Gdp fucose

Manufactured by Merck Group

Sourced in United States

GDP-fucose is a chemical compound that serves as a substrate for various enzymatic reactions. It plays a key role in the synthesis of fucosylated glycans, which are involved in various biological processes. This lab equipment product provides a reliable source of GDP-fucose to support research and development activities.

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11 protocols using gdp fucose

Exofucosylation of Human Mesenchymal Stem Cells

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For α(1,3)-exofucosylation, hMSCs were treated with 1µg of α(1,3)-linkage-specific human fucosyltransferase VII (FTVII) (obtained from R&D Systems) in HBSS without Ca²⁺/Mg²⁺ containing 10mM HEPES, 0.1% human serum albumin (HSA) and 1mM GDP-Fucose (Sigma Aldrich), at 37°C for 1h, as described previously [11 ]. In each experiment, we compared FTVII treatment (as described above) to controls which consisted of buffer treated cells (without enzyme in an equivalent volume of buffer under identical conditions). Recombinant human FTVI enzyme was created and used for hMSC exofucosylation as previously described [15 ].

Pachón-Peña G., Donnelly C., Ruiz-Cañada C., Katz A., Fernández-Veledo S., Vendrell J, & Sackstein R. (2017). A Glycovariant of Human CD44 is Characteristically Expressed on Human Mesenchymal Stem Cells. Stem cells (Dayton, Ohio), 35(4), 1080-1092.

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CD44 N-Glycan and Sialic Acid Removal

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To remove N-glycans, CD44 immunoprecipitates were treated with Peptide-N-Glycosidase (PNGase-F, New England Biolabs) according to the manufacturer’s instructions. Sialic acid residues were removed by treatment with 200 mU/mL Vibrio Cholerae sialidase (Roche Molecular Biochemicals) at 37°C for 1 hour. Cell surface proteins were cleaved with 0.1% bromelain (Sigma-Aldrich) for 1 hour at 37°C, and efficiency was assessed by flow cytometry staining for residual CD44 expression. For α(1,3)-exofucosylation, cells were treated with 0.07 mg/mL of fucosyltransferase VII (FTVII) (R&D systems) in HBSS buffer containing 10 mM HEPES, 0.1% human serum albumin and 1 mM GDP-fucose (Sigma-Aldrich) for 60 minutes at 37°C. As controls, cells were suspended in the same HBSS buffer but without addition of FTVII (buffer-treated).

Silva M., Fung R.K., Donnelly C.B., Videira P.A, & Sackstein R. (2017). Cell–specific Variation in E-selectin Ligand Expression Among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology. Journal of immunology (Baltimore, Md. : 1950), 198(9), 3576-3587.

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In Vitro Fucosylation of HSCs

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The fucosylation treatment was performed as described previously.⁸⁶ (link) Briefly, 18Lin^negCD34^neg CD133^posHSCs were harvested, washed twice with Hank’s Balanced Salt Solution (HBSS), and resuspended at a density of in FTVI reaction buffer [25 mM HEPES (pH 7.5) (Gibco Invitrogen), 0.1% human serum albumin (Sigma-Aldrich), 0.5 mM GDP-fucose (Sigma), and 5 mM MnCl₂] and 1 ug purified rhFTVI enzyme in HBSS. Cells were incubated at 37°C for 30 min. Buffer only controls without the rhFTVI enzyme were used as a negative control. After the reaction, the cells were washed twice with HBSS and 10 mM EDTA and used immediately for experiments.

Al-Amoodi A.S., Kai J., Li Y., Malki J.S., Alghamdi A., Al-Ghuneim A., Saera-Vila A., Habuchi S, & Merzaban J.S. (2024). α1,3-fucosylation treatment improves cord blood CD34 negative hematopoietic stem cell navigation. iScience, 27(2), 108882.

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NA Glycosylation and Mass Spectrometry

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Ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), iodomethane (CH₃I), sodium borohydride (NaBH₄), 2,5-dihydroxybenzoic acid (DHB), urea, cellulose (medium fibrous), trypsin, HPLC grade ethanol, 1-butanol, and GDP-fucose were from Sigma-Aldrich. Sep-Pak C18 SPE cartridge was from Waters. PNGase F was from New England Biolabs. The NuPAGE series of LDS sample buffer (4×), MOPS SDS running buffer (20×), SimplyBlue SafeStain and 4-12% Bis-Tris Gels were from Life Technologies. 10,000 MWCO centrifugal devices were obtained from Millipore. NA was expressed with N-terminal His tag in sf21 insect cells and purified using nickel-histidine affinity chromatography followed by gel filtration as previously described [11 (link)]. Recombinant FUT8, FUT11, GDP-azido-fucose, biotinylated alkyne, and streptavidin-HRP were from R&D Systems.

Wu Z.L., Zhou H., Ethen C.M, & Reinhold V. (2016). Core-6 Fucose and the Oligomerization of the 1918 Pandemic Influenza Viral Neuraminidase. Biochemical and biophysical research communications, 473(2), 524-529.

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α1,3-Fucosyltransferase Activity Assay

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α1,3-FUT activity was measured in whole tissue lysates. The assay mixture contained 50 mM Na/cacodylate buffer pH 6.5, 15 mM MnCl₂, 0.5% Triton X-100, 5 mM ATP, 0.1 mM unlabelled GDP-fucose from Sigma-Aldrich (Merck KGaA), 55000 dpm GDP-[¹⁴C] fucose (PerkinElmer, Inc.) and 300 µg fetuin (Sigma-Aldrich; Merck KGaA), theoretically corresponding to 0.8 mM Siaα2,3Galβ1,4GlcNAc-R acceptor sites. The enzyme reaction was performed in triplicate at 37°C for 2 h, and then the products were precipitated, washed and counted by liquid scintillation. Controls without the acceptor (fetuin) were run in parallel, and the incorporation was subtracted. Homogenates of COLO-205 cells (28 (link)) (kindly provided by Professor Fabio Dall'Olio, University of Bologna, Italy) were used as positive controls.

Ferreira I.G., Carrascal M., Mineiro A.G., Bugalho A., Borralho P., Silva Z., Dall'olio F, & Videira P.A. (2019). Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E-selectin ligand in non-small cell lung cancer. International Journal of Oncology, 55(5), 1033-1048.

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Fucosylation of Bone Marrow Stem Cells

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BMSCs were digested and washed twice with Ca²⁺- and Mg²⁺- free Hanks Balanced Salt Solution (HBSS) (Gibco, NY, USA). After centrifugation, the cell precipitates were treated with fucosyltransferase VII (FTVII) reaction buffer system, containing the 60 mU/mL FTVII (Cusabio, WuHan, China), 20 mM HEPES (Sigma, MissouriState, USA), 1% human serum albumin (HSA, Sigma, MissouriState, USA) and 1 mM GDP-fucose (Sigma, MissouriState, USA) in HBSS. The mixture was then incubated in a 5% CO₂ incubator at 37℃ for 60 min, and mixed once every 15 min. The cells were then washed with HBSS containing 1% HSA and 20 mM HEPES and suspended in 1× phosphate-buffered saline (PBS) for later use. Cell viability was assessed by trypan blue staining.

Shi H., Jiang C., Yao H., Zhang Y., Zhang Q., Hou X, & Lin R. (2021). CD44 fucosylation on bone marrow-derived mesenchymal stem cells enhances homing and promotes enteric nervous system remodeling in diabetic mice. Cell & Bioscience, 11, 118.

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Quantification of FTVI Enzyme Activity

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Recombinant FTVI enzyme was produced in CHO cells by established techniques [15 (link)], using cDNA encoding amino acids 35-359 of the FTVI protein sequence; this sequence omits the cytoplasmic and transmembrane regions of FTVI, and encompasses the entire stem and catalytic domain of the enzyme. The specific activity of the purified enzyme was determined using the Glycosyltransferase Activity Kit (R&D Systems), as per the manufacturer’s instructions. Briefly, 0.1 μg of recombinant FTVI, 1 μL of ENTPD3/CD39L3 phosphatase, 15 nmol of N-acetyl-D-lactosamine (V-labs Inc), and 4 nmol of GDP-Fucose (Sigma-Aldrich) were mixed in 50 μL reaction buffer (25 mM Tris, 10 mM CaCl₂ and 10 mM MnCl₂, pH 7.5) and incubated in a 96-well plate at 37°C for 20 minutes. A second reaction that contained the same components except the recombinant FTVI was performed as a negative control. Reactions were terminated by the addition of 30 μL of Malachite Green Reagent A and 100 μL of water to each well. Color was developed by the addition of 30 μL of Malachite Green Reagent B to each well followed by gentle mixing and incubation at room temperature for 20 minutes. The plate was read at 620 nm using a multi-well plate reader. Phosphate standards were used to generate a calibration curve, and the specific activity of the FTVI enzyme was determined to be 60 pmol/min/μg.

Dykstra B., Lee J., Mortensen L.J., Yu H., Wu Z.L., Lin C.P., Rossi D.J, & Sackstein R. (2016). Glycoengineering of E-selectin ligands by intracellular versus extracellular fucosylation differentially affects osteotropism of human mesenchymal stem cells. Stem cells (Dayton, Ohio), 34(10), 2501-2511.

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Fucosylation of Murine and Human MSCs

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Murine AdMSCs were derived from C57BL/6 mice, the recipient strain for MHC-mismatched HSCT, whereas hAdMSCs and hBMMSCs were isolated from healthy human donors. Fucose was stereoselectively installed onto sialyllactosaminyl glycans of CD44 using an α(1,3)-linkage-specific fucosyltransferase, fucosyltransferase VII (FTVII; obtained from R&D Systems), in presence of donor fucose substrate (GDP-fucose; Sigma Aldrich): MSCs were resuspended at 2 × 10⁷ cells/ml and incubated for 60 min at 37 °C in FTVII reaction buffer composed of Hank’s Balanced Salt Solution (HBSS) (without Ca²⁺ and Mg²⁺) (Lonza) containing 20 mM HEPES (Lonza), 0.1% human serum albumin (HSA) (Grifols), 30 μg/ml FTVII (R&D Systems), and 1 mM GDP-fucose (Fucosylation-modified, “FucmAdMSCs”). Controls consisted of MSCs treated with reaction buffer alone (i.e., Unmodified MSCs, “UmAdMSCs” or, “UhAdMSCs” and “UhBMMSCs”). Exofucosylation efficacy was measured by analysis of HECA452 antibody (10 μg/ml, BD Biosciences, Cat#555946) staining and murine E-selectin-human Fc chimera (mE-Ig; 5 μg/ml, R&D Systems, Cat#575-ES-100) binding by flow cytometry and western blot.

García-Bernal D., Blanquer M., Martínez C.M., García-Guillén A.I., García-Hernández A.M., Carmen Algueró M., Yáñez R., Lamana M.L., Moraleda J.M, & Sackstein R. (2022). Enforced mesenchymal stem cell tissue colonization counteracts immunopathology. NPJ Regenerative Medicine, 7, 61.

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Enzymatic Exofucosylation of Murine Pericytes

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Murine pericytes were modified by enzymatic exofucosylation as previously reported (Garcia-Bernal et al., 2020 (link)). Briefly, cells were resuspended at 2 × 10⁷ cells/ml in fucosyltransferase VII (FTVII) reaction buffer composed of Hanks Balanced Salt Solution (HBSS, Gibco) containing 30 μg/ml fucosyltransferase VII (FTVII, R&D Systems), 20 mM HEPES (Thermo Fisher Scientific), 0.1% human serum albumin (Grifols) and 1 mM guanosine 5′-diphospho-β-L-fucose sodium salt (GDP-fucose, Sigma Aldrich), and incubated for 60 min at 37°C and 5% CO₂. Unmodified controls pericytes were treated only with GDP-fucose (w/o FTVII) in the same conditions as above. Cell viability after exofucosylation was assessed by trypan blue exclusion (usually 95% live cells). Efficacy of exofucosylation was evaluated by analysis of HECA452 antibody (BD Biosciences) staining and calcium-dependent mouse E-selectin/human IgG chimera (R&D Systems) binding by flow cytometry.

Molina M.L., García-Bernal D., Salinas M.D., Rubio G., Aparicio P., Moraleda J.M., Martínez S, & Valdor R. (2022). Chaperone-Mediated Autophagy Ablation in Pericytes Reveals New Glioblastoma Prognostic Markers and Efficient Treatment Against Tumor Progression. Frontiers in Cell and Developmental Biology, 10, 797945.

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Enzymatic Characterization of ppGalNAcT

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The enzyme activity of ppGalNAcT and ΔppGalNAcT was determined in 20 μl reaction mixture containing 50 mM MES (2-(N-morpholino)ethanesulfonic acid), pH 7.0, 10 mM MnCl₂, 2 mM UDP-GalNAc (Sigma-Aldrich, Vienna, Austria), 10 nmol acceptor peptide (Cellmano Biotech Co., Ltd., Shanghai, China) and 2 μl enzyme solution (ppGalNAcT or ΔppGalNAcT, protein concentrations 5.0 μg/ml) at 37 °C for 90 min.
For determination of donor specificity UDP-Gal, UDP-GlcNAc, GDP-fucose, UDP-xylose, UDP-glucuronic acid (all from Sigma-Aldrich, Vienna, Austria) and CMP-neuraminic acid (Jennewein Biotechnologie GmBH, Germany) were used instead of UDP-GalNAc. For time courses the standard assay was conducted for 30 h and aliquots were taken at 1, 2, 4, 6, 8, 23, 26 and 30 h. For determination of the position of the transferred GalNAc-residues within the peptide, incubation was carried out with Muc1a, Muc1a’, Muc5Ac and Muc2, respectively (for sequences see Table 1), for 24 h analogously to the standard assay with additions of 10 nmol UDP-GalNAc and 2 μl of enzyme solution after 4, 8 and 20 h.
Each assay was carried out at least in duplicate with appropriate controls.

Taus C., Windwarder M., Altmann F., Grabherr R, & Staudacher E. (2014). UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferase from the snail Biomphalaria glabrata – substrate specificity and preference of glycosylation sites. Glycoconjugate Journal, 31(9), 661-670.

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