Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Oct 10;22(10):465.
doi: 10.3390/md22100465.

Novel Insights into Ethanol-Soluble Oyster Peptide-Zinc-Chelating Agents: Structural Characterization, Chelation Mechanism, and Potential Protection on MEHP-Induced Leydig Cells

Zhen Lu  1   2 , Qianqian Huang  1 , Xiaoming Qin  1   3 , Fujia Chen  2 , Enzhong Li  2 , Haisheng Lin  1   3
Affiliations

Novel Insights into Ethanol-Soluble Oyster Peptide-Zinc-Chelating Agents: Structural Characterization, Chelation Mechanism, and Potential Protection on MEHP-Induced Leydig Cells

Zhen Lu et al. Mar Drugs. .

Abstract

Numerous studies have reported that mono-(2-ethylhexyl) phthalate (MEHP) (bioactive metabolite of Di(2-ethylhexyl) phthalate) has inhibitory effects on Leydig cells. This study aims to prepare an oyster peptide-zinc complex (PEP-Zn) to alleviate MEHP-induced damage in Leydig cells. Zinc-binding peptides were obtained through the following processes: zinc-immobilized affinity chromatography (IMAC-Zn2+), liquid chromatography-mass spectrometry technology (LC-MS/MS) analysis, molecular docking, molecular dynamic simulation, and structural characterization. Then, the Zn-binding peptide (PEP) named Glu-His-Ala-Pro-Asn-His-Asp-Asn-Pro-Gly-Asp-Leu (EHAPNHDNPGDL) was identified. EHAPNHDNPGDL showed the highest zinc-chelating ability of 49.74 ± 1.44%, which was higher than that of the ethanol-soluble oyster peptides (27.50 ± 0.41%). In the EHAPNHDNPGDL-Zn complex, Asn-5, Asp-7, Asn-8, His-2, and Asp-11 played an important role in binding to the zinc ion. Additionally, EHAPNHDNPGDL-Zn was found to increase the cell viability, significantly increase the relative activity of antioxidant enzymes and testosterone content, and decrease malondialdehyde (MDA) content in MEHP-induced TM3 cells. The results also indicated that EHAPNHDNPGDL-Zn could alleviate MEHP-induced apoptosis by reducing the protein level of p53, p21, and Bax, and increasing the protein level of Bcl-2. These results indicate that the zinc-chelating peptides derived from oyster peptides could be used as a potential dietary zinc supplement.

Keywords: MEHP; TM3; apoptosis; in silico screening; zinc-chelating peptides.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Isolation of zinc-binding peptides using IMAC and determination of amino acidic profiles and molecular weight distribution. (A) A1, A2, A3 were eluted by IMAC-Zn2+ from OP-ES; (B) ratio of different amino acids of A1, A2, A3; (C) percentage of histidine amino acid of A1, A2, A3. MALDI-TOF-MS spectrum of peptides obtained from isolated fractions of IMAC ((D): A1, (E): A2, (F): A3). Different lowercase letters (a–c) on top of the bars in same amino acid specie denote significant difference (p < 0.05). Different capital letters (A–C) indicated the between-group significant differences (p < 0.05).
Figure 2
Figure 2
The protective effect of isolated and purified peptides and their zinc chelates in MEHP-TM3 cells. Effect of different IMAC fractions (A) and their zinc chelates (B) on the viability of TM3 cells; effect of different IMAC fractions (C) and their zinc chelates (D) on the viability of MEHP-injured TM3 cells. Effects of peptide–zinc chelates (A2Z, A3Z) on MEHP-induced oxidative stress and reproductive hormones in TM3 cells. (E) GSH level; (F) T-SOD level; (G) MDA level; (H) T level. Different letters represented the significant difference at p < 0.05. Values are mean ± standard error of at least 3 independent experiments.
Figure 3
Figure 3
Screening and identification of zinc-chelating peptides. (A) Zinc-chelating ability of synthetic peptides; mass spectra of the peptides EHAPNHDNPGDL (B), GHPGLPGDAGPEGPR (C), and HLDDILFS (D) identified in A3. Different letters represented the significant difference at p < 0.05.
Figure 4
Figure 4
Graphical representation of the molecular dynamics simulation studies conducted during 200 ns and binding mode of zinc-chelating peptides to Zn2+ after molecular dynamics simulation. (A) Root mean square deviation (RMSD); (B) radius of gyration (Rg) curves; (C) solvent-accessible surface area (SASA). (D) PEP6-Zn; (E) PEP3-Zn; (F) PEP10-Zn. The atoms represented by different colors are as follows: red: O; blue: N; pink: C; gray: Zn2+.
Figure 5
Figure 5
Morphological analysis and structural characterization of PEP and PEP-Zn. SEM analysis of peptides (PEP3, PEP6, PEP10) and peptide–zinc complexes (PEP3-Zn, PEP6-Zn, PEP10-Zn).
Figure 6
Figure 6
Structural characterization of PEP and PEP-Zn. FTIR spectroscopy (A), particle size (B), and zeta potential (C) of peptides (PEP3, PEP6, PEP10) and peptide–zinc complexes (PEP3-Zn, PEP6-Zn, PEP10-Zn). Different letters (a–e) represented the significant difference at p < 0.05.
Figure 7
Figure 7
Structural PEP-Zn attenuated MEHP-induced damage in TM3. (A) The effect of synthetic peptide–zinc chelate on the viability of TM3 cells; (B) the effect of synthetic peptide–zinc chelate on the viability of MEHP-TM3 cells; (C) MDA level; (D) T-SOD level; (E) CAT level; (F) GSH level; (G) zinc level; (H) T level. Different letters (a–j) represented the significant difference at p < 0.05.
Figure 8
Figure 8
The protein levels of apoptotic markers (A–D) after PEP6-Zn and MEHP exposure (n = 3). The results were expressed as the means ± standard deviations. Different letters (a–d) represented the significant difference at p < 0.05.
Figure 9
Figure 9
Observation of the effect of PEP6-Zn on the ultrastructural changes of MEHP-induced TM3 cells through transmission electron microscopy (scale bar: 1 μm).
See this image and copyright information in PMC

References

    1. Martínez M.A., Rovira J., Prasad Sharma R., Nadal M., Schuhmacher M., Kumar V. Comparing dietary and non-dietary source contribution of BPA and DEHP to prenatal exposure: A Catalonia (Spain) case study. Environ. Res. 2018;166:25–34. doi: 10.1016/j.envres.2018.05.008. - DOI - PubMed
    1. Hong Y., Zhou Y., Shen L., Wei Y., Long C., Fu Y., Wu H., Wang J., Wu Y., Wu S., et al. Exposure to DEHP induces testis toxicity and injury through the ROS/mTOR/NLRP3 signaling pathway in immature rats. Ecotoxicol. Environ. Saf. 2021;227:112889. doi: 10.1016/j.ecoenv.2021.112889. - DOI - PubMed
    1. Zhang Q., Sun Y., Zhang Q. Phthalate exposure in Chinese homes and its association with household consumer products. Sci. Total Environ. 2020;719:136965. doi: 10.1016/j.scitotenv.2020.136965. - DOI - PubMed
    1. Di Bella G., Saitta M., Lo Curto S., Salvo F., Licandro G., Dugo G. Production process contamination of citrus essential oils by plastic materials. J. Agric. Food Chem. 2001;49:3705–3708. doi: 10.1021/jf0100043. - DOI - PubMed
    1. Xu J., Wang L., Zhang L., Zheng F., Wang F., Leng J., Wang K., Héroux P., Shen H.M., Wu Y., et al. Mono-2-ethylhexyl phthalate drives progression of PINK1- parkin-mediated mitophagy via increasing mitochondrial ROS to exacerbate. Redox Biol. 2021;38:101776. doi: 10.1016/j.redox.2020.101776. - DOI - PMC - PubMed

LinkOut - more resources