, 14-5, and 15-2, which are directed against one of these epitopes, strongly inhibited the interaction between the purified Mannose receptor and t-PA. These Moab also inhibited Mannose receptor-mediated degradation of t-PA by cultured human macrophages. The low density lipoprotein receptor-related protein (LRP) mediated t-PA degradation was not affected by the Moab. It is concluded that the Moab are useful for studying the expression of the human Mannose receptor in Western blot and in immunohistochemistry, and for studying the interactions between the human Mannose receptor and the Mannose-containing ligand t-PA. Copyright © 1997 Schattauer Verlag. Chemicals/CAS: Antibodies, Monoclonal; Epitopes; Lectins, C-Type; Mannose receptor; Mannose-Binding Lectins; Receptors, Cell Surface; Tissue Plasminogen Activator, EC 3.4.21.68
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Lysine-based Cluster Mannosides That Inhibit Ligand Binding to the Human Mannose Receptor at Nanomolar Concentration
Journal of Biological Chemistry, 1996Co-Authors: Erik A L Biessen, Femke Noorman, M.m. Barrett-bergshoeff, Marco Van Teijlingen, Martin K Bijsterbosch, Dingeman C. Rijken, Johan Kuiper, Theo J C Van BerkelAbstract:In search of synthetic high affinity ligands for the Mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal α-D-Mannose groups that are connected with the backbone by flexible elongated spacers (16 A). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human Mannose receptor. The affinity of these cluster mannosides for the Mannose receptor was continuously enhanced from 18-23 μM to 0.5-2.6 nM, with Mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional α-D-Mannose group resulted in a 10-fold increase in its affinity for the Mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the Mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the Mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the Mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques. Chemicals/CAS: Biotin, 58-85-5; Lectins; Lectins, C-Type; Lysine, 56-87-1; Mannose receptor; Mannose-Binding Lectins; oligomannoside; Oligosaccharides; Receptors, Cell Surface; ribonuclease B, EC 3.1.27.-; Ribonucleases, EC 3.1.-; Tissue Plasminogen Activator, EC 3.4.21.68
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Isolation and characterization of the Mannose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator
Hepatology, 1992Co-Authors: Marlies Otter, M.m. Barrett-bergshoeff, Petra Žočková, Theo J C Van Berkel, Johan Kuiper, Dingeman C. RijkenAbstract:Various studies have shown that Mannose receptors rapidly eliminate glycoproteins and microorganisms bearing high Mannose–type carbohydrate chains from the blood circulation. The purpose of this study was to characterize the Mannose receptor in the liver, which in vivo is involved in the rapid clearance of tissue-type plasminogen activator from the circulation. Human liver membranes were solubilized in Triton X-100, and the solution was applied to a tissue-type plasminogen activator Sepharose column. Bound proteins were eluted with ethylenediaminetetraacetate (10 mmol/L). A second, similar purification step rendered a single liver protein of 175,000 daltons. A combination of ligand blotting and a chromogenic assay for tissue-type plasminogen activator demonstrated that the identified liver protein is a Mannose receptor because it bound tissue-type plasminogen activator, this tissue-type plasminogen activator binding being fully inhibited by 0.2 mol/L D-Mannose. Western-blot analysis revealed that the isolated liver protein is immunologically identical to the human Mannose receptor from placenta. Treatment of the liver protein and the placenta Mannose receptor with trypsin yielded the same pattern of proteolytic degradation products as identified on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. We conclude that the physiologically relevant Mannose receptor for tissue-type plasminogen activator clearance isolated from human liver is immunologically and structurally similar to or identical with the human Mannose receptor isolated from placenta. (HEPATOLOGY 1992;16:54–59.)
Stéphanie Dupoiron - One of the best experts on this subject based on the ideXlab platform.
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The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A toolbox for sequential plant n-glycan processing
Journal of Biological Chemistry, 2015Co-Authors: Stéphanie Dupoiron, Claudine Zischek, Laetitia Ligat, Julien Carbonne, Alice Boulanger, Thomas Duge De Bernonville, Martine Lautier, Pauline Rival, Matthieu Arlat, Elisabeth JametAbstract:N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three Mannoses, one fucose, and one xylose (N2M3FX) as a substrate. The removal of the α-1,3-Mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-Mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.
Sean Munro - One of the best experts on this subject based on the ideXlab platform.
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the saccharomyces cerevisiae protein mnn10p bed1p is a subunit of a golgi mannosyltransferase complex
Journal of Biological Chemistry, 1999Co-Authors: Joern Jungmann, Julian C Rayner, Sean MunroAbstract:Abstract In the yeast Saccharomyces cerevisiaemany of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large Mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 α-1,6-linked Mannoses to which are attached many branches consisting of α-1,2-linked and α-1,3-linked Mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of theMNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesisin vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the α-1,6-polymerizing activity of the complex.
Angela M. Gronenborn - One of the best experts on this subject based on the ideXlab platform.
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dissecting carbohydrate cyanovirin n binding by structure guided mutagenesis functional implications for viral entry inhibition
Protein Engineering Design & Selection, 2006Co-Authors: Laura G Barrientos, Fatima Lasala, Rafael Delgado, Elena Matei, Angela M. GronenbornAbstract:: The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-Mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-Mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the triMannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the Mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.
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Dissecting carbohydrate–Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition
Protein Engineering Design & Selection, 2006Co-Authors: Laura G Barrientos, Fatima Lasala, Rafael Delgado, Elena Matei, Angela M. GronenbornAbstract:: The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-Mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-Mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the triMannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the Mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.
