Elsevier

Food Chemistry

Volume 366, 1 January 2022, 130626
Food Chemistry

Active sites of peptides Asp-Asp-Asp-Tyr and Asp-Tyr-Asp-Asp protect against cellular oxidative stress

https://doi.org/10.1016/j.foodchem.2021.130626Get rights and content

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Abstract

The protective effects of the peptides Asp-Asp-Asp-Tyr (DDDY) and Asp-Tyr-Asp-Asp (DYDD) against AAPH-induced HepG2 cells are unclear. Our objective was to investigate the active sites of these peptides and their cellular antioxidant mechanism. DDDY and DYDD show a direct free radical scavenging effect in reducing ROS levels and maintained cellular antioxidant enzymes at normal levels. The quantum chemistry analysis of the electronic properties of antioxidant activity showed that DYDD has a greater energy in the highest occupied molecular orbital than DDDY, and O58-H59 and N10-H12 were identified as the active antioxidant sites in DYDD and DDDY, respectively, indicating that the inconsistent arrangement of amino acids affects the distribution of the highest occupied orbital energy as well as the active sites; thus, influences the antioxidant activity of peptides. It provide valuable insights into the antioxidant active sites of peptides.

Introduction

Reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, organic peroxides, and the hydroxyl radical, can cross cell membranes, leading to oxidative stress (Akhtar, Ahamed, Alhadlaq, & Alshamsan, 2017). Additionally, the antioxidant defense mechanisms of organisms to reduce ROS levels in vivo include both enzymatic and non-enzymatic systems. Specifically, enzymatic systems involve superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), whereas non-enzymatic systems mainly consists of exogenous antioxidants (Uribe, López-Giraldo, & Decker, 2018). Further, the mechanisms of free radical scavenging are primarily based on the transfer of hydrogen atoms or their supply to stop free radical reactions (Foti, Johnson, Vinqvist, Wright, Lrc, & Ingold, 2002).

It is also well known that antioxidant peptides can reduce free radical levels, and the antioxidant activities of such peptides have been determined primarily based on their amino acid compositions and peptide structures (Elias, Kellerby, & Decker, 2008). Specifically, amino acid structure (such as side chains and sequences) affects the antioxidant activities of peptides (Matsui et al., 2018), and compared with synthetic antioxidants, antioxidant peptides have fewer side effects and are readily digested and absorbed owing to their low molecular weights (500–1800 Da) and amino acid contents (2–20); they also have favorable nutritional and processing properties (Wattanasiritham, Theerakulkait, Wickramasekara, Maier, & Stevens, 2016). It has also been reported that some antioxidant peptides can be absorbed by the intestine-like Caco-2 cells; thus, it is probable that they affect the human body (Liu, Wu, & Wang, 2018). It has also been demonstrated that peptides derived from black soybean and chickpea exhibit free radical scavenging activities (Kou et al., 2013, Ren, 2010). In a previous study, we isolated the peptides Asp-Asp-Asp-Tyr (DDDY) and Asp-Tyr-Asp-Asp (DYDD) from DPP (Dendrobium aphyllum after Lactobacillus amylolyticus solid-state optimized fermentation peptides) (Liu, Ma, Yin, & Wu, 2018) and demonstrated that DPP shows chemical antioxidant activity. Therefore, we believe that DDDY and DYDD from DPP may have free radical-scavenging potential, indicating that they can be used as potential antioxidants. However, the mechanism of their antioxidant activity as well as their structure–related antioxidant activity are unclear.

Theoretical calculation-based analytical methods, such as the density functional theory (DFT), are tools that can be employed to explore the structure–activity relationships of chemical compounds (Chen, Xiao, Zheng, & Liang, 2015). Further, the quantum chemical parameters of antioxidants include the highest occupied and lowest unoccupied molecular orbital (HOMO and LUMO) energy, bond length, and molecular shape parameters (Ananda et al., 2012, Johns and Platts, 2014), and the active sites of antioxidant peptides can be identified by analyzing their HOMO energy and bond lengths (Cheng, Luo, Zeng, Li, Xiao, Bu, et al., 2015).

Given that DYDD and DDDY have a similar amino acid composition, it would be interesting to explore how their peptide sequences affect their antioxidant capacities. Therefore, in this study, we examined the mechanisms of the antioxidant activities of these peptides as follows: a) fluorescent-labeled peptides were used to determine dynamic changes; b) cytoprotective effects were evaluated by assessing oxidant-related signaling pathways; c) the structure–antioxidant activity relationships of the peptides were evaluated via quantum chemistry theoretical calculations and active site prediction; and d) the predicted active sites of the peptides were methylated, and ROS levels were determined for verification. The findings of this study clarify the underlying relationship between the structures of amino acid and their free radical scavenging activities. They may also facilitate the development of new antioxidant peptides with similar structures.

Section snippets

Materials

The peptides DYDD, DDDY, FITC-6-aminocaproic acid (acp)-DDDY, FITC-acp-DYDD, methylated-DDDY, and methylated-DYDD (Supplementary Figs. 1–4) were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China). The antioxidant enzymes assay kits were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China), and the ROS assay kits were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). TRIzol reagent was obtained from Life

Cell viability

Before determining the activities of the peptides DYDD and DDDY (each with purity greater than 98%), we first of all confirmed their non-toxic effects on HepG2 cells by conducting cell viability tests. Thus, it was observed that after all the treatments, the viability of the HepG2 cells remained above 90%. Reportedly, a cell viability above 80% is indicative of a non-cytotoxic effect of peptide treatment on HepG2 cells (J. Wu, Huo, Huang, Zhao, Luo, & Sun, 2017). In line with this previous

Conclusion

DDDY and DYDD showed the ability to directly scavenge free radicals to the end of protecting AAPH-treated HepG2 cells intracellularly and extracellularly, instead of the activation of the Nrf2/Keap1 signaling pathway. Based on quantum chemistry theoretical calculations, O58-H59 on DYDD and N10-H12 on DDDY were identified as antioxidant active sites (Fig. 4). Our findings provide insights into the structure–activity relationships of antioxidant peptides with different amino acid arrangements.

Funding sources

This study was funded by the Fundamental and Applied Basic Research Fund for Science and Technology Planning Project of Guangzhou (Project No. 202002030383), the Basic Research and Applied Basic Research Funding Project of Guangdong Province (Grant No. 2019A1515011283), Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology (Grant No. 2021B1212040013).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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