Characterization of Angiotensin-? Converting Enzyme Inhibiting Peptide from Venerupis philippinarum with Nano-Liquid Chromatography in Combination with Orbitrap Mass Spectrum Detection and Molecular Docking

The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-? converting enzyme(ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-Iinhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC_(50) of 5.75 μmol L^(-1). The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.

Molecular cloning and differential expression patterns of sigma and omega glutathione S-transferases from Venerupis philippinarum to heavy metals and benzo[a]pyrene exposure

Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. We identified two glutathione S-transferase isoforms (VpGSTS, sigma GST; VpGSTO, omega GST) from Venerupis philippinarum by RACE approaches. The open reading frames of VpGSTS and VpGSTO were of 612 bp and 729 bp, encoding 203 and 242 amino acids with an estimated molecular mass of 22.88 and 27.94 kDa, respectively. The expression profiles of VpGSTS and VpGSTO responded to heavy metals and benzo[a]pyrene (B[a]P) exposure were investigated by quantitative real-time RT-PCR. The expression of VpGSTS and VpGSTO were both rapidly up-regulated, however, they showed differential expression patterns to different toxicants. Cd displayed stronger induction of VpGSTS expression with an approximately 12-fold increase than that of VpGSTO with a maximum 6.4-fold rise. Cu exposure resulted in similar expression patterns for both VpGSTS and VpGSTO. For B[a]P exposure, the maximum induction of VpGSTO was approximately two times higher than that of VpGSTS. Altogether, these findings implied the involvement of VpGSTS and VpGSTO in host antioxidant responses, and highlighted their potential as a biomarker to Cd and B[a]P exposure.

Glutathione S-transferase(GST) gene expression profiles in two marine bivalves exposed to BDE-47 and their potential molecular mechanisms

Glutathione S-transferases （GSTs） are phase II enzymes that facilitate the detoxification of xenobioties and play important roles in antioxidant defense. We investigated the expression patterns of seven Venerupis philippinarum GSTs （VpGSTs） and four Mytilus galloprovincialis GSTs （MgGSTs） following exposure to BDE-47. Differential expressions of the seven VpGSTs and four MgGSTs transcripts were observed, with differences between the hepatopancreas and gills. Among these GSTs, the sigma classes （VpGSTS1, VpGSTS2, VpGSTS3, MgGST1, and MgGST3） were highly expressed in response to BDE-47 exposure, demonstrating their potential as molecular biomarkers for environmental biomonitoring studies. We obtained the three-dimensional crystal structures of VpGSTs and MgGSTs by homologous modeling. A model to elucidate the binding interactions between the ligands and receptors was defined by molecular docking, Hydrophobic and n were the most often observed interactions between BDE-47 and the GSTs.

Evaluating the bioavailability of heavy metals in natural-zeoliteamended aquatic sediments using thin-film diffusive gradients

The effectiveness of natural zeolite amendment of sediments as a restorative material was studied,as was the feasibility of utilizing thin-film gradient diffusion(DGT)as a proxy for the accumulation of heavy metals in Venerupis philippinaram.The results showed that addition of natural zeolite to sediment,significantly decreased the equilibrium partitioning of Cu,Pb,Cd,Cr and As between the sediment pore water and natural zeolite over 24 h by 67%,81%,72%,62%and 71%,respectively.Furthermore,the accumulation of Cu,Pb,Cd,Cr and As in V.philippinaram in the zeolite-amended sediments decreased by 44%,37%,54%,30%and 59%,respectively after 28 days and the absorption rates also declined.The amount of heavy metals enriched into the DGT film and V.philippinaram over 28 days showed a significant correlation(P<0.001)and indicated that DGT has the potential as a proxy to predict the bioaccumulation of heavy metals in benthic organisms in sediments amended by natural zeolite.Further studies focused on the modification of natural zeolite and the predictive ability of DGT in different sediments/organism scenarios are warranted.

Stabilization of heavy metals in sediments:A bioavailability-based assessment of carbon adsorbent efficacy using diffusive gradients in thin films

This study investigated the effectiveness of carbon adsorbents as remediation material for sediments contaminated with heavy metals and the feasibility of utilizing diffusive gradients in thin films(DGT)as a biomimetic tool to estimate the accumulation of heavy metals in Venerupis philippinaram(Manila clam).The results showed that carbon materials had significant inhibitory effects(14.0-53.0%)on the enrichment of heavy metals in organisms and the order of increasing overall inhibitory effect was:charcoal,peat,activated carbon,and biochar.There were significant correlations(P<0.0001)between the four heavy metals accumulated in Venerupis philippinaram and those accumulated in DGT devices after 28 days in the laboratory.Observed concentrations of heavy metals enriched in DGT(5.4-42.0%)were less than accumulations in Venerupis philippinaram.The results of in situ DGT applications showed significant correlations between the amount accumulated in DGT and the acid-soluble portion in sediments for both Cu and Pb,with positive linear correlations and R-squares of 0.97 and 0.92,respectively.These results supported the notion that a DGT device can be used as a biomimetic tool that predicts and monitors the accumulation of heavy metals in aquaculture ponds.Future studies should focus on improving the stabilization of heavy metals in sediments using different types of carbon sorbents,as well as minimizing simulation deviations using DGT.