FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment

ACS Catalysis ◽  
2021 ◽  
pp. 9052-9065
Author(s):  
Ana García-García ◽  
Sonia Serna ◽  
Zhang Yang ◽  
Ignacio Delso ◽  
Víctor Taleb ◽  
...  
Keyword(s):  
Glycan Structure ◽  
Protein Environment ◽  
Core Fucosylation

Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics

2018 ◽  
Author(s):  
Christopher Ashwood ◽  
Brian Pratt ◽  
Brendan MacLean ◽  
Nicolle H. Packer
Keyword(s):  
Retention Time ◽  
Internal Standard ◽  
Automated Assignment ◽  
Glycan Structure ◽  
Spectral Library ◽  
Reference Library ◽  
Glucose Unit ◽  
Unit Value ◽  
Area Variation ◽  
Core Fucosylation

<p>Porous graphitised carbon (PGC) based chromatographic separation of glycans achieves high-resolution separation of glycan mixtures released from glycoproteins, including structurally similar isomers. While there is some understanding of glycan separation on PGC, system-independent retention values have not been established.</p> <p> </p> <p>Using hydrolysed dextran as an internal standard, and Skyline software for post-acquisition normalisation, retention time and glycan peak area variation of replicate injections of glycan mixtures was significantly reduced. Normalisation of retention time to the dextran ladder allowed assignment of system-independent retention values, values that are applicable to all PGC-based separations regardless of chromatographic system. We have built a library of over 300 PGC-separated glycan structures with assigned normalised glucose unit (GU) PGC retention values.</p> <p> </p> <p>To further define the mechanism of glycan separation with PGC, we identified predictive models for the chromatographic effects resulting from addition and/or removal of core-fucosylation and bisecting GlcNAc based on the PGC normalised retention time library. A dextran ladder spectral library was also built to ensure correct retention time assignment of the internal standard added to glycan mixtures. Using the spectral matching feature in Skyline, isomeric discrimination between <i>O</i>-mannosylated glycans and the glucose-based dextran ladder was achieved.</p> <p> </p> <p>As a result, system-independent automated assignment of glycan structure based on precursor mass and glucose unit value, using a glycan structure reference library, can be achieved using PGC-LC-MS. </p>

scholarly journals The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation

2020 ◽  
Vol 295 (23) ◽  
pp. 7992-8004 ◽  
Author(s):  
Seita Tomida ◽  
Misaki Takata ◽  
Tetsuya Hirata ◽  
Masamichi Nagae ◽  
Miyako Nakano ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Interactions ◽  
Sh3 Domain ◽  
Glycan Structure ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Critical Residue ◽  
Core Fucosylation ◽  
In Cells

Core fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and in vitro and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.

Protein trafficking: Glycan structure sorts it out

2009 ◽  
Author(s):  
Catherine Goodman
Keyword(s):  
Protein Trafficking ◽  
Glycan Structure

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Biocatalytic oxidation of polycyclic aromatic hydrocarbons in media containing organic solvents

1997 ◽  
Vol 36 (10) ◽  
pp. 37-44 ◽  
Author(s):  
Eduardo Torres ◽  
Raunel Tinoco ◽  
Rafael Vazquez-Duhalt
Keyword(s):  
Cytochrome C ◽  
Organic Solvents ◽  
Oxidation Mechanism ◽  
Site Directed Mutagenesis ◽  
Solvent Mixtures ◽  
Reaction Products ◽  
Prosthetic Group ◽  
Mass Transfer Limitation ◽  
Polycyclic Aromatic ◽  
Protein Environment

Lignin peroxidase, cytochrome c and haemoglobin were tested for oxidation of polycyclic aromatic hydrocarbon (PAH) in the presence of hydrogen peroxide. The reaction mixture Contained water-miscible organic solvents in order to reduce the mass transfer limitation of hydrophobic substrates. The reaction products from all three haemoproteins were mainly quinones, suggesting the same oxidation mechanism for the three biocatalysts. The haeme prosthetic group must have located in a protein environment for it to catalyze these reactions, and only certain types of protein environment are able to induce this type of haemebased catalytic activity. The solvent hydrophobicity is a factor affecting the biocatalysis in organic media. Substrate partitioning between the active site (haeme) and the bulk solvent is the main factor of the biocatalytic behaviour in organic solvent mixtures. Site-directed mutagenesis of yeast cytochrome c significantly altered the kinetic behaviour of the protein. The Gly82;Thr 102 variant was 10 times more active and showed a catalytic efficiency 10-fold greater than the wild-type iso-1-cytochrome c. These results suggest that it is possible to design a new biocatalyst for environmental purposes.

Design, Synthesis and Bioactivity of Core 1 O-glycan and its Derivative on Human Gut Microbiota

2019 ◽  
Vol 16 (12) ◽  
pp. 1348-1353
Author(s):  
Huanhuan Qu ◽  
Baixue Li ◽  
Jingyi Yang ◽  
Huaiwen Liang ◽  
Meixia Li ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Initial Step ◽  
Building Blocks ◽  
Glycan Structure ◽  
Human Gut ◽  
Design Synthesis ◽  
The Core ◽  
Human Gut Microbiota ◽  
Biochemical Studies ◽  
Gut Symbionts

Background: Disaccharide core 1 (Galβ1-3GalNAc) is a common O-glycan structure in nature. Biochemical studies have confirmed that the formation of the core 1 structure is an important initial step in O-glycan biosynthesis and it is of great importance for human body. Objective: Our study will provide meaningful and useful sights for O-glycan synthesis and their bioassay. And all the synthetic glycosides would be used as intermediate building blocks in the scheme developed for oligosaccharide construction. Methods: In this article, we firstly used chemical procedures to prepare core 1 and its derivative, and a novel disaccharide was efficiently synthesized. The structures of the synthesized compounds were elucidated and confirmed by 1H NMR, 13C NMR and MS. Then we employed three human gut symbionts belonging to Bacteroidetes, a predominantphyla in the distal gut, as models to study the bioactivity of core 1 and its derivative on human gut microbiota. Results: According to our results, both core 1 and derivative could support the growth of B. fragilis, especially the core 1 derivative, while failed to support the growth of B. thetaiotaomicron and B. ovatus. Conclusion: This suggested that the B. fragilis might have the specificity glycohydrolase to cut the glycosidic bond for acquiring monosaccharide.

Glycan structure-based perspectives on the entry and release of glycoproteins in the calnexin/calreticulin cycle

2021 ◽  
Vol 502 ◽  
pp. 108273
Author(s):  
Taiki Kuribara ◽  
Ruchio Usui ◽  
Kiichiro Totani
Keyword(s):  
Glycan Structure

scholarly journals Core Fucosylation on T Cells, Required for Activation of T-Cell Receptor Signaling and Induction of Colitis in Mice, Is Increased in Patients With Inflammatory Bowel Disease

2016 ◽  
Vol 150 (7) ◽  
pp. 1620-1632 ◽  
Author(s):  
Hironobu Fujii ◽  
Shinichiro Shinzaki ◽  
Hideki Iijima ◽  
Kana Wakamatsu ◽  
Chizuru Iwamoto ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Bowel Disease ◽  
Cell Receptor ◽  
T Cell Receptor Signaling ◽  
Inflammatory Bowel ◽  
Core Fucosylation ◽  
Cell Receptor Signaling
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