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Abstract：
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.
摘要：
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。目前,GBP的聚糖特异性通常是从使用聚糖阵列生成的结合数据中推断出来的，其中GBP与固定在玻璃表面上的寡糖一起孵育。聚糖-GBP结合的检测通常是基于荧光的，涉及用荧光团或生物素标记GBP，与荧光标记的链霉亲和素结合，或使用识别GBP的荧光团标记的抗体。虽然已知GBP的共价标记可能会影响其结合特性，这些影响尚未得到很好的研究,在分析聚糖阵列数据时通常会被忽略.在本研究中,电喷雾质谱(ESI-MS)用于定量评价GBP标记对寡糖亲和力和特异性的影响。三种常见标签方法的影响，生物素化,用荧光染料标记并引入碘化试剂，评估了一系列人乳和血型寡糖对人半乳糖凝集素-3的C末端片段的亲和力。在所有情况下，标记导致寡糖亲和力的可测量降低，高达90%，变化的幅度对配体的性质敏感。这些发现表明，GBP标记可能会影响绝对和相对亲和力，因此，掩盖了真正的聚糖结合特性。这些结果还用于说明直接ESI-MS测定用于定量评估蛋白质标记对配体结合的影响的实用性。
