Multiple approaches to assess pectin binding to galectin-3
Multiple approaches to assess pectin binding to galectin-3
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评估果胶与 galectin-3 结合的多种方法
DOI：
10.1016/j.ijbiomac.2016.06.058
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发表时间：
2016-10-01
影响因子：
8.2
通讯作者：
Tai, Guihua
中科院分区：
文献类型：
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作者：
Zhang, Tao;Zheng, Yi;Tai, Guihua
尽管已经使用了几种方法来评估碳水化合物与凝集素的结合，但结果并不总是可比的，尤其是对于较大的多糖。在这里，我们定量评估和比较果胶衍生的多糖半乳糖凝集素-3（Gal-3）的结合使用五种方法：表面等离子体共振（SPR），生物层干涉（BLI），荧光偏振（FP），竞争性荧光联免疫吸附（cFLISA），和众所周知的基于细胞的血凝试验（G3 H）。我们的研究表明，虽然通过SPR和BLI确定的Gal-3-果胶结合参数是相当的，并且与来自G3 H测定的抑制效力相关，但是使用FP和cFLISA测定的结果是高度可变的，并且在很大程度上取决于多糖的探针和质量。例如，在cFLISA测定中，当使用DTAF标记的脱唾液酸胎球蛋白探针时，果胶没有显示出抑制，但当使用DTAF标记的果胶探针时，果胶显示出抑制。使用DTAF-乳糖探针的FP方法对多糖和大的半乳聚糖链不起作用，尽管它对较小的半乳聚糖很有效。然而，尽管所有这些方法得出的结果基本一致，但得出的K-D、IC 50和MIC值确实不同。我们的结果反映了使用各种技术的可变性，因此将是有用的研究人员开发果胶衍生的Gal-3拮抗剂作为抗癌药物。(C)2016爱思唯尔B. V.保留所有权利。
Although several approaches have been used to evaluate binding of carbohydrates to lectins, results are not always comparable, especially with larger polysaccharides. Here, we quantitatively assessed and compared binding of pectin-derived polysaccharides to galectin-3 (Gal-3) using five methods: surface plasmon resonance (SPR), bio-layer interferometry (BLI), fluorescence polarization (FP), competitive fluorescence-linked immunosorbance (cFLISA), and the well-known cell-based hemagglutination assay (G3H). Our studies revealed that whereas Gal-3-pectin binding parameters determined by SPR and BLI were comparable and correlated with inhibitory potencies from the G3H assay, results using FP and cFLISA assays were highly variable and depended greatly on the probe and mass of the polysaccharide. In the cFLISA assay, for example, pectins showed no inhibition when using the DTAF-labeled asialofetuin probe, but did when using a DTAF-Iabeled pectin probe. And the FP approach with the DTAF-lactose probe did not work on polysaccharides and large galactan chains, although it did work well with smaller galactans. Nevertheless, even though results derived from all of these methods are in general agreement, derived K-D, IC50, and MIC values do differ. Our results reflect the variability using various techniques and therefore will be useful to investigators who are developing pectin-derived Gal-3 antagonists as anti-cancer agents. (C) 2016 Elsevier B.V. All rights reserved.