Eriodictyol suppresses the malignant progression of colorectal cancer by downregulating tissue specific transplantation antigen P35B (TSTA3) expression to restrain fucosylation.

Eriodictyol is a natural flavonoid with many pharmacological effects, such as anti-oxidation, anti-inflammation, anti-tumor, and neuroprotection. Besides, it has been reported that flavonoids play an important role in protein glycosylation. The fucosylation structure is closely associated with processes of various tumor metastases. TSTA3 is involved in the de novo synthesis and can convert cellular GDP-D-mannose into GDP-L-fucose. It was predicted on the STITCH database that eriodictyol interacted with TSTA3. In addition, literature has confirmed that TSTA3 is upregulated in CRC and can regulate the proliferation and migration of breast cancer cells. Herein, the precise effects of eriodictyol on the clone-forming, proliferative, migratory and invasive abilities of CRC cells as well as EMT process were assessed. Moreover, the correlation among eriodictyol, TSTA3, and fucosylation in these malignant behaviors of CRC cells was evaluated, in order to elucidate the underlying mechanism. The current work discovered that eriodictyol inhibited the viability, clone-formation, proliferation, migration, invasion, and EMT of CRC cells, and that these inhibitory effects of eriodictyol on the malignant behavior of CRC cells were reversed by TSTA3 overexpression. Additionally, eriodictyol suppresses fucosylation by downregulating the TSTA3 expression. Results confirmed that fucosylation inhibitor (2-F-Fuc) inhibited clone formation, proliferation, migration, invasion, as well as EMT of CRC cells and eriodictyol treatment further reinforced the suppressing effects of 2-F-Fuc on the malignant behavior of CRC cells. We conclude that eriodictyol suppresses the clone-forming, proliferative, migrative and invasive abilities of CRC cells as well as represses the EMT process by downregulating TSTA3 expression to restrain fucosylation.

Homing Genes Expression in Fucosyltransferase VI-Treated Umbilical Cord Blood CD133+ Cells which Expanded on Protein-Coated Nanoscaffolds.

Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) are considered because of their self-renewing, differentiating, proliferating, and readily available properties. Moreover, HSCs' homing to the hematopoietic microenvironment is an important step in their transplantation process. But low content of progenitor cells in one unit of UCB and defect in the bone marrow (BM) homing limit their applications. Hence, we decided to correct this deficiency with ex vivo incubation of CD133+ cells using fucosyltransferase VI and GDP-fucose. Then C-X-C chemokines receptor-4 (CXCR4), very late activation antigen-4 (VLA4), very late activation antigen-5 (VLA5), lymphocyte function-associated antigen-1 (LFA-1), and E-cadherin (E-cad) genes expressions were investigated with the goal of homing evaluation. The purity of MACS isolated CD133+ cells and confirmation of fucosylation were done by flow cytometry, and the viability of cells seeded on protein-coated poly L-lactic acid (PLLA) scaffold was proven via MTT assay. Scanning electron microscopy (SEM), CFU assays, and expression assays of CXCR4, VLA4, VLA5, LFA-1 and E-cad by real-time PCR were performed, too. Flow cytometry data showed that isolated cells were suitable for fucosyltransferase VI (FT-VI) incubation and expansion on nanoscaffolds. MTT, CFU assays, and SEM micrographs demonstrated fibronectin (FN)-collagen-selectin (FCS)-coated scaffold serve as best environment for viability, clonogenicity, and cell attachment. High levels of homing genes expression were also observed in cells seeded on FCS-coated scaffolds. Also, CXCR4 flow cytometry analysis confirmed real-time data. FCS-PLLA scaffolds provided optimal conditions for viability of FT-VI-treated CD133+ cells, and clonogenicity with the goal of improving homing following UCB-HSCs transplantation.

Development of α1,6-fucosyltransferase inhibitors through the diversity-oriented syntheses of GDP-fucose mimics using the coupling between alkyne and sulfonyl azide.

We developed α1,6-fucosyltransferase (FUT8) inhibitors through a diversity-oriented synthesis. The coupling reaction between the fucose unit containing alkyne and the guanine unit containing sulfonyl azide under various conditions afforded a series of Guanosine 5'-diphospho-ß-l-fucose (GDP-fucose) analogs. The synthesized compounds displayed FUT8 inhibition activity. A docking study revealed that the binding mode of the inhibitor synthesized with FUT8 was similar to that of GDP-fucose.

Inhibition kinetics of carba- and C-fucosyl analogues of GDP-fucose against fucosyltransferase V: implication for the reaction mechanism.

Inhibition kinetics of two isosteric analogues of GDP-fucose (GDP-Fuc) were investigated against fucosyltransferase V using electrospray ionization mass spectrometry coupled to multiple reaction monitoring. The carba-Fuc analogue was found to be a competitive inhibitor with a K(i) value of 67.1+/-9.8 microM, similar to the K(m) value for GDP-Fuc (50.4+/-5.5 microM), while the C-Fuc analogue exhibited significantly weak competitive inhibition with a K(i) value of 889+/-93 microM.

Chemical modification of an alpha 3-fucosyltransferase; definition of amino acid residues essential for enzyme activity.

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) is dependent on the activity of an alpha 3-fucosyltransferase (EC 2.4.1.152, GDP-fucose:Gal beta (1-4)GlcNAc-R alpha (1-3)fucosyltransferase). This enzyme catalyses the transfer of fucose from GDP-beta-fucose to the 3-OH of N-acetylglucosamine present in lactosamine acceptors. In this report, we have investigated the amino acids essential for the activity of a recombinant alpha 3-fucosyltransferase (FucT-VI) through chemical modification of the enzyme with group-selective reagents. FucT-VI activity was found to be particularly sensitive to the histidine-selective reagent diethylpyrocarbonate and the cysteine reagent N-ethylmaleimide, with IC50 values of less than 200 microM. Reagents selective for arginine and lysine had no effect on enzyme activity. The inclusion of GDP-beta-fucose during preincubation with NEM reduces the rate of inactivation whereas inclusion of an acceptor saccharide for the enzyme, Gal beta (1-4)GlcNAc, had no effect. No protective effect with either GDP-beta-fucose or Gal beta (1-4)GlcNAc was observed on treatment of the enzyme with diethylpyrocarbonate. These data suggest that in addition to an NEM-reactive cysteine in, or adjacent to, the substrate-binding site of the enzyme, FucT-VI possesses histidine residue(s) that are essential for enzyme activity.

Presence of an essential lysine residue in a GDP-fucose protected site of the alpha 1----3fucosyltransferase from human small cell lung carcinoma NCl-H69 cells.

The NCI-H69 cell alpha 1----3fucosyltransferase has been purified from a 0.2% Triton X-100R solubilized enzyme fraction by GDP-hexanolamine-Sepharose affinity chromatography and Superose 12 gel filtration. Photoaffinity labeling experiments with 125I-GDP-hexanolaminyl-4-azidosalicylic acid present in concentrations equivalent to 0.5 and 1 times Ki of the inhibitor for the enzyme indicated that labeling of the 45-kDa protein band could be inhibited by addition of 400 microM GDP-fucose but was not effected by similar concentrations of either GDP-mannose or GDP-glucose. The purified enzyme was applied to studies intended to define catalytically essential amino acid residues of the protein. Incubation of the enzyme in the presence of increasing concentrations of pyridoxal 5'-phosphate was found to result in irreversible inactivation of the enzyme after NaBH4 reduction. The donor substrate, GDP-fucose, was found to protect the enzyme from inactivation. Little or no protection was found for either GDP-mannose or the acceptor substrate nLc4. Pyridoxal 5'-phosphate was shown to behave as a competitive inhibitor with respect to GDP-fucose with a Ki of 105 microM. Labeling with 3H-pyridoxal 5'-phosphate resulted in the incorporation of approximately 8 mol pyridoxal 5'-phosphate per mole subunit. Parallel experiments containing GDP-fucose indicated protection of one site per subunit correlated with GDP-fucose binding. Acid hydrolysis and chromatographic analysis of the 3H-pyridoxylated protein indicated greater than 95% of the 3H label was recovered as pyridoxyl-lysine irrespective of whether GDP-fucose was present or not during labeling. These studies indicate the presence of a catalytically essential lysine residue associated with GDP-fucose binding to this enzyme. This information will be of value in further studies of this and other alpha 1----3fucosyltransferases and may suggest a practical basis for modulation of enzyme activity in the cell.