Synthesis of the Thomsen-Friedenreich-antigen (TF-antigen) and binding of Galectin-3 to TF-antigen presenting neo-glycoproteins

Abstract

The Thomsen-Friedenreich-antigen, Gal(β1–3)GalNAc(α1-O-Ser/Thr (TF-antigen), is presented on the surface of most human cancer cell types. Its interaction with galectin 1 and galectin 3 leads to tumor cell aggregation and promotes cancer metastasis and T-cell apoptosis in epithelial tissue. To further explore multivalent binding between the TF-antigen and galectin-3, the TF-antigen was enzymatically synthesized in high yields with GalNAc(α1-EG3-azide as the acceptor substrate by use of the glycosynthase BgaC/Glu233Gly. Subsequently, it was coupled to alkynyl-functionalized bovine serum albumin via a copper(I)-catalyzed alkyne-azide cycloaddition. This procedure yielded neo-glycoproteins with tunable glycan multivalency for binding studies. Glycan densities between 2 and 53 glycan residues per protein molecule were obtained by regulated alkynyl-modification of the lysine residues of BSA. The number of coupled glycans was quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a trinitrobenzene sulfonic acid assay. The binding efficiency of the neo-glycoproteins with human galectin-3 and the effect of multivalency was investigated and assessed using an enzyme-linked lectin assay. Immobilized neo-glycoproteins of all modification densities showed binding of Gal-3 with increasing glycan density. However, multivalent glycan presentation did not result in a higher binding affinity. In contrast, inhibition of Gal-3 binding to asialofetuin was effective. The relative inhibitory potency was increased by a factor of 142 for neo-glycoproteins displaying 10 glycans/protein in contrast to highly decorated inhibitors with only 2-fold increase. In summary, the functionality of BSA-based neo-glycoproteins presenting the TF-antigen as multivalent inhibitors for Gal-3 was demonstrated.

Electrochemical Impedance Spectroscopy Biosensor Enabling Kinetic Monitoring of Fucosyltransferase Activity

“…The BSA and NGPs were functionalized with an NHS-PEGalkyne linker (tebu-bio GmbH, Le Perray-en-Yvelines, France) as previously described. 30 First, the linker was incubated (in excess, 1.1fold) with 30 μM BSA or NGP in phosphate-buffered saline (PBS, pH 7.4) for 1.5 h at room temperature. The modified proteins were separated from the remaining reagents by ultrafiltration.…”

“…After washing with PBST (PBS containing 0.02% Tween), alkyne-functionalized BSA or NGP (10 μM, 50 μL) in PBS (pH 7.4) supplemented with 5 mM sodium ascorbate, 0.5 mM THPTA, and 0.1 mM CuSO 4 for the CuAAC reaction was applied to the chips and incubated for another 30 min. 30,31 Sensor Fabrication. EIS biosensors were fabricated on 4″ borosilicate glass wafers (Siegert Wafer GmbH, Aachen, Germany).…”

“…The immobilization of proteins to the gold surface was performed using click chemistry via CuAAC. The BSA and NGPs were functionalized with an NHS-PEG-alkyne linker (tebu-bio GmbH, Le Perray-en-Yvelines, France) as previously described . First, the linker was incubated (in excess, 1.1-fold) with 30 μM BSA or NGP in phosphate-buffered saline (PBS, pH 7.4) for 1.5 h at room temperature.…”

“…Alkyne-functionalized BSA was further biotinylated as described by the manufacturer (EZ-Link Sulfo-LC-LC-NHS-Biotin, Thermo Fisher Scientific, Waltham, MA, USA) and served as a control for the CuAAC reaction. After washing with PBST (PBS containing 0.02% Tween), alkyne-functionalized BSA or NGP (10 μM, 50 μL) in PBS (pH 7.4) supplemented with 5 mM sodium ascorbate, 0.5 mM THPTA, and 0.1 mM CuSO 4 for the CuAAC reaction was applied to the chips and incubated for another 30 min. , …”

Galectins in Cancer and the Microenvironment: Functional Roles, Therapeutic Developments, and Perspectives

“…Evidence also shows that recombinant Gal-3C regulates the integrin/FAK/SRC/NDRG1 axis to suppress hepatocellular carcinoma progression [ 176 ]. Identifying peptides that can interfere with the carbohydrate recognition domain of specific Gals, such as Thomsen–Friedenreich antigen-specific peptide P-30, may disrupt Gal functions [ 177 , 178 ]. Lian and coworkers identified the Gal-binding peptide G3-C12-HPMA-KLA, which has dual effects on cancer cells and subcellular mitochondria [ 179 ].…”