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yeast glycosylation

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https://www.readbyqxmd.com/read/29143081/generation-in-yeast-and-antigenic-characterization-of-hepatitis-e-virus-capsid-protein-virus-like-particles
#1
Martynas Simanavicius, Paulius Lukas Tamosiunas, Rasa Petraityte-Burneikiene, Reimar Johne, Rainer G Ulrich, Aurelija Zvirbliene, Indre Kucinskaite-Kodze
Hepatitis E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products and with blood components through transfusions. There are no approved or golden standard serologic systems for HEV diagnostics. Commercially available HEV tests often provide inconsistent results which may differ among the assays. In this study, we describe generation in yeast and characterization of HEV genotype 3 (HEV-3) and rat HEV capsid proteins self-assembled into virus-like particles (VLPs) and the development of HEV-specific monoclonal antibodies (MAbs)...
November 15, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29109265/diminished-ost3-dependent-n-glycosylation-of-the-bip-nucleotide-exchange-factor-sil1-is-an-adaptive-response-to-reductive-er-stress
#2
Kofi L P Stevens, Amy L Black, Kelsi M Wells, K Y Benjamin Yeo, Robert F L Steuart, Colin J Stirling, Benjamin L Schulz, Carl J Mousley
BiP (Kar2 in yeast) is an essential Hsp70 chaperone and master regulator of endoplasmic reticulum (ER) function. BiP's activity is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, Sil1 and Lhs1. Both Sil1 and Lhs1 are glycoproteins, but how N-glycosylation regulates their function is not known. Here, we show that N-glycosylation of Sil1, but not of Lhs1, is diminished upon reductive stress. N-glycosylation of Sil1 is predominantly Ost3-dependent and requires a functional Ost3 CxxC thioredoxin motif...
November 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29108953/investigating-the-function-of-gdt1p-in-yeast-golgi-glycosylation
#3
Eudoxie Dulary, Shin-Yi Yu, Marine Houdou, Geoffroy de Bettignies, Valérie Decool, Sven Potelle, Sandrine Duvet, Marie-Ange Krzewinski-Recchi, Anne Garat, Gert Matthijs, Yann Guerardel, François Foulquier
The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca(2+)/Mn(2+) homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca(2+) concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination...
November 3, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29080432/base-modified-gdp-mannose-derivatives-and-their-substrate-activity-towards-a-yeast-mannosyltransferase
#4
Alice Collier, Gerd K Wagner
We have previously developed a new class of inhibitors and chemical probes for glycosyltransferases through base-modification of the sugar-nucleotide donor. The key feature of these donor analogues is the presence of an additional substituent at the nucleobase. To date, the application of this general concept has been limited to UDP-sugars and UDP-sugar-dependent glycosyltransferases. Herein, we report for the first time the application of our approach to a GDP-mannose-dependent mannosyltransferase. We have prepared four GDP-mannose derivatives with an additional substituent at either position 6 or 8 of the nucleobase...
November 27, 2017: Carbohydrate Research
https://www.readbyqxmd.com/read/29069140/a-new-acetylcholinesterase-inhibitor-from-green-glycosylation-of-trachyloban-19-oic-acid-by-mucor-plumbeus
#5
Gabriel F Dos Santos, Jacqueline A Takahashi
The in vitro metabolism of a widespread natural product, trachyloban-19-oic acid (1), by the fungal species Mucor plumbeus was studied in a sucrose-yeast liquid medium. Two products were isolated, and their structures were determined by spectroscopic means as 7β-hydroxytrachyloban-19-oic acid (5) and trachyloban-19-O-β-D-glucopyranosyl ester (6). To the best of our knowledge, compound 6 is herein reported by the first time in the literature. These compounds were assayed for acetylcholinesterase inhibition along with some related compounds...
October 23, 2017: Anais da Academia Brasileira de Ciências
https://www.readbyqxmd.com/read/29042445/analysis-of-substrate-specificity-of-trypanosoma-brucei-osts-by-functional-expression-of-domain-swapped-chimeras-in-yeast
#6
Kristina Poljak, Jörg Breitling, Robert Gauss, George Rugarabamu, Mauro Pellanda, Markus Aebi
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...
October 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29042437/posttranslational-processing-and-membrane-translocation-of-the-yeast-regulatory-mid1-subunit-of-the-cch1-vgcc-nalcn-cation-channel-family
#7
Kazuko Iida, Jinfeng Teng, Toshihiko Cho, Sato Yoshikawa-Kimura, Hidetoshi Iida
Saccharomyces cerevisiae Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na+ leak channel non-selective (NALCN) than to the α1 subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α2/δ regulatory subunits and NALCN-associated proteins. Unlike animal α2/δ subunits, Mid1 and NALCN-associated proteins are essential for the function of the pore-forming subunit...
October 17, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29042410/h-and-pi-byproducts-of-glycosylation-affect-ca-2-%C3%A2-homeostasis-and-are-retrieved-from-the-golgi-complex-by-homologs-of-tmem165-and-xpr1
#8
Nathan A Snyder, Christopher P Stefan, Camille T Soroudi, Adam Kim, Carlos Evangelista, Kyle W Cunningham
Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H(+)) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H(+) for cytoplasmic Ca(2+), respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H(+)/Ca(2+) exchangers, Gdt1 also promoted growth in high Ca(2+) environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated...
October 17, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28993419/quantitative-profiling-of-n-linked-glycosylation-machinery-in-yeast-saccharomyces-cerevisiae
#9
Kristina Poljak, Nathalie Selevsek, Elsy Ngwa, Jonas Grossmann, Marie Estelle Losfeld, Markus Aebi
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...
October 9, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28951601/modified-tunicamycins-with-reduced-eukaryotic-toxicity-that-enhance-the-antibacterial-activity-of-%C3%AE-lactams
#10
Neil Pj Price, Trina M Hartman, Jiakun Li, Kiran K Velpula, Todd A Naumann, Maheedhara R Guda, Biao Yu, Kenneth M Bischoff
Tunicamycins (TUN) are inhibitors of the UDP-HexNAc: polyprenol-P HexNAc-1-P transferase family of enzymes, which initiate the biosynthesis of bacterial peptidoglycan and catalyze the first step in eukaryotic protein N-glycosylation. The TUN are therefore general and potent toxins to both eukaryotes and prokaryotes. Screening a library of synthetic TUN against Bacillus and yeast identified TUN that are antibacterial, but have significantly reduced eukaryotic toxicity. One of these (Tun-15:0) differs from the native TUN control only by the lack of the conjugated double bond in the tunicaminyl N-acyl group...
November 2017: Journal of Antibiotics
https://www.readbyqxmd.com/read/28935113/directed-evolution-of-glycopeptides-using-mrna-display
#11
Satoru Horiya, Jennifer K Bailey, Isaac J Krauss
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28928140/selenoprotein-t-is-a-novel-ost-subunit-that-regulates-upr-signaling-and-hormone-secretion
#12
Abdallah Hamieh, Dorthe Cartier, Houssni Abid, André Calas, Carole Burel, Christine Bucharles, Cedric Jehan, Luca Grumolato, Marc Landry, Patrice Lerouge, Youssef Anouar, Isabelle Lihrmann
Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production...
November 2017: EMBO Reports
https://www.readbyqxmd.com/read/28924963/combining-metabolic-and-process-engineering-strategies-to-improve-recombinant-glycoprotein-production-and-quality
#13
Eric Karengera, Yves Durocher, Gregory De Crescenzo, Olivier Henry
Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes...
November 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28921426/production-of-full-length-antibody-by-pichia-pastoris
#14
Adam Nylen, Ming-Tang Chen
The methylotrophic yeast Pichia pastoris has become an increasingly popular host for recombinant protein expression in recent times. MRL pioneered a glycoengineered humanized P. pastoris expression system that could produce glycoproteins with glycosylation profiles similar to mammalian systems. Therapeutic glycoproteins produced by the humanized P. pastoris platform have shown comparable folding, stability, and in vitro and in vivo efficacies in preclinical models to their counterparts produced from the CHO cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28857681/expression-and-purification-of-the-extracellular-domains-of-human-glycoprotein-vi-gpvi-and-the-receptor-for-advanced-glycation-end-products-rage-from-rattus-norvegicus-in-leishmania-tarentolae
#15
Thomas Langer, Carsten Corvey, Katja Kroll, Oliver Boscheinen, Thomas Wendrich, Werner Dittrich
Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive...
August 31, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28810660/recent-advancements-in-understanding-mammalian-o-mannosylation
#16
M Osman Sheikh, Stephanie M Halmo, Lance Wells
The post-translational glycosylation of select proteins by O-linked mannose (O-mannose or O-man) is a conserved modification from yeast to humans and has been shown to be necessary for proper development and growth. The most well studied O-mannosylated mammalian protein is α-dystroglycan (α-DG). Hypoglycosylation of α-DG results in varying severities of congenital muscular dystrophies, cancer progression and metastasis, and inhibited entry and infection of certain arenaviruses. Defects in the gene products responsible for post-translational modification of α-DG, primarily glycosyltransferases, are the basis for these diseases...
September 1, 2017: Glycobiology
https://www.readbyqxmd.com/read/28797224/the-adaptive-landscape-of-wildtype-and-glycosylation-deficient-populations-of-the-industrial-yeast-pichia-pastoris
#17
Josef W Moser, Iain B H Wilson, Martin Dragosits
BACKGROUND: The effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed...
August 10, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28794948/glucosomes-glycosylated-vesicle-in-vesicle-aggregates-in-water-from-ph-responsive-microbial-glycolipid
#18
Niki Baccile, Patrick Le Griel, Sylvain Prévost, Bernd Everaert, Inge N A Van Bogaert, Sophie Roelants, Wim Soetaert
Vesicle-in-vesicle self-assembled containers, or vesosomes, are promising alternatives to liposomes because of their possible hierarchical encapsulation and high stability. We report herein the first example of sugar-based vesicles-in-vesicles, which we baptize glucosomes. These were prepared by using a natural microbial glycolipid (branched C22 sophorolipid) extracted from the culture medium of the yeast Pseudohyphozyma bogoriensis. Glucosomes spontaneously formed in water between pH 6 and pH 4 at room temperature, without the requirement of any additive...
August 2017: ChemistryOpen
https://www.readbyqxmd.com/read/28759578/heterologous-expression-of-the-antimyotoxic-protein-dm64-in-pichia-pastoris
#19
Saulo Martins Vieira, Surza Lucia Gonçalves da Rocha, Ana Gisele da Costa Neves-Ferreira, Rodrigo Volcan Almeida, Jonas Perales
Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures...
July 2017: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/28735452/characterization-of-polymeric-substance-classes-in-cereal-based-beverages-using-asymmetrical-flow-field-flow-fractionation-with-a-multi-detection-system
#20
Georg Krebs, Thomas Becker, Martina Gastl
Cereal-based beverages contain a complex mixture of various polymeric macromolecules including polysaccharides, peptides, and polyphenols. The molar mass of polymers and their degradation products affect different technological and especially sensory parameters of beverages. Asymmetrical flow field-flow fractionation (AF4) coupled with multi-angle light scattering (MALS) and refractive index detection (dRI) or UV detection (UV) is a technique for structure and molar mass distribution analysis of macromolecules commonly used for pure compound solutions...
July 22, 2017: Analytical and Bioanalytical Chemistry
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