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.
Highlights
A carbohydrate structure of particular importance in O-glycosylation of mammalian glycoproteins is the ThomsenFriedenreich-antigen (TF-antigen; Gal(β1–3)GalNAc(α1-OSer/Thr; core 1)
We reported on the generation of the galactosynthase BgaC/Glu233Gly by rational design based on the crystal structure of BgaC from B. circulans and its use in combination with glycosyltransferases for the synthesis of various type 1 and type 2 poly-LacNAc structures [23, 24]
The purified product 6 was used for the synthesis of BSAbased neo-glycoproteins (NGPs 12) (Scheme 1B)
Summary
A carbohydrate structure of particular importance in O-glycosylation of mammalian glycoproteins is the ThomsenFriedenreich-antigen (TF-antigen; Gal(β1–3)GalNAc(α1-OSer/Thr; core 1). Deriving from its precursor GalNAc(α-OSer/Thr (Tn-antigen), the TF-antigen serves as scaffold for longer and more complex mucin-type O-glycan structures [1]. High levels of Tn- and TF-antigen occur in 70–90% of all human carcinomas [1, 2]. They are mostly present on the cell-surface bound glycoprotein mucin-1 (MUC1) and considered as “pancarcinoma antigens” [3]. Gal-3 is expressed on the outer cell membrane of endothelial cells as well as carcinoma cells and binds to the TF-antigen presented on MUC1 under static and flow conditions [9].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
