Glycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan-GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP. While it is known that covalent labeling of a GBP may influence its binding properties, these effects have not been well studied and are usually overlooked when analyzing glycan array data. In the present study, electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively evaluate the impact of GBP labeling on oligosaccharide affinities and specificities. The influence of three common labeling approaches, biotinylation, labeling with a fluorescent dye and introducing an iodination reagent, on the affinities of a series of human milk and blood group oligosaccharides for a C-terminal fragment of human galectin-3 was evaluated. In all cases labeling resulted in a measurable decrease in oligosaccharide affinity, by as much as 90%, and the magnitude of the change was sensitive to the nature of the ligand. These findings demonstrate that GBP labeling may affect both the absolute and relative affinities and, thereby, obscure the true glycan binding properties. These results also serve to illustrate the utility of the direct ESI-MS assay for quantitatively evaluating the effects of protein labeling on ligand binding.

中文翻译：

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标记对聚糖亲和力和聚糖结合蛋白特异性的影响。涉及人半乳凝素3 C端片段的案例研究。

聚糖与聚糖结合蛋白（GBP）的相互作用在多种细胞过程中起着至关重要的作用。当前，GBP的聚糖特异性最经常从使用聚糖阵列产生的结合数据推断，其中GBP与固定在玻璃表面上的寡糖一起温育。聚糖-GBP结合的检测通常是基于荧光的，包括用荧光团或生物素标记GBP，该生物素与荧光团标记的链霉亲和素结合，或使用能够识别GBP的荧光团标记的抗体。虽然众所周知，GBP的共价标记可能会影响其结合特性，但尚未对这些作用进行深入研究，而在分析聚糖阵列数据时通常忽略了这些作用。在目前的研究中，电喷雾电离质谱（ESI-MS）用于定量评估GBP标记对寡糖亲和力和特异性的影响。评估了三种常见的标记方法，生物素化，荧光染料标记和加碘试剂，对一系列人乳和血型低聚糖对人半乳糖凝集素3的C末端片段的亲和力的影响。在所有情况下，标记都会导致可测量的寡糖亲和力下降多达90％，并且变化的幅度对配体的性质敏感。这些发现表明，GBP标记可能同时影响绝对亲和力和相对亲和力，从而掩盖了真正的聚糖结合特性。