Binding characteristics of galectin-3 fusion proteins
Binding characteristics of galectin-3 fusion proteins
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DOI：
10.1093/glycob/cwx007
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发表时间：
2017-05-01
期刊：
影响因子：
4.3
通讯作者：
Elling, Lothar
中科院分区：
文献类型：
--
作者：
Boecker, Sophia;Elling, Lothar
Galectin-3通过含有碳水化合物配体的特殊结合和交联半乳糖苷来调节细胞黏附和信号事件。金属蛋白水解酶在体内产生N端截短的Galectin-3，仍然带有碳水化合物识别结构域。截短的Galectin-3在体内被证明是Galectin-3的负抑制物，因为它对碳水化合物配体有更高的亲和力。我们在这里介绍了我们对一系列12个人Galectin-3蛋白结构的研究。截短的Galectin-3(匕首1-62和匕首1-116)以及与SNAP-Tag和/或黄色荧光蛋白(YFP)的融合在固相酶联免疫吸附试验(ELISA)和表面等离子共振(SPR)结合试验中显示出与积雪草素(ASF)的结合效率(最大结合信号与表观亲和常数K-d之比)的改变。在ELISA和SPR实验中，Galectin-3(Delta 1-62)和全长(天然)Galectin-3分别与ASF有最高的亲和力，而Galectin-3(Delta 1-116)仅表现出弱结合。我们在这里首次证明了Galectin-3和截短Galectin-3蛋白的SNAP-Tag和YFP融合提高了与ASF的结合效率。与天然Galectin-3相比，Snap标记的Galectin-3、Galectin-3(Delta 1-62)和Galectin-3(Delta 1-116)与SPR的结合效率显著(3-6倍)高。截短的Galectin-3与YFP的融合使结合特性类似于天然Galectin-3，而与SNAP-Tag结合可获得更好的结合特性。我们的结果强调了人Galectin-3的N-末端结构域对配体结合的重要性。最重要的是，结合适合于诊断和治疗工具设计的融合蛋白，可以有益地调整结合特性。由此产生的新型蛋白质工具可能有利于Galectin-3在肿瘤诊断和治疗中的潜在应用。
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 (a dagger 1-62 and a dagger 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 K-d) to asialofetuin (ASF) in solid-phase enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) binding assays. Galectin-3(Delta 1-62) and full-length (native) galectin-3 have highest affinity to ASF in ELISA and SPR experiments, respectively, whereas galectin-3(Delta 1-116) 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(Delta 1-62) and galectin-3(Delta 1-116) 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.