Böcker Sophia, Elling Lothar
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 20, 52074 Aachen, Germany.
Glycobiology. 2017 May 1;27(5):457-468. doi: 10.1093/glycob/cwx007.
Galectin-3 modulates cell adhesion and signaling events by specific binding and cross-linking galactoside containing carbohydrate ligands. Proteolytic cleavage by metalloproteinases yields in vivo N-terminally truncated galectin-3 still bearing the carbohydrate recognition domain. Truncated galectin-3 has been demonstrated to act in vivo as a negative inhibitor of galectin-3 due to higher affinity for carbohydrate ligands. We here present our studies on a series of 12 human galectin-3 protein constructs. Truncated galectin-3 (∆1-62 and ∆1-116) and fusions with SNAP-tag and/or yellow fluorescent protein (YFP) display altered binding efficiencies (ratio of maximum binding signal and apparent affinity constant Kd) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Δ1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Δ1-116) shows only weak binding. We demonstrate here for the first time that SNAP-tag and YFP fusions of galectin-3 and truncated galectin-3 proteins improve binding efficiencies to ASF. SNAP-tagged galectin-3, galectin-3(Δ1-62) and galectin-3(Δ1-116) are found with significant (3- to 6-fold) higher binding efficiencies in SPR when compared with native galectin-3. Fusion of truncated galectin-3 with YFP renders binding properties similar to native galectin-3, whereas in combination with SNAP-tag improved binding characteristics are obtained. Our results emphasize the importance of the N-terminal domain of human galectin-3 for ligand binding. Most importantly, in combination with fusion proteins suitable for the design of diagnostic and therapeutic tools binding properties can be beneficially tuned. The resulting novel protein tools may be advantageous for potential galectin-3 directed applications in tumor diagnostics and therapy.
半乳糖凝集素-3通过特异性结合和交联含半乳糖苷的碳水化合物配体来调节细胞黏附和信号转导事件。金属蛋白酶的蛋白水解切割在体内产生N端截短的半乳糖凝集素-3，其仍带有碳水化合物识别结构域。由于对碳水化合物配体具有更高的亲和力，截短的半乳糖凝集素-3已被证明在体内作为半乳糖凝集素-3的负性抑制剂发挥作用。我们在此展示了对一系列12种人半乳糖凝集素-3蛋白构建体的研究。截短的半乳糖凝集素-3（∆1-62和∆1-116）以及与SNAP标签和/或黄色荧光蛋白（YFP）的融合体在固相酶联免疫吸附测定（ELISA）和表面等离子体共振（SPR）结合测定中对去唾液酸胎球蛋白（ASF）显示出改变的结合效率（最大结合信号与表观亲和常数Kd的比值）。在ELISA和SPR实验中，半乳糖凝集素-3(Δ1-62)和全长（天然）半乳糖凝集素-3分别对ASF具有最高亲和力，而半乳糖凝集素-3(Δ1-116)仅显示出弱结合。我们在此首次证明半乳糖凝集素-3和截短的半乳糖凝集素-3蛋白的SNAP标签和YFP融合体提高了对ASF的结合效率。与天然半乳糖凝集素-3相比，在SPR中发现带有SNAP标签的半乳糖凝集素-3、半乳糖凝集素-3(Δ1-62)和半乳糖凝集素-3(Δ1-116)具有显著更高（3至6倍）的结合效率。截短的半乳糖凝集素-3与YFP融合后的结合特性与天然半乳糖凝集素-3相似，而与SNAP标签结合则获得了改善的结合特性。我们的结果强调了人半乳糖凝集素-3的N端结构域对配体结合的重要性。最重要的是，与适合设计诊断和治疗工具的融合蛋白相结合，可以有益地调节结合特性。由此产生的新型蛋白工具可能对潜在的半乳糖凝集素-3导向的肿瘤诊断和治疗应用具有优势。
