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N-glycosylation

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https://www.readbyqxmd.com/read/28649776/improving-the-baculovirus-expression-vector-system-with-vankyrin-enhanced-technology
#1
Kendra H Steele, Barbara J Stone, Kathleen M Franklin, Xiufeng Zhang, Haobo Jiang, Bruce A Webb, Christoph Geisler, Angelika Fath-Goodin
The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein...
June 26, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28645725/cell-surface-n-glycan-alteration-in-hepad38-cell-lines-expressing-hepatitis-b-virus
#2
Surya Agung Priyambada, Ryo Misaki, Toru Okamoto, Yuta Okamoto, Takao Ohashi, Keiji Ueda, Yoshiharu Matsuura, Kazuhito Fujiyama
Hepatitis B virus (HBV) is the smallest partially double-stranded DNA virus known to infect humans. Worldwide, more than 50% of hepatocellular carcinoma (HCC) cases are related to chronic Hepatitis B. Development of HCC from normal liver cells is characterized by changes in cell surface N-glycans, which can promote the invasive behavior of tumor cells, leading ultimately to the progression of cancer. However, little is understood about the cell surface N-glycans of HBV-infected liver cells. We try to address this by taking advantage of the HepAD38 cell line, which can replicate HBV in the absence of tetracycline [tet(-)] in growth medium...
June 20, 2017: Virus Research
https://www.readbyqxmd.com/read/28640838/next-generation-sequencing-of-the-clonal-igh-rearrangement-detects-ongoing-mutations-and-interfollicular-trafficking-in-in-situ-follicular-neoplasia
#3
Perikles Kosmidis, Irina Bonzheim, Claudia Dufke, Sema Colak, Thomas Hentrich, Christopher Schroeder, Peter Bauer, Patrick Adam, Falko Fend
Follicular lymphoma (FL) is characterized genetically by a significant intraclonal diversity of rearranged immunoglobulin heavy chain (IGH) genes and a substantial cell migration activity (follicular trafficking). Recently, in situ follicular neoplasia (ISFN), characterized by accumulations of immunohistochemically strongly BCL2-positive, t(14;18)+ clonal B cells confined to germinal centers in reactive lymph nodes, has been identified as a precursor lesion of FL with low risk of progression to manifest FL...
2017: PloS One
https://www.readbyqxmd.com/read/28640745/the-amino-terminal-subdomain-of-glycoprotein-gc-of-schmallenberg-virus-disulfide-bonding-and-structural-determinants-of-neutralization
#4
Gleyder Roman-Sosa, Axel Karger, Franziska Kraatz, Andrea Aebischer, Kerstin Wernike, Pavlo Maksimov, Christopher H Lillig, Ilona Reimann, Emiliana Brocchi, Markus Keller, Martin Beer
Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus (SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes...
June 22, 2017: Journal of General Virology
https://www.readbyqxmd.com/read/28637675/structure-functional-aspects-of-the-human-riboflavin-transporter-3-slc52a3-role-of-the-predicted-glycosylation-and-substrate-interacting-sites
#5
Veedamali S Subramanian, Subrata Subai, Trevor Teafatiller, Jennifer A Bohl, Hamid M Said
The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF...
June 21, 2017: American Journal of Physiology. Cell Physiology
https://www.readbyqxmd.com/read/28636329/metallic-element-chelated-tag-labeling-mectl-for-quantitation-of-n-glycans-in-maldi-ms
#6
Lijun Yang, Ye Peng, Jing Jiao, Tao Tao, Jun Yao, Ying Zhang, Haojie Lu
N-glycosylation plays an important role in chief biological and pathological processes. Quantifying the N-glycan is important since glycan alterations are related to many diseases. In this study, we developed a novel N-glycan quantitation approach using metallic element chelated tag labeling through reductive amination (MeCTL). The MeCTL strategy is of high labeling efficiency and accurate in quantitation with high reproducibility (CV<17.03%) and good linearity (R2>0.99) within two orders of magnitude of dynamic range...
June 21, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28630087/paucimannose-rich-n-glycosylation-of-spatiotemporally-regulated-human-neutrophil-elastase-modulates-its-immune-functions
#7
Ian Loke, Ole Østergaard, Niels H H Heegaard, Nicolle H Packer, Morten Thaysen-Andersen
Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilised to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays...
June 19, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28627758/spectral-library-based-glycopeptide-analysis-detection-of-circulating-galectin-3-binding-protein-in-pancreatic-cancer
#8
Eslam N Nigjeh, Ru Chen, Yasuko Allen-Tamura, Randall E Brand, Teresa A Brentnall, Sheng Pan
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by its late diagnosis, poor prognosis and rapid development of drug resistance. Using the data independent acquisition (DIA) technique, we applied a spectral library-based proteomic approach to analyze N-glycosylated peptides in human plasma, in the context of pancreatic cancer study. EXPERIMENTAL DESIGN: We extended the application of DIA to the quantification of N-glycosylated peptides enriched from plasma specimens from a clinically well-defined cohort that consists of patients with early stage PDAC, chronic pancreatitis and healthy subjects...
June 19, 2017: Proteomics. Clinical Applications
https://www.readbyqxmd.com/read/28620376/the-n-glycosylation-of-mouse-immunoglobulin-g-igg-fragment-crystallizable-differs-between-igg-subclasses-and-strains
#9
Noortje de Haan, Karli R Reiding, Jasminka Krištić, Agnes L Hipgrave Ederveen, Gordan Lauc, Manfred Wuhrer
N-linked glycosylation of the fragment crystallizable (Fc)-region of immunoglobulin G (IgG) is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation. However, while human IgG Fc-glycosylation has been extensively characterized in both health and disease, this is not the case for mice...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28617578/antibodies-against-specific-muc16-glycosylation-sites-inhibit-ovarian-cancer-growth
#10
Thapi Dharma Rao, Alberto Fernández-Tejada, Abram Axelrod, Nestor Rosales, Xiujun Yan, Sahityasri Thapi, Amy Wang, Kay J Park, Brandon Nemieboka, Jingyi Xiang, Jason S Lewis, Nariso Olvera, Douglas A Levine, Samuel J Danishefsky, David Spriggs
Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors co-localized on lipid rafts...
June 15, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28614667/n-glycosylation-of-asparagine-130-in-the-extracellular-domain-of-the-human-calcitonin-receptor-significantly-increases-peptide-hormone-affinity
#11
Sang-Min Lee, Jason M Booe, Joseph J Gingell, Virginie Sjoelund, Debbie L Hay, Augen A Pioszak
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays...
June 26, 2017: Biochemistry
https://www.readbyqxmd.com/read/28598353/cellular-consequences-of-diminished-protein-o-mannosyltransferase-activity-in-baker-s-yeast
#12
Ewa Zatorska, Lihi Gal, Jaro Schmitt, Daniela Bausewein, Maya Schuldiner, Sabine Strahl
O-Mannosylation is a type of protein glycosylation initiated in the endoplasmic reticulum (ER) by the protein O-mannosyltransferase (PMT) family. Despite the vital role of O-mannosylation, its molecular functions and regulation are not fully characterized. To further explore the cellular impact of protein O-mannosylation, we performed a genome-wide screen to identify Saccharomyces cerevisiae mutants with increased sensitivity towards the PMT-specific inhibitor compound R3A-5a. We identified the cell wall and the ER as the cell compartments affected most upon PMT inhibition...
June 9, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28580641/melatonin-reduces-endoplasmic-reticulum-stress-and-corneal-dystrophy-associated-tgfbip-through-activation-of-endoplasmic-reticulum-associated-protein-degradation
#13
Seung-Il Choi, Eunhee Lee, Begum Akuzum, Jang Bin Jeong, Yong-Sun Maeng, Tae-Im Kim, Eung Kweon Kim
Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress-induced death than were wild-type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase-3 activation, and poly (ADP-ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin...
June 5, 2017: Journal of Pineal Research
https://www.readbyqxmd.com/read/28576849/proteome-wide-identification-of-glycosylation-dependent-interactors-of-galectin-1-and-galectin-3-on-mesenchymal-retinal-pigment-epithelial-cells
#14
Jara Obermann, Claudia S Priglinger, Juliane Merl-Pham, Arie Geerlof, Sigfried Priglinger, Magdalena Götz, Stefanie M Hauck
Identification of interactors is a major goal in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here we aim to identify novel interacting binding partners for the β-galactoside-binding proteins Galectin-1 (Gal-1) and Galectin-3 (Gal-3) relevant in the context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion and epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes...
June 2, 2017: Molecular & Cellular Proteomics: MCP
https://www.readbyqxmd.com/read/28575723/glycoform-of-a-newly-identified-pollen-allergen-cha-o-3-from-chamaecyparis-obtusa-japanese-cypress-hinoki
#15
Toshihiro Osada, Megumi Maeda, Chinatsu Tanabe, Kaori Furuta, Christopher J Vavricka, Eiji Sasaki, Mitsuhiro Okano, Yoshinobu Kimura
Cha o 3 is a newly found glycosylated allergen from Chamaecyparis obtusa (Japanese cypress) pollen. The deduced amino acid sequence of Cha o 3 indicates that this glycoallergen contains a cellulase domain and a number of putative N-glycosylation sites. However, the structures of N -glycans linked to Cha o 3 remain to be determined. In this study, therefore, we analyzed the glycoform of Cha o 3 and found that this glycoallergen carries exclusively plant complex-type N-glycans; major structures were GlcNAc2Man3Xyl1Fuc1GlcNAc2 (39%), Gal1Fuc1GlcNAc2Man3Xyl1Fuc1GlcNAc2 (14%), and Gal2Fuc2GlcNAc2Man3Xyl1Fuc1GlcNAc2 (25%)...
May 24, 2017: Carbohydrate Research
https://www.readbyqxmd.com/read/28575298/chemo-enzymatic-synthesis-of-lipid-linked-glcnac2man5-oligosaccharides-using-recombinant-alg1-alg2-and-alg11-proteins
#16
Ana S Ramírez, Jérémy Boilevin, Chia-Wei Lin, Bee Ha Gan, Daniel Janser, Markus Aebi, Tamis Darbre, Jean-Louis Reymond, Kaspar P Locher
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...
June 1, 2017: Glycobiology
https://www.readbyqxmd.com/read/28556996/semi-high-throughput-isolation-and-n-glycan-analysis-of-human-fibrinogen-using-monolithic-supports-bearing-monoclonal-anti-human-fibrinogen-antibodies
#17
Urška Vidic, Irena Trbojević-Akmačić, Urh Černigoj, Malena Albers, Jernej Gašperšič, Maja Pučić-Baković, Jana Vidič, Aleš Štrancar, Gordan Lauc
Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples...
May 30, 2017: Electrophoresis
https://www.readbyqxmd.com/read/28554670/serine-protease-isoforms-in-gloydius-intermedius-venom-full-sequences-molecular-phylogeny-and-evolutionary-implications
#18
Zhang-Min Yang, Hui Yu, Zhen-Zhen Liu, Jian-Zhu Pei, Yu-E Yang, Su-Xian Yan, Cui Zhang, Wen-Long Zhao, Zhe-Zhi Wang, Ying-Ming Wang, Inn-Ho Tsai
Nine distinct venom serine proteases (vSPs) of Gloydius intermedius were studied by transcriptomic, sub-proteomic and phylogenetic analyses. Their complete amino acid sequences were deduced after Expression Sequence Tag (EST) analyses followed by cDNA cloning and sequencing. These vSPs appear to be paralogs and contain the catalytic triads and 1-4 potential N-glycosylation sites. Their relative expression levels evaluated by qPCR were grossly consistent with their EST hit-numbers. The major vSPs were purified by HPLC and their N-terminal sequences matched well to the deduced sequences, while fragments of the minor vSPs were detected by LC-MS/MS identification...
May 26, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/28554385/co-overexpression-of-mgat1-and-mgat4-in-cho-cells-for-production-of-highly-sialylated-albumin-erythropoietin
#19
Hyun-Myoung Cha, Jin-Hyuk Lim, Jung-Heum Yeon, Jeong-Min Hwang, Dong-Il Kim
Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity...
August 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28551118/synthesis-of-hydrazide-functionalized-hydrophilic-polymer-hybrid-graphene-oxide-for-highly-efficient-n-glycopeptide-enrichment-and-identification-by-mass-spectrometry
#20
Haihong Bai, Yiting Pan, Cong Guo, Xinyuan Zhao, Bingquan Shen, Xinghe Wang, Zeyuan Liu, Yuanguo Cheng, Weijie Qin, Xiaohong Qian
Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite...
August 15, 2017: Talanta
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