Laaf Dominic, Bojarová Pavla, Pelantová Helena, Křen Vladimír, Elling Lothar
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University , Pauwelsstrasse 20, 52074 Aachen, Germany.
Institute of Microbiology, Czech Academy of Sciences , Vídeňská 1083, 14220 Prague, Czech Republic.
Bioconjug Chem. 2017 Nov 15;28(11):2832-2840. doi: 10.1021/acs.bioconjchem.7b00520. Epub 2017 Oct 18.
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
半乳糖凝集素-3（Gal-3）是β-半乳糖苷结合凝集素家族的成员，是一种肿瘤生物标志物，参与肿瘤血管生成和转移。因此，Gal-3被认为是早期癌症诊断和抗癌治疗的一个有前景的靶点。我们在此展示了一系列定制的多价新糖蛋白文库的合成，并评估了它们与Gal-3的结合特性。通过糖基转移酶和化学酶促反应的组合使用，我们首先合成了一组基于N-乙酰乳糖胺（Galβ1,4GlcNAc；2型乳糖胺）的寡糖，分别具有五种不同的末端糖基化表位。通过将方酸二烷基酯与赖氨酸残基偶联，实现了牛血清白蛋白（BSA）的新糖基化，从而得到了一系列确定的多价新糖蛋白文库。用固定化新糖蛋白进行的固相结合试验揭示了多价聚糖表位与Gal-3结合的不同亲和力和特异性。特别是，用N'，N″-二乙酰乳糖胺（GalNAcβ1,4GlcNAc；乳糖二胺）表位修饰的新糖蛋白表现出高选择性，并被证明能以高亲和力从人血清中捕获Gal-3。此外，具有末端生物素化乳糖胺聚糖基序的新糖蛋白可作为夹心酶联免疫吸附测定形式的Gal-3检测剂。我们得出结论，与基于抗体的捕获步骤相比，所展示的新糖蛋白对于高选择性和高亲和力地检测功能完整的Gal-3非常有用。我们还利用定制的多价新糖蛋白对Gal-3的结合亲和力有了新的认识，这些新糖蛋白在癌症相关生物医学研究中具有应用潜力。
