Purification And Bioinformatic Analysis Of Lectin From Black Turtle Bean And Determination Of Its Conformation And Functional Properties

Lectins are the glycoproteins with important biological activities showing in blood typing, cell signaling, anti-malignancy, immune responses and drug targeting. Meanwhile, lectins are also considered as the anti-nutritional factor in the food. It is well known that the legumes are rich of lectins. Black turtle bean, which is widely cultivated, was found in higher levels of lectin. However, there were very few reports about the the structure and biological properties of black tu rtle bean lectin. In this study, the black turtle bean lectin was extracted and purified, and the relationship between physical and chemical characteristics and protein conformation was investigated, for the reference in the development of legumes lectins.The reverse micellar extraction(RME) was applied to purify lectin from black turtle beans in the crude extraction of the bean, and the effects of the RME on the activity and structure of the lectin were investigated. The optimum parameters obtained by the response surface optimization were determined as 77.6 mmol/L Na Cl, p H 5.7, AOT 127.4 mmol/L for the forward extraction, and 593.0 mmol/L KCl, p H 8.0 for the backward extraction. The yield was 63.2 ± 2.4 mg protein/g bean meal with a purification factor of 8.8 ± 0.2 under the con ditions. It was shown that the activity of the lectin was not changed by RME. The results of FTIR and CD spectra showed that the protein structure of lectin might still be retained after the purification. One-step ion exchange chromatography(DE-52) was used for the further purification. And the final yield increased by 70% after the chromatography purification compared with the conventional method using ion exchange and size-exclusion chromatography.The purified lectin was identified by the LC-MS/MS analysis, while it seemed to be widely conserved to PHA-E gene. Genomic DNA from the black turtle bean was extracted, and the black turtle bean lectin c DNA was obtained by the PCR method following the pair of primers designed according to the c DNA of PHA-E in Gen Bank. The multiple alignment and phylogenetic analysis clearly indicated that the lectin belonged to the PHA family, and the lectin might evolve from the Arc7 variant. Based on the bioinformatics analysis, the lectin was proved to be a hydrophilic protein, and had a high stability containing a hydrophobic core region. Meanwhile, the lectin might be in a tetrameric state at the natural p H, and might have a highly conserved Lectin_leg B functional domains containing Ca2+ and Mn2+ binding sites. The analysis also indicated that the lectin could have affinity with N-glycans. The B-lymphocyte epitopes and T lymphocyte epitopes were also found in the sequence of the lectin, which indicated that the lectin might have anti- malignant cells activity.The molecular weight of the lectin was determined at around 31 k Da in the monomeric state, while it was around 116.97 k Da in the tetrameric state. The isoelectric point of the lectin was found to be around 5.27 in the monomeric state, 6.03 in the dimeric state, and 6.91 in the tetrameric state. N-acetyl-D-glucosamine was found to be the specific sugar of the lectin using the fluorescent spectrometry, while the affinity constant was determined at about 0.45 mmol/L, which showed that it was a moderate affinity. The results of the mental ion-dependent analysis using activity recovery method showed that the Mn2+ and Ca2+ were the essential components to maintain the activity of the lectin. The two-way analysis of variance indicated that the heating temperature and treatment time significantly affected the stability of the lectin during thermal treatments. The analysis of kinetic data suggested that the thermal stability of the lectin followed first-order reaction kinetics. The assays also indicated that the thermal inactivation was associated with the unfolding of the protein. The results of UV, fluorescence and DSC showed that the structure of the lectin was stable from p H 2.0 to 10.0.An appropriate exponent kinetic model was developed to quantify the lectin bands on SDS-PAGE gels by densitometry, to overcome the shortcomings associated with the evaluation for digestion results in vitro study. It was found that the native lectin was relatively stable in both SGF(half-life = 22.71 min) and tryptic digestion(half-life ≥ 90 min), the susceptibility of the protein to hydrolysis by proteases was markedly increased by preheating and also enhanced by demetallization, while an unfolding state of the preheated lectin was observed by UV and fluorescence spectroscopy. The results of the cytotoxicity test of the lectin to the simulated intestinal epithelial cells showed that there were no obvious toxic effects during the short incubation time(2 h). In the allergic reaction analysis, it was found that the low dose(6 mg lectin/120 g rat) of lectin could not induce allergic manifestations. It was observed that the lectin could trigger the release both of IL-4 and interferon-γ, the antagonism of the cytokines might adjust the expression of Ig E antibodies.The results of experiments showed that the lectin could obviously inhibit the growth of the Hela cell, while the IC50 was 9.85 μg/m L, and the effect was in a does-dependent and time-dependent manner. The morphological observation suggested that high doses of lectin could cause apoptosis or necrosis of the Hela cell. The flow cytometry analysis showed that most of the cells were in the apoptosis state when the lectin concentrations was lower than the IC50 value, and in the late apoptosis and death state when the lecin concentrations was hi gher than the IC50 value. It was also found that the cell cycle was significantly affected by the lectin, the distribution from G1 phase to S phase was observed to be blocked, while the lectin was also found that could arrest Hela cells in G2/M phase.