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.

Quantum Dot Labelling of Tepary Bean (Phaseolus acutifolius) Lectins by Microfluidics.

Lectins are bioactive proteins with the ability to recognize cell membrane carbohydrates in a specific way. Diverse plant lectins have shown diagnostic and therapeutic potential against cancer, and their cytotoxicity against transformed cells is mediated through the induction of apoptosis. Previous works have determined the cytotoxic activity of a Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) and its anti-tumorigenic effect on colon cancer. In this work, lectins from the TBLF were additionally purified by ionic-exchange chromatography. Two peaks with agglutination activity were obtained: one of them was named TBL-IE2 and showed a single protein band in two-dimensional electrophoresis; this one was thus selected for coupling to quantum dot (QD) nanoparticles by microfluidics (TBL-IE2-QD). The microfluidic method led to low sample usage, and resulted in homogeneous complexes, whose visualization was achieved using multiphoton and transmission electron microscopy. The average particle size (380 nm) and the average zeta potential (-18.51 mV) were determined. The cytotoxicity of the TBL-IE2 and TBL-IE2-QD was assayed on HT-29 colon cancer cells, showing no differences between them (p ≤ 0.05), where the LC50 values were 1.0 × 10-3 and 1.7 × 10-3 mg/mL, respectively. The microfluidic technique allowed control of the coupling between the QD and the protein, substantially improving the labelling process, providing a rapid and efficient method that enabled the traceability of lectins. Future studies will focus on the potential use of the QD-labelled lectin to recognize tumor tissues.

Effects of Tepary bean (Phaseolus acutifolius) protease inhibitor and semipure lectin fractions on cancer cells.

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes' extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.

Hybridization between Crotalus aquilus and Crotalus polystictus Species: A Comparison of Their Venom Toxicity and Enzymatic Activities.

Hybridization is defined as the interbreeding of individuals from two populations distinguishable by one or more heritable characteristics. Snake hybridization represents an interesting opportunity to analyze variability and how genetics affect the venom components between parents and hybrids. Snake venoms exhibit a high degree of variability related to biological and biogeographical factors. The aim of this work is to analyze the protein patterns and enzymatic activity of some of the main hemotoxic enzymes in snake venoms, such as serine proteases (trypsin-like, chymotrypsin-like, and elastase-like), metalloproteases, hyaluronidases, and phospholipase A2. The lethal dose of 50 (LD50) of venom from the Crotalus aquilus (Cabf) and Crotalus polystictus (Cpbm) parents and their hybrids in captivity was determined, and phenetic analysis is also conducted, which showed a high similarity between the hybrids and C. polystictus. The protein banding patterns and enzymatic activity analyze by zymography resulted in a combination of proteins from the parental venoms in the hybrids, with variability among them. In some cases, the enzymatic activity is higher in the hybrids with a lower LD50 than in the parents, indicating higher toxicity. These data show the variability among snake venoms and suggest that hybridization is an important factor in changes in protein concentration, peptide variability, and enzymatic activity that affect toxicity and lethality.

Properties of a Non-canonical Complex Formed Between a Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and α-Chymotrypsin.

Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.

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.

Characterization of a Hydrophobic Amylase Inhibitor from Corn (Zea mays) Seeds with Activity Against Amylase from Fusarium verticillioides.

ABSTRACT A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering.

New Amylase Inhibitor Present in Corn Seeds Active In Vitro Against Amylase from Fusarium verticillioides.

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 µg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Cloning, sequencing, purification, and crystal structure of Grenache (Vitis vinifera) polyphenol oxidase.

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.