Structure-allergenicity Relationship Of Protonation Induced Black Turtle Bean(Phaseolus Vulgaris L.) Lectin

In the study,lectin from black turtle bean（Phaseolus vulgaris L.）was modified by protonation induced treatments.Lectin was prepared in a series of pH buffers below the isoelectric point（pI）,respectively,and structure-allergenicity relationship of protonation induced lectin was investigated.Furthermore,the quality of black turtle bean protein isolate subjected to protonation induced treatments,following neutralized to pH 7.2,were further explored.1.The three-dimensional structure of P.vulgaris L.lectin was modeled by the SWISS-MODEL,the B and T-cell epitopes were predicted by various bioinformatics tools,and subsequently validated by ELISA,lymphocyte proliferation and cytokine profle analyses.Four B-cell epitope regions（B1:NVNDNGEPTLSS,B2:VGSEPKDKGG,B3:NNYKYDSNAHT,B4:LYNVHWDPKPRH）and two T-cell epitope regions（T1:LQRDATVSS,T2:FNIDVPNNS）were identified,and positively charged amino acid residues with high hydrophobicity were more likely to be in the lectin epitope region.2.The P.vulgaris L.lectin was incubated in low acidic buffers,a progressive unfolding process were confirmed,and the treated lectin attained its stable state at 8 h.Intrinsic/extrinsic fluorescence spectra assays,SDS-PAGE,dynamic light scattering（DLS）,size exclusion chromatography（SEC）and differential scanning calorimetry（DSC）analyses suggested lectin appeared unfolding and monomerization in the protonation solutions.These structural alterations might bury,destroy or degrade surface epitopes,and expose cleavage sites to protease.3.The BALB/c mice were intraperitoneally sensitized with protonation treated lectin for 5 weeks using complete Freund’s adjuvant（CFA）as adjuvant,following the mice were challenged orally.The results indicated that,the BALB/c mice administrated treated lectin showed significant reduction in clinical anaphylactic symptoms compared with the native group,and the levels of IgE and IgG₁,mMCPT-1,His and cytokines（IL-4,IL-5,IL-13 and IFN-γ）decreased significantly.Circular dichroism（CD）,Fourier transform infrared（FT-IR）and molecular dynamic（MD）simulation demonstrated a significant decrease inα-helix content with the increase ofβ-sheet content,and lectin protein unfolding with more exposure the cleavage sites for trypsin were confirmed in treated lectin.4.The black turtle bean（P.vulgaris L.）protein isolate was treated by protonation induced treatments for 8 h,following the treated sample solutions were freeze-dried after adjusting the solutions to neutral condition（i.e.,protonation reduction induced treatment）,and the powder of P.vulgaris L.protein was obtained.The structure analyses indicated that theα-helix content of the bean protein isolate significantly decreased with theβ-sheet content increasing after protonation reduction induced treatments.The structure of protein isolate unfolding,more hydrophobic amino acid residues exposed,and the reduction of total sulfhydryl groups（-SH）and free sulfhydryl groups with the increase of disulfide bond（-S-S-）content were found.Significant increase in emulsifying properties（EAI and ESI）,foaming capacity（FC）and foaming stabilities（FS）,fat-holding capacities（FHC）and gel properties of the black turtle bean protein isolate after protonation reduction induced treatments were observed,but the treated protein samples displayed a decrease in solubility and water-holding capacity（WHC）.In addition,the P.vulgaris L.protein powder displayed significant reduction in allergenicity and digestion stability.