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.

中文翻译：

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Thomsen-Friedenreich抗原（TF抗原）的合成以及Galectin-3与TF抗原呈递的新糖蛋白的结合。

Thomsen-Friedenreich抗原，Gal（β1-3）GalNAc（α1- O-Ser / Thr（TF抗原）存在于大多数人类癌细胞类型的表面。它与半乳凝素1和半乳凝素3的相互作用导致肿瘤细胞聚集，并促进上皮组织中的癌症转移和T细胞凋亡。为了进一步探索TF抗原和galectin-3之间的多价结合，利用糖合酶BgaC / Glu233Gly以GalNAc（α1-EG3-叠氮化物为受体底物）高产率地酶促合成了TF抗原。通过铜（I）催化的炔-叠氮化物环加成反应与炔基官能化的牛血清白蛋白偶联，该程序生成了具有可调聚糖多价性的新糖蛋白，用于结合研究，通过调节，可得到每个蛋白分子2至53个聚糖残基的聚糖密度BSA赖氨酸残基的炔基修饰。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和三硝基苯磺酸测定定量偶联的聚糖的数量。使用酶联凝集素测定法研究和评估了新糖蛋白与人galectin-3的结合效率和多价效应。固定化的所有修饰密度的新糖蛋白均显示出Gal-3的结合与聚糖密度的增加有关。然而，多价聚糖呈递并未导致更高的结合亲和力。相反，抑制Gal-3与去唾液酸铁蛋白的结合是有效的。与新糖蛋白表现出10个聚糖/蛋白的相对抑制力相比，其抑制能力提高了142倍，而装饰性强的抑制剂仅增加了2倍。综上所述，