Centro de Investigación y de Estudios Avanzados. Unidad Irapuato. Departamento de Biotecnología y Bioquímica., Libramiento Norte. Carretera Irapuato-León. Km. 9.6, 36824, Irapuato, Guanajuato, México.
Universidad Autónoma de Querétaro. Campus Juriquilla. Facultad de Ciencias Naturales., Av. de las Ciencias s/n, Juriquilla, 76230, Santiago de Querétaro, Querétaro, México.
Glycoconj J. 2023 Feb;40(1):69-84. doi: 10.1007/s10719-022-10091-7. Epub 2022 Nov 17.
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.
我们展示了从 Tepary 豆（Phaseolus acutifolius）种子中分离出的两种最丰富的凝集素同型体的纯化和特性，它们已被证明会对不同癌细胞的存活产生不同的影响。它们通过伴刀豆球蛋白 A 亲和层析分离。纯化后，为了释放 N-聚糖，用内切糖苷酶 PNGase 和 Glycanase A 进行消化。水解产物的馏分用于分析其碳水化合物组成。质谱数据表明，两种同型体都含有高甘露糖聚糖，其中甘露糖 6 是最丰富的形式。此外，根据序列 Ans-X-Ser/Thr，其中 X 是脯氨酸以外的任何氨基酸，在天冬酰胺 36 上确定了一个糖基化位点。当确定它们保留生物活性的金属需求时，凝集素表现出差异。虽然凝集素 A（LA）的凝集活性在镁存在下最佳，但凝集素 B（LB）在钙存在下最佳。此外，只有 LA 对人 A 型红细胞表现出亲和力。尽管两种凝集素在性质上略有差异，但通过同源建模过程生成了两种凝集素的相同结构模型。此外，还实现了配体结合位点的分析和计算机糖基化。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-聚糖结合区不仅限于碳水化合物结合域，而是其表面的扩展部分，这可能为解释其生物学活性提供新的策略。
