We present the purification and characterization of the two most abundant isoforms of lectins isolated from Tepary bean (Phaseolus acutifolius) seeds, which have been shown to differentially affect the survival of different cancer cells. They were separated by concanavalin A-affinity chromatography. After purification, to release the N-glycans, they were digested with the endoglycosidases PNGase and Glycanase A. Fractions resulted from the hydrolysis products were analyzed to determine their carbohydrate composition. Mass spectrometry data indicated that both isoforms contained high mannose glycans being mannose 6 the most abundant form. Furthermore, based on sequence Ans-X-Ser/Thr, where X is any amino acid except proline, a glycosylation site was determined on asparagine 36. When their metal requirement to preserve their biological activity was determined, the lectins showed differences. While lectin A (LA) agglutination activity was best in the presence of magnesium, lectin B (LB) was best with calcium. Additionally, only LA exhibited affinity to human type-A erythrocytes. Although both lectins showed small differences in their properties, an identical structure-model for both lectins was generated by the homology modelling process. Also, the analysis of ligand binding sites and in silico glycosylation were achieved. Molecular docking with colon adenocarcinoma associated-N-glycans revealed some highly possible interactions and, on the other hand, that N-glycan interaction zones of Tepary bean lectins is not restricted to the carbohydrate binding domain but to an extended part of their surface, which could lead new strategies to explain their biological activity.

Graphical Abstract

中文翻译：

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两种非胎球蛋白结合的 Tepary 豆凝集素与癌相关聚糖的比较研究、同源建模和分子对接

我们介绍了从 Tepary 豆 ( Phaseolu s acutifolius ) 种子中分离出的两种最丰富的凝集素亚型的纯化和表征，这两种亚型已被证明对不同癌细胞的存活有不同的影响。它们通过刀豆球蛋白 A 亲和层析分离。纯化后释放N-聚糖，它们被内切糖苷酶 PNGase 和聚糖酶 A 消化。分析水解产物产生的级分以确定它们的碳水化合物组成。质谱数据表明，两种亚型都含有高甘露糖聚糖，甘露糖 6 是最丰富的形式。此外，基于 Ans-X-Ser/Thr 序列，其中 X 是除脯氨酸以外的任何氨基酸，确定了天冬酰胺 36 上的糖基化位点。当确定了它们保持生物活性的金属需求时，凝集素显示出差异。凝集素 A (LA) 的凝集活性在镁存在时最佳，而凝集素 B (LB) 在​​钙存在时最佳。此外，只有 LA 表现出对人类 A 型红细胞的亲和力。尽管这两种凝集素在它们的特性上表现出微小的差异，同源建模过程生成了两种凝集素的相同结构模型。此外，配体结合位点的分析和实现了计算机糖基化。与结肠腺癌相关的N-聚糖的分子对接揭示了一些高度可能的相互作用，另一方面， Tepary 豆凝集素的N-聚糖相互作用区不限于碳水化合物结合域，而是限于其表面的延伸部分，这可能会导致新的策略来解释它们的生物活性。

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