235 results on '"Markus Aebi"'
Search Results
2. Glycosylation network mapping and site-specific glycan maturation in vivo
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Marie-Estelle Losfeld, Ernesto Scibona, Chia-wei Lin, and Markus Aebi
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Cell biology ,Integrative aspects of cell biology ,Mathematical biosciences ,Science - Abstract
Summary: Glycoprotein processing along a complex highly compartmentalized pathway is a hallmark of eukaryotic cells. We followed the kinetics of intracellular, site-specific glycan processing of a model protein with five distinct N-glycosylation sites and deduced a mathematical model of the secretory pathway that describes a complex set of processing reactions localized in defined intracellular compartments such as the endoplasmic reticulum the Golgi, or the lysosome. The model was able to accommodate site-specific N-glycan processing and we identified phosphorylated glycan structures of the mannose-6-phosphate pathway responsible for the lysosomal sorting of the glycoprotein. Importantly, our model protein can take different routes of the cellular secretory pathway, resulting in an increased glycan complexity of the secreted protein.
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- 2022
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3. Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies
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Hanne L. P. Tytgat, Chia-wei Lin, Mikail D. Levasseur, Markus B. Tomek, Christoph Rutschmann, Jacqueline Mock, Nora Liebscher, Naohiro Terasaka, Yusuke Azuma, Michael Wetter, Martin F. Bachmann, Donald Hilvert, Markus Aebi, and Timothy G. Keys
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Science - Abstract
Established bacterial glycoengineering platforms limit access to protein and glycan substrates. Here the authors design a cytoplasmic protein glycosylation system, Glycoli, to generate a variety of multivalent glycostructures.
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- 2019
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4. The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.
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Paul Schmid-Hempel, Markus Aebi, Seth Barribeau, Toshihiko Kitajima, Louis du Plessis, Regula Schmid-Hempel, and Stefan Zoller
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Medicine ,Science - Abstract
Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.
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- 2018
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5. Inhibition of Haemonchus contortus larval development by fungal lectins
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Christian Heim, Hubertus Hertzberg, Alex Butschi, Silvia Bleuler-Martinez, Markus Aebi, Peter Deplazes, Markus Künzler, and Saša Štefanić
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Haemonchus contortus ,Fungal lectins ,Nematotoxicity ,Glycan targets ,Vaccine development ,Larval development test (LDT) ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Lectins are carbohydrate-binding proteins that are involved in fundamental intra- and extracellular biological processes. They occur ubiquitously in nature and are especially abundant in plants and fungi. It has been well established that certain higher fungi produce lectins in their fruiting bodies and/or sclerotia as a part of their natural resistance against free-living fungivorous nematodes and other pests. Despite relatively high diversity of the glycan structures in nature, many of the glycans targeted by fungal lectins are conserved among organisms of the same taxon and sometimes even among different taxa. Such conservation of glycans between free-living and parasitic nematodes is providing us with a useful tool for discovery of novel chemotherapeutic and vaccine targets. In our study, a subset of fungal lectins emanating from toxicity screens on Caenorhabditis elegans was tested for their potential to inhibit larval development of Haemonchus contortus. Methods The effect of Coprinopsis cinerea lectins - CCL2, CGL2, CGL3; Aleuria aurantia lectin – AAL; Marasmius oreades agglutinin - MOA; and Laccaria bicolor lectin – Lb-Tec2, on cultivated Haemonchus contortus larval stages was investigated using a larval development test (LDT). To validate the results of the toxicity assay and determine lectin binding capacity to the nematode digestive tract, biotinylated versions of lectins were fed to pre-infective larval stages of H. contortus and visualized by fluorescent microscopy. Lectin histochemistry on fixed adult worms was performed to investigate the presence and localisation of lectin binding sites in the disease-relevant developmental stage. Results Using an improved larval development test we found that four of the six tested lectins: AAL, CCL2, MOA and CGL2, exhibited a dose-dependent toxicity in LDT, as measured by the number of larvae developing to the L3 stage. In the case of AAL, CGL2 and MOA lectin, doses as low as 5 μg/ml caused >95 % inhibition of larval development while 40 μg/ml were needed to achieve the same inhibition by CCL2 lectin. MOA was the only lectin tested that caused larval death while other toxic lectins had larvistatic effect manifesting as L1 growth arrest. Using lectin histochemistry we demonstrate that of all lectins tested, only the four toxic ones displayed binding to the larvae’s gut and likewise were found to interact with glycans localized to the gastrodermal tissue of adults. Conclusion The results of our study suggest a correlation between the presence of target glycans of lectins in the digestive tract and the lectin-mediated toxicity in Haemonchus contortus. We demonstrate that binding to the structurally conserved glycan structures found in H. contortus gastrodermal tissue by the set of fungal lectins has detrimental effect on larval development. Some of these glycan structures might represent antigens which are not exposed to the host immune system (hidden antigens) and thus have a potential for vaccine or drug development. Nematotoxic fungal lectins prove to be a useful tool to identify such targets in parasitic nematodes.
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- 2015
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6. The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering
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Jon Cuccui, Vanessa S. Terra, Janine T. Bossé, Andreas Naegeli, Sherif Abouelhadid, Yanwen Li, Chia-Wei Lin, Prerna Vohra, Alexander W. Tucker, Andrew N. Rycroft, Duncan J. Maskell, Markus Aebi, Paul R. Langford, and Brendan W. Wren
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n-linked glycosylation ,actinobacillus pleuropneumoniae ,adhesion ,Biology (General) ,QH301-705.5 - Abstract
Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.
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- 2017
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7. Parasite Glycobiology: A Bittersweet Symphony.
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Joao A Rodrigues, Alvaro Acosta-Serrano, Markus Aebi, Michael A J Ferguson, Françoise H Routier, Irene Schiller, Simão Soares, Daniel Spencer, Alexander Titz, Iain B H Wilson, and Luis Izquierdo
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2015
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8. Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals.
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Katrin Stutz, Andres Kaech, Markus Aebi, Markus Künzler, and Michael O Hengartner
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Medicine ,Science - Abstract
Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals.
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- 2015
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9. Correction: Plasticity of the β-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System.
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Mario Schubert, Silvia Bleuler-Martinez, Alex Butschi, Martin A. Wälti, Pascal Egloff, Katrin Stutz, Shi Yan, Mayeul Collot, Jean-Maurice Mallet, Iain B. H. Wilson, Michael O. Hengartner, Markus Aebi, Frédéric H.-T. Allain, and Markus Künzler
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Published
- 2012
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10. Plasticity of the β-trefoil protein fold in the recognition and control of invertebrate predators and parasites by a fungal defence system.
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Mario Schubert, Silvia Bleuler-Martinez, Alex Butschi, Martin A Wälti, Pascal Egloff, Katrin Stutz, Shi Yan, Mayeul Collot, Jean-Maurice Mallet, Iain B H Wilson, Michael O Hengartner, Markus Aebi, Frédéric H-T Allain, and Markus Künzler
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity.
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- 2012
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11. Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus.
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Thierry Fontaine, Aurélie Delangle, Catherine Simenel, Bernadette Coddeville, Sandra J van Vliet, Yvette van Kooyk, Silvia Bozza, Silvia Moretti, Flavio Schwarz, Coline Trichot, Markus Aebi, Muriel Delepierre, Carole Elbim, Luigina Romani, and Jean-Paul Latgé
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
A new polysaccharide secreted by the human opportunistic fungal pathogen Aspergillus fumigatus has been characterized. Carbohydrate analysis using specific chemical degradations, mass spectrometry, ¹H and ¹³C nuclear magnetic resonance showed that this polysaccharide is a linear heterogeneous galactosaminogalactan composed of α1-4 linked galactose and α1-4 linked N-acetylgalactosamine residues where both monosacharides are randomly distributed and where the percentage of galactose per chain varied from 15 to 60%. This polysaccharide is antigenic and is recognized by a majority of the human population irrespectively of the occurrence of an Aspergillus infection. GalNAc oligosaccharides are an essential epitope of the galactosaminogalactan that explains the universal antibody reaction due to cross reactivity with other antigenic molecules containing GalNAc stretches such as the N-glycans of Campylobacter jejuni. The galactosaminogalactan has no protective effect during Aspergillus infections. Most importantly, the polysaccharide promotes fungal development in immunocompetent mice due to its immunosuppressive activity associated with disminished neutrophil infiltrates.
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- 2011
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12. Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.
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Alex Butschi, Alexander Titz, Martin A Wälti, Vincent Olieric, Katharina Paschinger, Katharina Nöbauer, Xiaoqiang Guo, Peter H Seeberger, Iain B H Wilson, Markus Aebi, Michael O Hengartner, and Markus Künzler
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.
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- 2010
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13. Structure–function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity
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Silvia Bleuler-Martinez, Annabelle Varrot, Vincent Olieric, Mario Schubert, Eva Vogt, Céline Fetz, Therese Wohlschlager, David Fernando Plaza, Martin Wälti, Yannick Duport, Guido Capitani, Markus Aebi, and Markus Künzler
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Binding Sites ,nematode ,defense ,fucose ,mushroom ,toxin ,Carbohydrates ,Crystallography, X-Ray ,Biochemistry ,Fungal Proteins ,Structure-Activity Relationship ,X-Ray Diffraction ,Lectins ,Scattering, Small Angle ,Animals ,Agaricales ,Caenorhabditis elegans - Abstract
Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes., Glycobiology, 32 (7), ISSN:0959-6658
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- 2022
14. Glycan–protein interactions determine kinetics of N-glycan remodeling
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R. Gregor Weiß, Christoph Giese, Markus Aebi, Sereina Riniker, Marie-Estelle Losfeld, Corina Mathew, Rudi Glockshuber, and Chia-Wei Lin
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chemistry.chemical_classification ,0303 health sciences ,Glycan ,Glycosylation ,010304 chemical physics ,biology ,Chemistry ,Kinetics ,Oligosaccharide ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Biochemistry ,Protein tertiary structure ,Protein–protein interaction ,carbohydrates (lipids) ,03 medical and health sciences ,chemistry.chemical_compound ,Chemistry (miscellaneous) ,0103 physical sciences ,biology.protein ,Protein disulfide-isomerase ,Glycoprotein ,Molecular Biology ,030304 developmental biology - Abstract
A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways.
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- 2021
15. Architecture and function of human uromodulin filaments in urinary tract infections
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Markus Aebi, Dawid Zyla, Rudi Glockshuber, Johannes Trück, Jessica J Stanisich, Olivier Devuyst, Chia-Wei Lin, Maximilian M. Sauer, Jonathan Eras, Martin Pilhofer, Gregor L. Weiss, University of Zurich, UCL - SSS/IREC/NEFR - Pôle de Néphrologie, and UCL - (SLuc) Service de néphrologie
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0301 basic medicine ,Glycosylation ,Tamm–Horsfall protein ,610 Medicine & health ,macromolecular substances ,030204 cardiovascular system & hematology ,Biology ,Ligands ,Pilus ,Epitope ,10052 Institute of Physiology ,Protein filament ,03 medical and health sciences ,0302 clinical medicine ,Uromodulin ,Humans ,Adhesins, Bacterial ,1000 Multidisciplinary ,Multidisciplinary ,Cryoelectron Microscopy ,Adhesion ,In vitro ,3. Good health ,Cell biology ,Bacterial adhesin ,030104 developmental biology ,Urinary Tract Infections ,biology.protein ,570 Life sciences ,biology ,Function (biology) - Abstract
Uromodulin is the most abundant protein in human urine, and it forms filaments that antagonize the adhesion of uropathogens; however, the filament structure and mechanism of protection remain poorly understood. We used cryo–electron tomography to show that the uromodulin filament consists of a zigzag-shaped backbone with laterally protruding arms. N-glycosylation mapping and biophysical assays revealed that uromodulin acts as a multivalent ligand for the bacterial type 1 pilus adhesin, presenting specific epitopes on the regularly spaced arms. Imaging of uromodulin-uropathogen interactions in vitro and in patient urine showed that uromodulin filaments associate with uropathogens and mediate bacterial aggregation, which likely prevents adhesion and allows clearance by micturition. These results provide a framework for understanding uromodulin in urinary tract infections and in its more enigmatic roles in physiology and disease. ISSN:0036-8075 ISSN:1095-9203
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- 2020
16. Characterization of the single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei using synthetic peptides and lipid-linked oligosaccharide analogs
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Ana S. Ramírez, Bee Ha Gan, Kaspar P. Locher, Jérémy Boilevin, Tamis Darbre, Daniel Janser, Markus Aebi, Jean-Louis Reymond, and Rasomoy Biswas
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0301 basic medicine ,Lipopolysaccharides ,Glycan ,Stereochemistry ,Trypanosoma brucei brucei ,Protozoan Proteins ,N-glycosylation ,Chitobiose ,Trypanosoma brucei ,Disaccharides ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,N-linked glycosylation ,enzyme kinetics ,540 Chemistry ,Chemical Biology ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Oligosaccharyltransferase ,Membrane Proteins ,Sequon ,oligosaccharyltransferase ,Oligosaccharide ,biology.organism_classification ,Original articles ,lipid-linked oligosaccharide ,Enzyme kinetics ,Lipid-linked oligosaccharide ,030104 developmental biology ,chemistry ,Hexosyltransferases ,Membrane protein complex ,biology.protein ,570 Life sciences ,Peptides - Abstract
The initial transfer of a complex glycan in protein N-glycosylation is catalyzed by oligosaccharyltransferase (OST), which is generally a multisubunit membrane protein complex in the endoplasmic reticulum but a single-subunit enzyme (ssOST) in some protists. To investigate the reaction mechanism of ssOST, we recombinantly expressed, purified and characterized the STT3A protein from Trypanosoma brucei (TbSTT3A). We analyzed the in vitro activity of TbSTT3A by synthesizing fluorescently labeled acceptor peptides as well as lipid-linked oligosaccharide (LLO) analogs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25) and with different double bond stereochemistry. We found that in addition to proline, charged residues at the +1 position of the sequon inhibited glycan transfer. An acidic residue at the −2 position significantly increased catalytic turnover but was not essential, in contrast to the bacterial OST. While all synthetic LLO analogs were processed by TbSTT3A, the length of the polyprenyl tail, but not the stereochemistry of the double bonds, determined their apparent affinity. We also synthesized phosphonate analogs of the LLOs, which were found to be competitive inhibitors of the reaction, although with lower apparent affinity to TbSTT3A than the active pyrophosphate analogs., Glycobiology, 27 (6), ISSN:0959-6658
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- 2021
17. N-Glycosylation Enhances Conformational Flexibility of Protein Disulfide Isomerase Revealed by Microsecond Molecular Dynamics and Markov State Modeling
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Markus Aebi, Sereina Riniker, Marie-Estelle Losfeld, and R. Gregor Weiß
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chemistry.chemical_classification ,Glycan ,Glycosylation ,biology ,Protein dynamics ,Protein Disulfide-Isomerases ,Protein superfamily ,Molecular Dynamics Simulation ,Surfaces, Coatings and Films ,carbohydrates (lipids) ,chemistry.chemical_compound ,chemistry ,N-linked glycosylation ,Chaperone (protein) ,Materials Chemistry ,biology.protein ,Biophysics ,Humans ,Physical and Theoretical Chemistry ,Glycoprotein ,Protein disulfide-isomerase - Abstract
Secreted proteins of eukaryotes are decorated with branched carbohydrate oligomers called glycans. This fact is only starting to be considered for in silico investigations of protein dynamics. Using all-atom molecular dynamics (MD) simulations and Markov state modeling (MSM), we unveil the influence of glycans on the conformational flexibility of the multidomain protein disulfide isomerase (PDI), which is a ubiquitous chaperone in the endoplasmic reticulum (ER). Yeast PDI (yPDI) from Saccharomyces cerevisiae is glycosylated at asparagine side chains and the knowledge of its five modified sites enables a realistic computational modeling. We compare simulations of glycosylated and unglycosylated yPDI and find that the presence of glycan-glycan and glycan-protein interactions influences the flexibility of PDI in different ways. For example, glycosylation reduces interdomain interactions, shifting the conformational ensemble toward more open, extended structures. In addition, we compare our results on yPDI with structural information of homologous proteins such as human PDI (hPDI), which is natively unglycosylated. Interestingly, hPDI lacks a surface recess that is present in yPDI. We find that glycosylation of yPDI facilitates its catalytic site to reach close to this surface recess. Hence, this might point to a possible functional relevance of glycosylation in yeast to act on substrates, while glycosylation seems redundant for the human homologous protein. We conclude that glycosylation is fundamental for protein dynamics, making it a necessity for a truthful representation of the flexibility and function in in silico studies of glycoproteins.
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- 2021
18. Prion-inducing domain 2–114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments
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CHIH-YEN KING, PETER TITTMANN, HEINZ GROSS, ROLAND GEBERT, MARKUS AEBI, and KURT WÜTHRICH
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- 2021
19. Glycan-protein interactions determine kinetics of
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Corina, Mathew, R Gregor, Weiß, Christoph, Giese, Chia-Wei, Lin, Marie-Estelle, Losfeld, Rudi, Glockshuber, Sereina, Riniker, and Markus, Aebi
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carbohydrates (lipids) ,Chemistry - Abstract
A hallmark of N-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemical in vitro analysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of the N-glycans for their processing enzymes derived from the MD simulations, N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure of the glycoprotein PDI affected its N-glycan remodeling in a site-specific way. We propose that the observed differential N-glycan reactivities depend on the surrounding protein tertiary structure and lead to different glycan structures in the same protein through kinetically controlled processing pathways., Atomistic glycoprotein simulations reveal a site-specific availability of glycan substrates in time-resolved mass spectrometry of maturating enzyme kinetics.
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- 2021
20. Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases
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Neuhaus Jd, Jillianne Eyring, Chia-Wei Lin, Kaspar P. Locher, Markus Aebi, Rossitza N. Irobalieva, Rebekka Wild, Julia Kowal, Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), University of Zurich, Eyring, Jillianne, and Aebi, Markus
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1303 Biochemistry ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Protein subunit ,thioredoxin domain ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,cryo-electron microscopy ,Saccharomyces cerevisiae ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,oligosaccharyltransferase complex ,Asparagine ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,substrate recognition ,030302 biochemistry & molecular biology ,Oligosaccharyltransferase ,fungi ,Cryoelectron Microscopy ,Membrane Proteins ,Oligosaccharide ,peptide binding ,Transmembrane domain ,Enzyme ,chemistry ,Hexosyltransferases ,570 Life sciences ,biology ,Thioredoxin - Abstract
The oligosaccharyltransferase (OST) is the central enzyme in theN-glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analysed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzymein vitroand the more severe hypoglycosylation in Ost3p lacking strainsin vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.
- Published
- 2021
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21. Glycan-Protein Interactions Determine Kinetics ofN-Glycan Remodeling
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Markus Aebi, Sereina Riniker, C. Giese, G. Weiss, C. Mathew, Marie-Estelle Losfeld, Rudi Glockshuber, and Chia-Wei Lin
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chemistry.chemical_classification ,Glycan ,Glycosylation ,biology ,Chemistry ,Kinetics ,Context (language use) ,Oligosaccharide ,Protein tertiary structure ,Protein–protein interaction ,carbohydrates (lipids) ,chemistry.chemical_compound ,Biochemistry ,biology.protein ,Protein disulfide-isomerase - Abstract
A hallmark ofN-linked glycosylation in the secretory compartments of eukaryotic cells is the sequential remodeling of an initially uniform oligosaccharide to a site-specific, heterogeneous ensemble of glycostructures on mature proteins. To understand site-specific processing, we used protein disulfide isomerase (PDI), a model protein with five glycosylation sites, for molecular dynamics (MD) simulations and compared the result to a biochemicalin vitroanalysis with four different glycan processing enzymes. As predicted by an analysis of the accessibility of theN-glycans for their processing enzymes derived from the MD simulations,N-glycans at different glycosylation sites showed different kinetic properties for the processing enzymes. In addition, altering the tertiary structure context ofN-glycan substrates affectedN-glycan remodeling in a site-specific way. We propose that differential, tertiary structure context dependentN-glycan reactivities lead to different glycan structures in the same protein through kinetically controlled processing pathways.
- Published
- 2020
22. Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms
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Jillianne, Eyring, Chia-Wei, Lin, Elsy Mankah, Ngwa, Jérémy, Boilevin, Giorgio, Pesciullesi, Kaspar P, Locher, Tamis, Darbre, Jean-Louis, Reymond, and Markus, Aebi
- Subjects
Saccharomyces cerevisiae Proteins ,glycosylation ,FDR, false discovery rate ,TAMRA, tetramethylrhodamine ,DDM, n-dodecyl-β-D-maltopyranoside ,Saccharomyces cerevisiae ,carbohydrate structure ,glycosylation inhibitor ,yeast ,Substrate Specificity ,glycoprotein biosynthesis ,XIC, extracted ion chromatography ,ER, endoplasmic reticulum ,enzyme kinetics ,Protein Isoforms ,OST, oligosaccharyltransferase ,FA, formic acid ,OST ,protein complex ,LLO, lipid-linked oligosaccharide ,Membrane Proteins ,oligosaccharyltransferase ,ACN, acetonitrile ,Kinetics ,Hexosyltransferases ,HCD, higher-energy collisional dissociation ,CHS, cholesteryl hemisuccinate ,membrane enzyme ,Research Article - Abstract
Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.
- Published
- 2020
23. Structure and mechanism of the ER-based glucosyltransferase ALG6
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Tamis Darbre, Markus Aebi, Jean-Louis Reymond, Kamil Nosol, Giorgio Pesciullesi, Rossitza N. Irobalieva, Kaspar P. Locher, Jérémy Boilevin, Anthony A. Kossiakoff, and Joël S. Bloch
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Models, Molecular ,Glycan ,Saccharomyces cerevisiae Proteins ,Mannose ,Saccharomyces cerevisiae ,In Vitro Techniques ,010402 general chemistry ,Endoplasmic Reticulum ,01 natural sciences ,Article ,Conserved sequence ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Catalytic Domain ,Glycosyltransferase ,540 Chemistry ,Conserved Sequence ,030304 developmental biology ,Dolichol Phosphates ,0303 health sciences ,Polyisoprenyl Phosphate Monosaccharides ,Multidisciplinary ,biology ,Endoplasmic reticulum ,Cryoelectron Microscopy ,Active site ,Glycosyltransferases ,Membrane Proteins ,500 Science ,Lipids ,Transmembrane protein ,0104 chemical sciences ,Glucose ,Biochemistry ,chemistry ,Mutation ,biology.protein ,Biocatalysis ,570 Life sciences ,Glucosyltransferase ,Dolichol Monophosphate Mannose ,Protein Binding - Abstract
In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes—ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10—are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0 A resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9 A resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms. Analyses reveal a previously undescribed transmembrane protein fold in the endoplasmic reticulum-based glucosyltransferase ALG6 and provide a structural basis for understanding the glucose transfer mechanism.
- Published
- 2020
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- View/download PDF
24. Mechanistic reconstruction of glycoprotein secretion through monitoring of intracellular N-glycan processing
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Hervé Broly, Chia-Wei Lin, Ernesto Scibona, Jonathan Souquet, Markus Aebi, Ilaria Arigoni-Affolter, and David Brühlmann
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Glycosylation ,CHO Cells ,01 natural sciences ,03 medical and health sciences ,chemistry.chemical_compound ,Cricetulus ,N-glycan processing ,Biosynthesis ,Polysaccharides ,Animals ,Research Articles ,Secretory pathway ,Glycoproteins ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Chemistry ,Chinese hamster ovary cell ,010401 analytical chemistry ,SciAdv r-articles ,Cell Biology ,0104 chemical sciences ,Glycoproteomics ,Cell biology ,carbohydrates (lipids) ,Carbohydrate Metabolism ,Glycoprotein ,Intracellular ,Research Article - Abstract
N-linked glycosylation plays a fundamental role in determining the thermodynamic stability of proteins and is involved in multiple key biological processes. The mechanistic understanding of the intracellular machinery responsible for the stepwise biosynthesis of N-glycans is still incomplete due to limited understanding of in vivo kinetics of N-glycan processing along the secretory pathway. We present a glycoproteomics approach to monitor the processing of site-specific N-glycans in CHO cells. On the basis of a model-based analysis of structure-specific turnover rates, we provide a kinetic description of intracellular N-glycan processing along the entire secretory pathway. This approach refines and further extends the current knowledge on N-glycans biosynthesis and provides a basis to quantify alterations in the glycoprotein processing machinery., Science Advances, 5 (11), ISSN:2375-2548
- Published
- 2019
25. Quantitative Profiling of N-linked Glycosylation Machinery in Yeast Saccharomyces cerevisiae
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Marie-Estelle Losfeld, Markus Aebi, Nathalie Selevsek, Elsy Mankah Ngwa, Kristina Poljak, and Jonas Grossmann
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0301 basic medicine ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Protein subunit ,Saccharomyces cerevisiae ,Proteomics ,Biochemistry ,Mass Spectrometry ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,N-linked glycosylation ,Stable isotope labeling by amino acids in cell culture ,Lipid biosynthesis ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Research ,Oligosaccharyltransferase ,Sequon ,030104 developmental biology ,chemistry ,Isotope Labeling ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Asparagine ,Protein Processing, Post-Translational - Abstract
Asparagine-linked glycosylation is a common posttranslational protein modification regulating the structure, stability and function of many proteins. The N-linked glycosylation machinery involves enzymes responsible for the assembly of the lipid-linked oligosaccharide (LLO), which is then transferred to the asparagine residues on the polypeptides by the enzyme oligosaccharyltransferase (OST). A major goal in the study of protein glycosylation is to establish quantitative methods for the analysis of site-specific extent of glycosylation. We developed a sensitive approach to examine glycosylation site occupancy in Saccharomyces cerevisiae by coupling stable isotope labeling (SILAC) approach to parallel reaction monitoring (PRM) mass spectrometry (MS). We combined the method with genetic tools and validated the approach with the identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of OST. Based on the observations that alternations in LLO substrate structure and OST subunits activity differentially alter the systemic output of OST, we conclude that sequon recognition is a direct property of the catalytic subunit Stt3p, auxiliary subunits such as Ost3p and Ost6p extend the OST substrate range by modulating interfering pathways such as protein folding. In addition, our proteomics approach revealed a novel regulatory network that connects isoprenoid lipid biosynthesis and LLO substrate assembly.
- Published
- 2018
26. Analysis of substrate specificity of Trypanosoma brucei oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeast
- Author
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Mauro Pellanda, George Rugarabamu, Markus Aebi, Robert Gauss, Kristina Poljak, Jörg Breitling, University of Zurich, and Aebi, Markus
- Subjects
0301 basic medicine ,Glycan ,1303 Biochemistry ,Saccharomyces cerevisiae Proteins ,Trypanosoma brucei brucei ,Saccharomyces cerevisiae ,Glycobiology and Extracellular Matrices ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,Trypanosoma brucei ,Biochemistry ,Substrate Specificity ,1307 Cell Biology ,03 medical and health sciences ,Protein Domains ,N-linked glycosylation ,1312 Molecular Biology ,Asparagine ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chimera ,Chemistry ,fungi ,Oligosaccharyltransferase ,Membrane Proteins ,Cell Biology ,biology.organism_classification ,Glycoproteomics ,030104 developmental biology ,Hexosyltransferases ,biology.protein ,570 Life sciences ,Heterologous expression - Abstract
N-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single-protein OSTs called TbSTT3A, TbSTT3B, and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST, making it an ideal experimental system to study the fundamental properties of OST activity. We characterized the LLO and polypeptide specificity of all three TbOST isoforms and their chimeric forms in the heterologous expression host S. cerevisiae where we were able to apply yeast genetic tools and newly developed glycoproteomics methods. We demonstrated that TbSTT3A accepted LLO substrates ranging from Man5GlcNAc2 to Man7GlcNAc2. In contrast, TbSTT3B required more complex precursors ranging from Man6GlcNAc2 to Glc3Man9GlcNAc2 structures, and TbSTT3C did not display any LLO preference. Sequence differences between the isoforms cluster in three distinct regions. We have swapped the individual regions between different OST proteins and identified region 2 to influence the specificity toward the LLO and region 1 to influence polypeptide substrate specificity. These results provide a basis to further investigate the molecular mechanisms and contribution of single amino acids in OST interaction with its substrates.
- Published
- 2017
27. Engineering protein glycosylation in prokaryotes
- Author
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Timothy G. Keys and Markus Aebi
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Glycan ,Glycosylation ,Glycoprotein synthesis ,Applied Mathematics ,030106 microbiology ,Oligosaccharyltransferase ,Protein engineering ,Biology ,biology.organism_classification ,Campylobacter jejuni ,General Biochemistry, Genetics and Molecular Biology ,3. Good health ,Computer Science Applications ,carbohydrates (lipids) ,03 medical and health sciences ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Modeling and Simulation ,Drug Discovery ,biology.protein ,Glycoprotein ,Function (biology) - Abstract
Controlling the specific glycan structure(s) present on a glycoprotein is a key challenge for both understanding its function and for developing effective next generation medical reagents. A decade ago the first engineered glycoproteins were produced in bacteria using the N-glycosylation machinery of Campylobacter jejuni . From the extensive development of this system, we learn the requisite features of a successful glycoengineering platform, as well as the factors that limit application in both glycan and protein structural space. From this perspective we review recent developments in the field of prokaryotic protein glycosylation. We highlight the emergence of cytoplasmic glycosylation systems as likely candidates to fuel the next wave of targeted glycoprotein synthesis with applications in research and medicine.
- Published
- 2017
28. Influence of protein/glycan interaction on site‐specific glycan heterogeneity
- Author
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Massimo Morbidelli, Ernesto Scibona, Markus Aebi, Chia-Wei Lin, Robert Gauss, Thomas K. Villiger, and Marie-Estelle Losfeld
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0301 basic medicine ,Glycan ,Glycosylation ,Golgi Apparatus ,Oligosaccharides ,CHO Cells ,Endoplasmic Reticulum ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,symbols.namesake ,Cricetulus ,Polysaccharides ,Genetics ,Animals ,Protein disulfide-isomerase ,Molecular Biology ,Glycoproteins ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Glycobiology ,Chinese hamster ovary cell ,Endoplasmic reticulum ,Golgi apparatus ,Recombinant Proteins ,carbohydrates (lipids) ,030104 developmental biology ,biology.protein ,symbols ,Glycoprotein ,Biotechnology - Abstract
To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity.
- Published
- 2017
29. Chemo-enzymatic synthesis of lipid-linked GlcNAc2Man5 oligosaccharides using recombinant Alg1, Alg2 and Alg11 proteins
- Author
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Ana S. Ramírez, Daniel Janser, Kaspar P. Locher, Jérémy Boilevin, Tamis Darbre, Bee Ha Gan, Markus Aebi, Chia-Wei Lin, and Jean-Louis Reymond
- Subjects
0301 basic medicine ,mannosylation ,Mannose ,N-glycans ,Trypanosoma brucei ,Chitobiose ,Biochemistry ,law.invention ,mannosyltransferase ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,law ,540 Chemistry ,Chemical Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Endoplasmic reticulum ,Oligosaccharyltransferase ,Original articles ,biology.organism_classification ,lipid-linked oligosaccharide ,030104 developmental biology ,Cytoplasm ,Recombinant DNA ,570 Life sciences ,lipids (amino acids, peptides, and proteins) - Abstract
The biosynthesis of eukaryotic lipid-linked oligosaccharides (LLOs) that act as donor substrates in eukaryotic protein N-glycosylation starts on the cytoplasmic side of the endoplasmic reticulum and includes the sequential addition of five mannose units to dolichol-pyrophosphate-GlcNAc2. These reactions are catalyzed by the Alg1, Alg2 and Alg11 gene products and yield Dol-PP-GlcNAc2Man5, an LLO intermediate that is subsequently flipped to the lumen of the endoplasmic reticulum. While the purification of active Alg1 has previously been described, Alg11 and Alg2 have been mostly studied in vivo. We here describe the expression and purification of functional, full length Alg2 protein. Along with the purified soluble domains Alg1 and Alg11, we used Alg2 to chemo-enzymatically generate Dol-PP-GlcNAc2Man5 analogs starting from synthetic LLOs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25). We found that while the addition of the first mannose unit by Alg1 was successful with all of the LLO molecules, the Alg2-catalyzed reaction was only efficient if the acceptor LLOs contained a sufficiently long lipid tail of four or five isoprenyl units (C20 and C25). Following conversion with Alg11, the resulting C20 or C25 -containing GlcNAc2Man5 LLO analogs were successfully used as donor substrates of purified single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei. Our results provide a chemo-enzymatic method for the generation of eukaryotic LLO analogs and are the basis of subsequent mechanistic studies of the enigmatic Alg2 reaction mechanism.
- Published
- 2017
30. Cytoplasmic glycoengineering enables biosynthesis of nanoscale glycoprotein assemblies
- Author
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Martin F. Bachmann, Markus B. Tomek, Christoph Rutschmann, Yusuke Azuma, Naohiro Terasaka, Markus Aebi, Chia-Wei Lin, Donald Hilvert, Jacqueline Mock, Hanne L. P. Tytgat, Michael Wetter, Mikail D. Levasseur, Nora Liebscher, and Timothy G. Keys
- Subjects
0301 basic medicine ,Cytoplasm ,Glycosylation ,Glycobiology ,General Physics and Astronomy ,01 natural sciences ,Epitopes ,Synthetic biology ,chemistry.chemical_compound ,Microbiologie ,Promoter Regions, Genetic ,lcsh:Science ,610 Medicine & health ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Chemistry ,Monosaccharides ,Recombinant Proteins ,Cell biology ,Metabolic Engineering ,Glucosyltransferases ,Glycan ,Science ,Green Fluorescent Proteins ,010402 general chemistry ,Microbiology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Polysaccharides ,Glycosyltransferase ,Escherichia coli ,Life Science ,Glycoproteins ,General Chemistry ,Periplasmic space ,Benzazepines ,Nanostructures ,0104 chemical sciences ,carbohydrates (lipids) ,Glucose ,030104 developmental biology ,biology.protein ,lcsh:Q ,Glycoprotein - Abstract
Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications., Nature Communications, 10 (1), ISSN:2041-1723
- Published
- 2019
31. Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC–ESI-MS/MS Analysis
- Author
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Markus Aebi, Chia-Wei Lin, Micha A. Haeuptle, University of Zurich, and Aebi, Markus
- Subjects
0301 basic medicine ,Spectrometry, Mass, Electrospray Ionization ,Glycan ,Glycosylation ,Resolution (mass spectrometry) ,Glycoconjugate ,Electrospray ionization ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Nanotechnology ,Monosaccharide ,Benzyl Alcohols ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,1602 Analytical Chemistry ,Chromatography ,biology ,Glycopeptides ,Glycopeptide ,Molecular Weight ,030104 developmental biology ,chemistry ,Reagent ,biology.protein ,570 Life sciences - Abstract
Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.
- Published
- 2016
32. Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeonHaloferax volcanii
- Author
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Markus Aebi, Chia-Wei Lin, Yann Guérardel, Jerry Eichler, Lina Kandiba, Ben-Gurion University of the Negev (BGU), Institute of Microbiology [Zurich], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), University of Zurich, Eichler, Jerry, and Université de Lille-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,N-linked glycosylation ,Glycan ,Glycosylation ,1303 Biochemistry ,Protein Conformation ,Oligosaccharides ,Mannose ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,Biochemistry ,Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Haloferax volcanii ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Membrane Glycoproteins ,biology ,S-layer glycoprotein ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,Archaea ,[CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry ,nuclear magnetic resonance ,030104 developmental biology ,chemistry ,biology.protein ,570 Life sciences ,Glycoprotein - Abstract
N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.
- Published
- 2016
33. Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae *
- Author
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Ewa Zatorska, Sabine Strahl, Henrik Clausen, Martin Loibl, Markus Aebi, Adnan Halim, Ida Signe Bohse Larsen, Joan Castells-Ballester, Martin Zauser, Andreas Essig, Hiren J. Joshi, and Patrick Neubert
- Subjects
0301 basic medicine ,Models, Molecular ,Proteomics ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Protein domain ,Saccharomyces cerevisiae ,Mannose ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Protein Structure, Secondary ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Molecular Biology ,Secretory pathway ,Glycoproteins ,chemistry.chemical_classification ,Binding Sites ,Research ,biology.organism_classification ,carbohydrates (lipids) ,030104 developmental biology ,chemistry ,Membrane protein ,Glycoprotein - Abstract
O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals., Molecular & Cellular Proteomics, 15 (4), ISSN:1535-9476, ISSN:1535-9484
- Published
- 2016
34. Precisely heterogeneous ‐ the making of N‐glycoproteins
- Author
-
Markus Aebi
- Subjects
Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2018
35. SnapShot: O-Glycosylation Pathways across Kingdoms
- Author
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Markus Aebi, Katrine T. Schjoldager, Hanne L. P. Tytgat, Henrik Clausen, Adnan Halim, Hiren J. Joshi, and Yoshiki Narimatsu
- Subjects
0301 basic medicine ,Glycosylation ,Bacteria ,Nematoda ,Fungi ,Computational biology ,Biology ,Plants ,General Biochemistry, Genetics and Molecular Biology ,carbohydrates (lipids) ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Post translational ,Protein processing ,Vertebrates ,Snapshot (computer storage) ,Animals ,Drosophila ,Protein Processing, Post-Translational - Abstract
O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of O-glycans found in different organisms and their principle biosynthetic pathways. To view this SnapShot, open or download the PDF.
- Published
- 2018
36. Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria
- Author
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Annageldi Tayyrov, Andreas Essig, Anja Kombrink, Pauli T. Kallio, Natalia Dürig, John Hintze, Sebastian Micheller, Markus Aebi, Yasemin van Heuvel, Markus Künzler, Chia-Wei Lin, and Martina Stöckli
- Subjects
Proteomics ,Bacillus subtilis ,Fungus ,medicine.disease_cause ,Microbiology ,Article ,Defensins ,Fungal Proteins ,03 medical and health sciences ,Fungal biology ,Antibiotics ,medicine ,Escherichia coli ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,0303 health sciences ,Mushroom ,biology ,030306 microbiology ,Effector ,fungi ,biology.organism_classification ,Anti-Bacterial Agents ,Coprinopsis cinerea ,Microbial Interactions ,Muramidase ,Antibacterial activity ,Agaricales ,Peptides ,Transcriptome ,Bacteria - Abstract
Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.
- Published
- 2018
37. Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation
- Author
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Julia Kowal, Jillianne Eyring, Elsy Mankah Ngwa, Kaspar P. Locher, Rebekka Wild, and Markus Aebi
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0301 basic medicine ,Multidisciplinary ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Stereochemistry ,Protein subunit ,Endoplasmic reticulum ,Oligosaccharyltransferase ,Cryoelectron Microscopy ,Membrane Proteins ,Translocon ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,N-linked glycosylation ,chemistry ,Oligosaccharyltransferase complex ,Hexosyltransferases ,Membrane protein complex ,Multienzyme Complexes ,Catalytic Domain ,Oxidation-Reduction ,Conserved Sequence - Abstract
Science, 359 (6375), ISSN:0036-8075, ISSN:1095-9203
- Published
- 2017
38. Glycosylation profiles determine extravasation and disease-targeting properties of armed antibodies
- Author
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Christian Hess, Markus Aebi, Chia-Wei Lin, Dario Neri, and Dario Venetz
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0303 health sciences ,Glycan ,Biodistribution ,Multidisciplinary ,Glycosylation ,biology ,Chemistry ,Disease ,Biological Sciences ,Extravasation ,3. Good health ,law.invention ,carbohydrates (lipids) ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Biochemistry ,law ,030220 oncology & carcinogenesis ,Recombinant DNA ,biology.protein ,Interleukin 9 ,Antibody ,030304 developmental biology - Abstract
Significance Therapeutic antibodies represent the largest and fastest growing class of biopharmaceuticals. There is a trend in moving from intact antibodies toward “armed” antibody products, in which the antibody moiety serves as pharmacodelivery vehicle. The impact of glycosylation on the targeting performance of armed antibodies is still largely unknown. Our article sheds light on the surprising finding that relatively small variations in glycostructures and sialic acid content can have dramatic effects on therapeutic agent performance. A better understanding of the impact of glycosylation on pharmaceutical activity is likely to be relevant not only for future antibody development activities, but also for changes in current manufacturing processes and for the development of biosimilar products.
- Published
- 2015
39. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins
- Author
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Sathish Kumar Lakshmipathy, Robert Gauss, Ernst Jarosch, Markus Aebi, Martin Mehnert, Thomas Sommer, Maren Berger, and Franziska Sommermeyer
- Subjects
Models, Molecular ,Protein Folding ,Cancer Research ,Saccharomyces cerevisiae Proteins ,Biosynthesis and Biodegradation ,Immunoblotting ,Saccharomyces cerevisiae ,Protein degradation ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Fungal Proteins ,03 medical and health sciences ,0302 clinical medicine ,HSP70 Heat-Shock Proteins ,Molecular Biology ,Secretory pathway ,030304 developmental biology ,0303 health sciences ,Membrane Glycoproteins ,biology ,Endoplasmic reticulum ,Membrane Proteins ,Articles ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,HSP40 Heat-Shock Proteins ,Protein Structure, Tertiary ,Cell biology ,Secretory protein ,Membrane protein ,Chaperone (protein) ,Mutation ,Unfolded Protein Response ,biology.protein ,Unfolded protein response ,030217 neurology & neurosurgery ,Molecular Chaperones ,Protein Binding - Abstract
Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1., Molecular Biology of the Cell, 26 (2), ISSN:1939-4586, ISSN:1059-1524
- Published
- 2015
- Full Text
- View/download PDF
40. Posttranslational modifications of intact proteins detected by NMR spectroscopy: application to glycosylation
- Author
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Markus Aebi, Michal J. Walczak, Mario Schubert, and Gerhard Wider
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Glycan ,Glycosylation ,Chemical structure ,010402 general chemistry ,Proteomics ,01 natural sciences ,Catalysis ,Glycomics ,03 medical and health sciences ,chemistry.chemical_compound ,Polysaccharides ,Nuclear Magnetic Resonance, Biomolecular ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Proteins ,General Medicine ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,0104 chemical sciences ,Structure and function ,Carbohydrate Sequence ,chemistry ,Biochemistry ,biology.protein ,Glycoprotein ,Protein Processing, Post-Translational - Abstract
Posttranslational modifications (PTMs) are an integral part of the majority of proteins. The characterization of structure and function of PTMs can be very challenging especially for glycans. Existing methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications, the inability to identify linkage types and thus chemical structure. We present a direct, robust, and simple NMR spectroscopy method for the detection and identification of PTMs in proteins. No isotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approach is well suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.
- Published
- 2015
- Full Text
- View/download PDF
41. An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast
- Author
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Alexander D. Frey and Markus Aebi
- Subjects
Glycosylation ,Saccharomyces cerevisiae Proteins ,animal structures ,Acid Phosphatase ,ta220 ,Saccharomyces cerevisiae ,macromolecular substances ,Biochemistry ,03 medical and health sciences ,N-linked glycosylation ,Lipid-linked oligosaccharide ,Oligosaccharyltransferase ,Protein N-glycosylation ,Reporter assay ,Yeast acid phosphatase ,Protein-fragment complementation assay ,Secretion ,yeast acid phosphatase ,protein N-glycosylation ,ta216 ,ta215 ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Reporter gene ,biology ,reporter assay ,030302 biochemistry & molecular biology ,Acid phosphatase ,oligosaccharyltransferase ,Yeast ,High-Throughput Screening Assays ,3. Good health ,lipid-linked oligosaccharide ,carbohydrates (lipids) ,Enzyme ,chemistry ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Protein Processing, Post-Translational - Abstract
N-Glycosylation efficiency is a key parameter when studying components of the protein N-glycosylation pathway, but was recently also recognized as an important factor in the production of glycosylated proteins. We have developed a novel assay to quantify N-glycosylation efficiency of proteins. This assay is based on the secreted activity of yeast acid phosphatase, the proper folding and hence secretion of which is strongly dependent on its N-glycosylation status. The results show that the reporter yields a quantitative measure for protein N-glycosylation in yeast, which is in good agreement with classically used assay based on protein migration patterns on SDS-PAGE. However, the assay is less laborious and is adaptable to high-throughput screening approaches as exemplified.
- Published
- 2015
- Full Text
- View/download PDF
42. Copsin, a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis
- Author
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Andreas Essig, Daniela Hofmann, Pauli T. Kallio, Markus Aebi, Gerhard Wider, Markus Künzler, Daniela Münch, Savitha Gayathri, Hans-Georg Sahl, and Tanja Schneider
- Subjects
Models, Molecular ,Protein Conformation ,Molecular Sequence Data ,Antimicrobial peptides ,Peptidoglycan ,Biology ,Microbiology ,Biochemistry ,Defensins ,Fungal Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Amino Acid Sequence ,Molecular Biology ,Defensin ,030304 developmental biology ,0303 health sciences ,Fungal protein ,Bacteria ,Lipid II ,030306 microbiology ,fungi ,Copsin ,Cell Biology ,Lantibiotics ,biology.organism_classification ,Coculture Techniques ,Anti-Bacterial Agents ,Coprinopsis cinerea ,chemistry ,Agaricales - Abstract
Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.
- Published
- 2014
43. Glycomimicry: Display of the GM3 sugar epitope on Escherichia coli and Salmonella enterica sv Typhimurium
- Author
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Karin Ilg, Elif Yavuz, Carola Maffioli, Markus Aebi, and Bernard Priem
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DNA, Bacterial ,Lipopolysaccharides ,Salmonella typhimurium ,Glycoengineering ,Neisseria meningitidis ,medicine.disease_cause ,Biochemistry ,Epitope ,Microbiology ,Bacterial genetics ,chemistry.chemical_compound ,Epitopes ,Gangliosides ,medicine ,Escherichia coli ,G(M3) Ganglioside ,Bacterial glycosylation ,Chimeric LPS ,Galactosyltransferase ,N-Acylneuraminate Cytidylyltransferase ,biology ,Chemistry ,Oxo-Acid-Lyases ,Salmonella enterica ,Pathogenic bacteria ,biology.organism_classification ,N-Acetylneuraminic Acid ,Neisseria gonorrhoeae ,Sialic acid ,carbohydrates (lipids) ,Lipid A ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Bacteria - Abstract
Glycobiology, 20 (10), ISSN:0959-6658
- Published
- 2017
44. Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro
- Author
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Markus Aebi, Anne Grundschober, Florian M. Freimoser, and Urs Tuor
- Subjects
Hyphal growth ,Insecta ,food.ingredient ,sporulation ,Nitrogen ,Hyphae ,Fungus ,Entomophthora ,In Vitro Techniques ,hyphal growth ,Microbiology ,CMM, conditioned medium from a culture that had formed mycelium ,food ,entomophthorales ,Sporogenesis ,Genetics ,Animals ,Molecular Biology ,zygomycetes ,Mycelium ,biology ,Obligate ,fungi ,nitrogen limitation ,differentiation ,Spores, Fungal ,Pathogenic fungus ,biology.organism_classification ,Spore ,FBS, foetal bovine serum - Abstract
FEMS Microbiology Letters, 222 (2), ISSN:0378-1097, ISSN:1574-6968, ISSN:0168-6496, ISSN:0920-8534, ISSN:0168-6445
- Published
- 2017
45. Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases
- Author
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Johannes H. Hegemann, Markus Aebi, Jens Beinhauer, Claude A. Jakob, Patricie Burda, University of Zurich, and Aebi, M
- Subjects
Mannosyltransferase ,Glycosylation ,1303 Biochemistry ,Mutant ,Molecular Sequence Data ,Mannose ,Oligosaccharides ,610 Medicine & health ,Saccharomyces cerevisiae ,Endoplasmic Reticulum ,Biochemistry ,142-005 142-005 ,Mannosyltransferases ,Substrate Specificity ,chemistry.chemical_compound ,Biosynthesis ,Glycosyltransferase ,DNA Primers ,chemistry.chemical_classification ,biology ,Base Sequence ,Chemistry ,Endoplasmic reticulum ,Oligosaccharide ,Lipids ,Carbohydrate Sequence ,biology.protein - Abstract
The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.
- Published
- 2017
46. A biosynthetic route for polysialylating proteins in Escherichia coli
- Author
-
Manuela Mally, Ivan Hang, Jörg Schneider, Markus Aebi, Michael Wetter, Fabian Müller, Christoph Rutschmann, Simona Russo, Michael Steffen, Chia-Wei Lin, Matthias Zuppiger, Timothy G. Keys, and Amirreza Faridmoayer
- Subjects
0301 basic medicine ,Glycosylation ,Green Fluorescent Proteins ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,law.invention ,Glycoengineering ,N-glycosyltransferase ,Polysialic acid ,DARPin ,Stealth polymer ,03 medical and health sciences ,0302 clinical medicine ,law ,Glycosyltransferase ,medicine ,Escherichia coli ,biology ,Immunogenicity ,Recombinant Proteins ,030104 developmental biology ,Biochemistry ,Cytoplasm ,biology.protein ,Recombinant DNA ,Sialic Acids ,Target protein ,030217 neurology & neurosurgery ,Biotechnology ,Protein Modification, Translational - Abstract
Polysialic acid (polySia) is a posttranslational modification found on only a handful of proteins in the central nervous and immune systems. The addition of polySia to therapeutic proteins improves pharmacokinetics and reduces immunogenicity. To date, polysialylation of therapeutic proteins has only been achieved in vitro by chemical or chemoenzymatic strategies. In this work, we develop a biosynthetic pathway for site-specific polysialylation of recombinant proteins in the cytoplasm of Escherichia coli. The pathway takes advantage of a bacterial cytoplasmic polypeptide-glycosyltransferase to establish a site-specific primer on the target protein. The glucose primer is extended by glycosyltransferases derived from lipooligosaccharide, lipopolysaccharide and capsular polysaccharide biosynthesis from different bacterial species to synthesize long chain polySia. We demonstrate the new biosynthetic route by modifying green fluorescent proteins and a therapeutic DARPin (designed ankyrin repeat protein).
- Published
- 2017
47. Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase
- Author
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Tamis Darbre, Markus Aebi, Maja Napiórkowska, Kaspar P. Locher, Jérémy Boilevin, Jean-Louis Reymond, and Tina Sovdat
- Subjects
0301 basic medicine ,Lipopolysaccharides ,Models, Molecular ,Glycan ,Glycosylation ,Stereochemistry ,Carbohydrates ,Glycobiology ,Peptide ,Campylobacter lari ,Crystallography, X-Ray ,03 medical and health sciences ,Bacterial Proteins ,Structural Biology ,Transferases ,540 Chemistry ,Asparagine ,Amino Acid Sequence ,Molecular Biology ,Ternary complex ,x-ray crystallography ,chemistry.chemical_classification ,biology ,fungi ,Oligosaccharyltransferase ,Rational design ,Active site ,Membrane Proteins ,Oligosaccharide ,030104 developmental biology ,chemistry ,Hexosyltransferases ,biology.protein ,570 Life sciences ,Protein Binding - Abstract
Nature Structural & Molecular Biology, 24, ISSN:1545-9993, ISSN:1545-9985
- Published
- 2017
- Full Text
- View/download PDF
48. Structural Basis of Substrate Specificity of Human Oligosaccharyl Transferase Subunit N33/Tusc3 and Its Role in Regulating Protein N-Glycosylation
- Author
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Goran Malojčić, Markus Aebi, Robin L. Owen, Rudi Glockshuber, Maurice S. Brozzo, and Elisabeth Mohorko
- Subjects
Models, Molecular ,Protein Folding ,Glycosylation ,Protein subunit ,Molecular Sequence Data ,Gene Expression ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Protein Structure, Secondary ,Substrate Specificity ,chemistry.chemical_compound ,N-linked glycosylation ,Structural Biology ,Catalytic Domain ,Humans ,Transferase ,Amino Acid Sequence ,Disulfides ,Molecular Biology ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Tumor Suppressor Proteins ,Endoplasmic reticulum ,Membrane Proteins ,Recombinant Proteins ,Protein Structure, Tertiary ,Protein Subunits ,chemistry ,Biochemistry ,Protein Multimerization ,Peptides ,Glycoprotein ,Hydrophobic and Hydrophilic Interactions ,Protein Binding ,Cysteine - Abstract
N-linked glycosylation of proteins in the endoplasmic reticulum (ER) is essential in eukaryotes and catalyzed by oligosaccharyl transferase (OST). Human OST is a hetero-oligomer of seven subunits. The subunit N33/Tusc3 is a tumor suppressor candidate, and defects in the subunit N33/Tusc3 are linked with nonsyndromic mental retardation. Here, we show that N33/Tusc3 possesses a membrane-anchored N-terminal thioredoxin domain located in the ER lumen that may form transient mixed disulfide complexes with OST substrates. X-ray structures of complexes between N33/Tusc3 and two different peptides as model substrates reveal a defined peptide-binding groove adjacent to the active site that can accommodate peptides in opposite orientations. Structural and biochemical data show that N33/Tusc3 prefers peptides bearing a hydrophobic residue two residues away from the cysteine forming the mixed disulfide with N33/Tusc3. Our results support a model in which N33/Tusc3 increases glycosylation efficiency for a subset of human glycoproteins by slowing glycoprotein folding.
- Published
- 2014
- Full Text
- View/download PDF
49. Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains
- Author
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Johannes Hutzler, Markus Aebi, Martin Loibl, Lina Wunderle, Benjamin L. Schulz, and Sabine Strahl
- Subjects
Sec61 ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Endoplasmic reticulum ,Glycobiology and Extracellular Matrices ,Context (language use) ,Translation (biology) ,Saccharomyces cerevisiae ,Cell Biology ,Biology ,Translocon ,Mannosyltransferases ,Biochemistry ,Cell biology ,Transport protein ,carbohydrates (lipids) ,Protein Transport ,SEC63 ,Protein folding ,Molecular Biology - Abstract
O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.
- Published
- 2014
50. HR-MAS NMR reveals a pH-dependent LPS alteration by de-O-acetylation at abequose in the O-antigen of Salmonella enterica serovar Typhimurium
- Author
-
Giorgia Zandomeneghi, George Rugarabamu, Markus Aebi, Beat H. Meier, and Karin Ilg
- Subjects
Lipopolysaccharides ,Salmonella typhimurium ,Salmonella ,Magnetic Resonance Spectroscopy ,Rhamnose ,Molecular Sequence Data ,Mannose ,medicine.disease_cause ,Biochemistry ,Epitope ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,medicine ,Hexoses ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Chemistry ,Organic Chemistry ,Galactose ,O Antigens ,Acetylation ,General Medicine ,Nuclear magnetic resonance spectroscopy ,Hydrogen-Ion Concentration ,biology.organism_classification ,Carbohydrate Sequence ,Salmonella enterica - Abstract
NMR spectroscopy can detect biomolecules like lipopolysaccharide directly on the surface of the cell, thus avoiding isolation and purification, and providing a more realistic description than the one derived from in vitro studies. Here we present a high-resolution magic-angle spinning NMR study of the O-antigen of Salmonella enterica serovar Typhimurium (S. Typhimurium) performed directly on the cells showing the alteration of its acetylation state over time. The O-antigen region of S. Typhimurium consists of the repeating unit [→2)-α-d-Manp-(1→4)-α-l-Rhap-(1→3)-α-d-Galp-(1→] where Man stands for mannose, Rha for rhamnose, and Gal for galactose. Man is substituted with abequose (Abe) O-acetylated at carbon 2. Our studies revealed that the appearance of de-O-acetylated O-antigen in the stationary growth phase is due to the de-O-acetylation of already synthesized O-acetylated O-antigen and that this reaction is caused by the metabolism-induced basic pH of the growth medium. The labile O-acetylation of the O-antigen we observed in S. Typhimurium generates non-stoichiometric O-acetylation states and therefore changes the nature of an immunogenic epitope.
- Published
- 2013
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