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Akira Togayachi, Azusa Tomioka, Mika Fujita, Masako Sukegawa, Erika Noro, Daisuke Takakura, Michiyo Miyazaki, Toshihide Shikanai, Hisashi Narimatsu, Hiroyuki Kaji
To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE)...
April 19, 2018: Journal of the American Society for Mass Spectrometry
Zaid Amso, Sean W Bisset, Sung-Hyun Yang, Paul W R Harris, Tom H Wright, Claudio D Navo, Mark L Patchett, Gillian E Norris, Margaret A Brimble
Glycocin F (GccF) is a unique diglycosylated bacteriocin peptide that possesses potent and reversible bacteriostatic activity against a range of Gram-positive bacteria. GccF is a rare example of a 'glycoactive' bacteriocin, with both the O -linked N -acetylglucosamine (GlcNAc) and the unusual S -linked GlcNAc moiety important for antibacterial activity. In this report, glycocin F was successfully prepared using a native chemical ligation strategy and folded into its native structure. The chemically synthesised glycocin appeared to be slightly more active than the recombinant material produced from Lactobacillus plantarum ...
February 14, 2018: Chemical Science
Elena Solovieva, Toshihide Shikanai, Noriaki Fujita, Hisashi Narimatsu
BACKGROUND: Inherited mutations in glyco-related genes can affect the biosynthesis and degradation of glycans and result in severe genetic diseases and disorders. The Glyco-Disease Genes Database (GDGDB), which provides information about these diseases and disorders as well as their causative genes, has been developed by the Research Center for Medical Glycoscience (RCMG) and released in April 2010. GDGDB currently provides information on about 80 genetic diseases and disorders caused by single-gene mutations in glyco-related genes...
April 18, 2018: Journal of Biomedical Semantics
Pil Seok Chae, Muhammad Ehsan, Manabendra Das, Valerie Stern, Yang Du, Jonas Mortensen, Parameswaran Hariharan, Bernadette Byrne, Claus Loland, Brian Kobilka, Lan Guan
Membrane proteins allow effective communication between cells and organelles and their external environments. Maintaining membrane protein stability in a non-native environment is the major bottleneck to their structural study. Detergents are widely used to extract membrane proteins from the membrane and keep the extracted protein in a stable state for downstream characterization. In the current study, three sets of steroid-based amphiphiles, glyco-diosgenin analogs (GDNs), steroid-based penta-saccharides either lacking a linker (SPSs) or with a linker (SPS-Ls), were developed as novel chemical tools for membrane protein research...
April 16, 2018: Chembiochem: a European Journal of Chemical Biology
Chu-Wei Kuo, Shih-Yun Guu, Kay-Hooi Khoo
High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies...
April 11, 2018: Journal of the American Society for Mass Spectrometry
Yimeng Li, Shu Shen, Liangbo Hu, Fei Deng, Just M Vlak, Zhihong Hu, Hualin Wang, Manli Wang
gp41 , one of the baculovirus core genes, encodes the only recognized tegument (O-glyco) protein of the occlusion-derived virion (ODV) phenotype so far. A previous study using a temperature-sensitive Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutant showed that GP41 plays a crucial role in budded virion (BV) formation. However, the precise function of GP41 in the baculovirus replication cycle remains unclear. In this study, AcMNPV GP41 was found to accumulate around the ring zone region (RZ) within the infected nucleus and finally assembled into both BV and ODV...
April 11, 2018: Journal of Virology
I-Hsuan Chen, Hillary Andaluz Aguilar, Juan Sebastian Paez Paez, Xiaofeng Wu, Li Pan, Michael K Wendt, Anton B Iliuk, Ying Zhang, W Andy Tao
Glycoproteins comprise more than half of current FDA-approved protein cancer markers but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array, and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood...
April 9, 2018: Analytical Chemistry
Huan Wang, Ying Liu, Chao Xu, Xi Wang, Guo-Rong Chen, Tony D James, Yi Zang, Jia Li, Xiang Ma, Xiao-Peng He
We have developed a supramoleuclar imaging probe based on thin-layer manganese dioxide functionalized with a fluorescent, multivalent glyco-poly-cycolodextrin for the targeted, stimulus-responsive bioimaging of cancer cells.
April 5, 2018: Chemical Communications: Chem Comm
Yukihiro Nomura, Hiroyuki Murata, Hiroaki Sasai, Akihiko Kimura, Takao Kurosawa, Takahiro Sasaki, Tsuyoshi Murai
Unusual bile acids (1β-hydroxylated bile acids), particularly 1β-hydroxyl-cholic acid (CA-1β-ol) and 1β-hydroxyl-chenodeoxycholic acid (CDCA-1β-ol), have been detected in the urine of infants. These acids are conjugated with amino acids, such as taurine, and are then excreted mainly via the urine. CA-1β-ol and CDCA-1β-ol are the predominant bile acids during infancy and are present in relatively large amounts in the urine. However, the biosynthetic pathway of 1β-hydroxylated bile acids in infants remains unclear...
2018: Biological & Pharmaceutical Bulletin
Ashish A Prabhu, Bibari Boro, Biju Bharali, Shuchishloka Chakraborty, V Venkata Dasu
BACKGROUND: Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. RESULT AND CONCLUSION: In the present review, we have summarized the recent developments in the expression of foreign proteins in P...
March 28, 2018: Current Pharmaceutical Biotechnology
Alessia Cozzolino, Tiziana Feola, Ilaria Simonelli, Giulia Puliani, Carlotta Pozza, Elisa Giannetta, Daniele Gianfrilli, Patrizio Pasqualetti, Andrea Lenzi, Andrea M Isidori
Introduction: Somatostatin analogs (SSAs) effectivelycontrol growth hormone secretion in first and second line treatmentof acromegaly. Their effect onglucose metabolism is still debated. Aim: to address the following questions: 1) Do SSAs affect fasting plasma glucose (FPG), fasting plasma insulin (FPI), glycosylated hemoglobin (HbA1c), glucose load (2h-OGTT), HOMA-I, HOMA-β, triglycerides (TGD), weight (W) or body mass index (BMI)? 2) Do lanreotide (LAN) and octreotide LAR (OCT) affect metabolism differently? 3)Does their effect depend on disease control? Methods: We performed a meta-analysis of prospective interventional trialstreating acromegaly with SSAs...
March 23, 2018: Journal of Clinical Endocrinology and Metabolism
B L Farrugia, M S Lord, J M Whitelock, J Melrose
The development of bioscaffolds that incorporate chondroitin sulphate (CS) and their applications with progenitor and stem cells in cartilage, bone, cornea, skin, and neural repair are reviewed. CS is a heterogeneous structure due to the organisation of multiple CS disaccharide sulphation motifs, giving rise to a vast range of CS chain structures, and hence the wide range of biological activity. The incorporation of this biological molecule represents a significant advance in bioscaffold design and performance in tissue repair strategies...
March 21, 2018: Biomaterials Science
Na Chen, Juan Xie
Nucleic acids and carbohydrates are essential biomolecules involved in numerous biological and pathological processes. Development of multifunctional building blocks based on nucleosides and sugars is in high demand for the generation of novel oligonucleotide mimics and glycoconjugates for biomedical applications. Recently, aminooxyl-functionalized compounds have attracted increasing research interest because of their easy derivatization through oxime ligation or N -oxyamide formation reactions. Various biological applications have been reported for O -amino carbohydrate- and nucleoside-derived compounds...
March 12, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Vojtech Franc, Jing Zhu, Albert J R Heck
The human complement hetero-trimeric C8αβγ (C8) protein assembly (~ 150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~ 20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions...
March 12, 2018: Journal of the American Society for Mass Spectrometry
Yan Weng, Tetsuya Ishino, Annette Sievers, Saswata Talukdar, Jeffrey R Chabot, Amy Tam, Weili Duan, Kelvin Kerns, Eric Sousa, Tao He, Alison Logan, Darwin Lee, Dongmei Li, Yingjiang Zhou, Barbara Bernardo, Alison Joyce, Mania Kavosi, Denise M O'Hara, Tracey Clark, Jie Guo, Craig Giragossian, Mark Stahl, Roberto A Calle, Ron Kriz, Will Somers, Laura Lin
Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration...
March 9, 2018: Scientific Reports
Benjamin G Kremkow, Kelvin H Lee
Glyco-Mapper is a novel systems biology product quality prediction tool created using a new framework termed: Discretized Reaction Network Modeling using Fuzzy Parameters (DReaM-zyP). Within Glyco-Mapper, users fix the nutrient feed composition and the glycosylation reaction fluxes to fit the model glycoform to the reference experimental glycoform, enabling cell-line specific glycoform predictions as a result of cell engineering strategies. Glyco-Mapper accurately predicts published genetically altered glycoforms resulting in the appearance or disappearance of one or more glycans between 1999 and 2014 with an accuracy, sensitivity, and specificity of 96%, 85%, and 97%, respectively...
March 6, 2018: Metabolic Engineering
Pornpimol Phuengmaung, Daisuke Fujiwara, Wasana Sukhumsirichart, Tatsuji Sakamoto
In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a β-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins...
May 2018: Enzyme and Microbial Technology
Pornpimol Phuengmaung, Yuika Kunishige, Wasana Sukhumsirichart, Tatsuji Sakamoto
We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa...
May 2018: Enzyme and Microbial Technology
Kutty Selva Nandakumar
Rheumatoid arthritis (RA) is a polygenic and multifactorial syndrome. Many complex immunological and genetic interactions are involved in the final outcome of the clinical disease. Autoantibodies (rheumatoid factors, anti-citrullinated peptide/protein antibodies) are present in RA patients' sera for a long time before the onset of clinical disease. Prior to arthritis onset, in the autoantibody response, epitope spreading, avidity maturation, and changes towards a pro-inflammatory Fc glycosylation phenotype occurs...
February 28, 2018: International Journal of Molecular Sciences
Mai Ishikawa, Mayu Kawasaki, Yoshihito Shiono, Takuya Koseki
α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) hydrolyzes the glycosidic linkage between the disaccharide 6-O-α-L-rhamnosyl-β-D-glucoside (rutinose) and the aglycone. We identified a hypothetical protein (annotated as AO090012000917) encoded in the Aspergillus oryzae genome that exhibits sequence similarity with Aspergillus niger rutinosidase. The recombinant enzyme was expressed in Pichia pastoris GS115 and purified as a glyco-protein with apparent molecular mass of 65-75 kDa by SDS-PAGE. After N-deglycosylation, we observed a 42- and 40-kDa band, representing proteins before and after N-terminal signal peptide processing, respectively...
February 23, 2018: Applied Microbiology and Biotechnology
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