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yeast glycosylation

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https://www.readbyqxmd.com/read/29339117/expression-activation-and-processing-of-a-novel-plant-milk-clotting-aspartic-protease-in-pichia-pastoris
#1
Lucía Feijoo-Siota, José Luis R Rama, Angeles Sánchez-Pérez, Tomás G Villa
Galium verum, also known as Lady's Bedstraw or Cheese Rennet, is a herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae...
January 12, 2018: Journal of Biotechnology
https://www.readbyqxmd.com/read/29331014/biochemical-characterization-of-recombinant-thermostable-cohnella-sp-a01-%C3%AE-glucanase
#2
Meysam Rezaie, Saeed Aminzadeh, Farid Heidari, Masoud Mashhadi Akbar Boojar, Ali Asghar Karkhane
Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are release by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3)...
January 13, 2018: Iranian Biomedical Journal
https://www.readbyqxmd.com/read/29329339/accessibility-and-contribution-to-glucan-masking-of-natural-and-genetically-tagged-versions-of-yeast-wall-protein-1-of-candida-albicans
#3
Bruce L Granger
Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized...
2018: PloS One
https://www.readbyqxmd.com/read/29311294/glycoengineering-of-antibody-herceptin-through-yeast-expression-and-in-vitro-enzymatic-glycosylation
#4
Chiu-Ping Liu, Tsung-I Tsai, Ting Cheng, Vidya S Shivatare, Chung-Yi Wu, Chung-Yi Wu, Chi-Huey Wong
Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P...
January 8, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29301962/structure-of-the-yeast-oligosaccharyltransferase-complex-gives-insight-into-eukaryotic-n-glycosylation
#5
Rebekka Wild, Julia Kowal, Jillianne Eyring, Elsy M Ngwa, Markus Aebi, Kaspar P Locher
Oligosaccharyltransferase (OST) is an essential membrane protein complex in the Endoplasmic Reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate-activated donor to glycosylation sites of secretory proteins. We here describe the atomic structure of yeast OST determined by cryo-EM, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, while two non-catalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide...
January 4, 2018: Science
https://www.readbyqxmd.com/read/29299842/structural-analysis-of-n-o-glycans-assembled-on-proteins-in-yeasts
#6
Eun Jung Thak, Jungho Kim, Dong-Jik Lee, Jeong Yoon Kim, Hyun Ah Kang
Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells...
January 2018: Journal of Microbiology / the Microbiological Society of Korea
https://www.readbyqxmd.com/read/29295913/o-mannosylation-of-proteins-enables-histoplasma-yeast-survival-at-mammalian-body-temperatures
#7
Andrew L Garfoot, Kristie D Goughenour, Marcel Wüthrich, Murugesan V S Rajaram, Larry S Schlesinger, Bruce S Klein, Chad A Rappleye
The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen Histoplasma capsulatum, elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection...
January 2, 2018: MBio
https://www.readbyqxmd.com/read/29273674/alternative-routes-for-synthesis-of-n-linked-glycans-by-alg2-mannosyltransferase
#8
Sheng-Tao Li, Ning Wang, Xin-Xin Xu, Morihisa Fujita, Hideki Nakanishi, Toshihiko Kitajima, Neta Dean, Xiao-Dong Gao
Asparagine (N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth step of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg2 adds both an α1,3- and α1,6-mannose onto ManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. The biochemical properties of Alg2 are controversial and remain undefined...
December 22, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/29215010/characterization-of-a-membrane-bound-c-glucosyltransferase-responsible-for-carminic-acid-biosynthesis-in-dactylopius-coccus-costa
#9
Rubini Kannangara, Lina Siukstaite, Jonas Borch-Jensen, Bjørn Madsen, Kenneth T Kongstad, Dan Staerk, Mads Bennedsen, Finn T Okkels, Silas A Rasmussen, Thomas O Larsen, Rasmus J N Frandsen, Birger Lindberg Møller
Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail...
December 7, 2017: Nature Communications
https://www.readbyqxmd.com/read/29164392/maturation-and-cytokine-pattern-of-human-dendritic-cells-in-response-to-different-yeasts
#10
Silvia Boschi Bazan, Barbara Walch-Rückheim, Manfred J Schmitt, Frank Breinig
Activated dendritic cells (DC) induce and polarize T-cell responses by expression of distinct maturation markers and cytokines. This study systematically investigated the capacity of different biotechnically relevant yeast species and strains including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Candida glabrata to initiate maturation of human DC. As important prerequisite for T-cell activation, all yeasts were shown to effectively induce, though to a different extent, the expression of the activation marker CD83, the co-stimulatory molecules CD80, CD86, CD54, CD58, and CD40, as well as the antigen-presenting molecules MHCs I and II...
November 21, 2017: Medical Microbiology and Immunology
https://www.readbyqxmd.com/read/29143081/generation-in-yeast-and-antigenic-characterization-of-hepatitis-e-virus-capsid-protein-virus-like-particles
#11
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
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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