Comparative study, homology modelling and molecular docking with cancer associated glycans of two non-fetuin-binding Tepary bean lectins.

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.

Tolerability assessment of a lectin fraction from Tepary bean seeds (Phaseolus acutifolius) orally administered to rats.

Our previous studies have shown that a lectin rich fraction (TBLF) extracted from Tepary bean seeds differentially inhibits cancer cells proliferation in vitro. Before testing the in vivo anticancer effect, the acute and subchronic toxicological assays in rats were conducted, where an oral dose of 50 mg/body weight kg was determined as the NOAEL. This study evaluated the resistance to digestion and complete blood count (CBC) after 24 h of the orally administered 50 mg/kg TBLF. The digestion resistance test showed lectins activity retention after 72 h and the CBC study showed a high level of eosinophils, suggesting an allergic-like response. Tolerability was assayed after 6 weeks of treatment by dosing with an intragastric cannula every third day per week. It was observed a transient reduction in food intake and body weight in the first weeks, resulting in body weight gain reduction of 10% respect to the control group at the end of the study. Additionally, organs weight, histopathological analysis and blood markers for nutritional status and for liver, pancreas and renal function were not affected. Our results suggest that 50 mg/kg TBLF administered by oral route, exhibit no toxicity in rats and it was well tolerated. Further studies will focus on long-term studies.