Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße. 20, 52074, Aachen, Germany.
Institute for Bioorganic Chemistry, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich, 52426, Jülich, Germany.
Glycoconj J. 2020 Aug;37(4):457-470. doi: 10.1007/s10719-020-09926-y. Epub 2020 May 4.
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.
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表面。它与半乳糖凝集素 1 和半乳糖凝集素 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 抗原与半乳糖凝集素 3 之间的多价结合，使用糖苷合成酶 BgaC/Glu233Gly 以 GalNAc(α1-EG3-叠氮化物作为受体底物，以高产量酶促合成 TF 抗原。随后，通过铜（I）催化的炔基-叠氮化物环加成将其偶联到炔基功能化的牛血清白蛋白上。该程序产生了具有可调节聚糖多价性的新型糖蛋白，用于结合研究。通过调节 BSA 赖氨酸残基的炔基修饰，获得了每蛋白分子 2 至 53 个糖基残基的糖密度。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测定偶联的糖数量。使用酶联凝集素测定法研究了新型糖蛋白与人类半乳糖凝集素 3 的结合效率及其多价性的影响。所有修饰密度的固定化新型糖蛋白均显示出随着糖密度增加与 Gal-3 的结合。然而，多价糖呈现并没有导致更高的结合亲和力。相反，抑制 Gal-3 与无唾液酸胎球蛋白的结合是有效的。与仅增加 2 倍的高度修饰抑制剂相比，显示 10 个糖/蛋白的新型糖蛋白的相对抑制效力增加了 142 倍。总之，证明了基于 BSA 的新型糖蛋白作为 Gal-3 的多价抑制剂呈现 TF 抗原的功能。
