Elsevier

Process Biochemistry

Volume 98, November 2020, Pages 151-159
Process Biochemistry

Preparation and purification of an immunoregulatory peptide from Stolephorus chinensis of the East Sea of China

https://doi.org/10.1016/j.procbio.2020.08.011Get rights and content

Highlights

  • An immunomodulatory peptide was isolated from Stolephorus chinensis.

  • The amino acid sequence of immunomodulatory peptide was identified as Tyr-Val-Met-Arg-Phe.

  • Fraction with molecular weight of < 1 kDa showed maximum RPR toward RAW 264.7 cells (70.03 %).

Abstract

Immunomodulatory peptides derived from marine organisms are potential sources of new immunomodulating drugs. This study aimed to investigate the optimal enzymatic hydrolysis conditions of immunoregulatory peptides from Stolephorus chinensis. The relative proliferation rate (RPR) of RAW264.7 macrophages was set as the screening index. The immunoregulatory peptides from S. chinensis was prepared via process optimization, ultrafiltration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HLPC). The amino acid sequence of the immunoregulatory peptide from S. chinensis (IPSC) was identified to be Tyr-Val-Met-Arg-Phe (YVMRF; MW 715.4 Da) using Edman degradation and mass spectrometry. In addition, the macrophages became larger with more pseudopods after IPSC treatment, indictating that IPSC stimulated RAW 264.7 differentiation. IPSC also increased productions of nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Our results provide a theoretical basis for preparing immunomodulatory functional food from S. chinensis in future.

Introduction

The immune system consists of immune organs, immune cells, and immune-active substances, which are involved in immunological defense and surveillance, and immunomodulation, and are related to the etiology of various diseases [1]. Interactions between the innate immune and specific immune systems are required for maintaining human health [2], infections and various diseases may occur if the immune system is dysfunctional or damaged. Therefore, development of compounds that improve the immunoregulatory activity of the human body is important.

Immunomodulation refers to the ability of the immune system to control various life-threatening diseases, such as cancer, human immunodeficiency virus infection, and aging [[3], [4], [5]]. Macrophages are immune cells that participate in the body's non-specific and specific immunity. They play important roles in the body's immune regulation by secreting active substances, and by presenting and phagocytosing antigens [6]. Proliferation is the premise and basis for the role of macrophages in immune regulation.

Immunomodulators can be divided into immunoadjuvants, immunostimulants, and immunosuppressants [7]. Currently, many drugs have been used clinically to regulate the body's immune function, including levamisole, cyclophosphamide, prostaglandin, imiquimod, pidotimod, macrolides, and azathioprine [[8], [9], [10], [11], [12], [13], [14]]. However, most synthetic immunomodulatory drugs are toxic (induce nausea, bone marrow toxicity, and liver toxicity) and have side effects; these drugs may also increase the risk of infection as they act on the immune system [15]. The various adverse reactions of synthetic drugs indicates that caution should be exercised while using immunomodulatory drugs. From an economic and social point of view, most naturally-derived immunomodulators are inexpensive, and beneficial and safe for human health, indicating that they can be used for immunotherapy.

Bioactive peptides refer to compounds that are beneficial for biological activity or exert positive effects on human health [16]. They are usually composed of 2∼20 amino acids [17], and regulate important body functions via various activities, including immunoregulation [18,19], antihypertensive [20], antioxidant [21,22], anti-diabetic [23], anti-infection [24], and hepatoprotective properties [25]. Therefore, the use of bioactive peptides as health and functional food [26] has attracted attention. Over the past few years, several immunoregulatory peptides that reduce or prevent chronic disease risk and provide immune protection have been isolated and identified. For example, Li et al. [27] isolated a pentadecapeptide (Arg-Val-Ala-Pro-Glu-Glu-His-Pro-Val-Glu-Gly-Arg-Tyr-Leu-Val) from the proteolysis of Cyclina sinensis, which enhanced the phagocytic function of macrophages and increased the production of NO, TNF-α, IL-6, and IL-1β. Wu et al. purifed a hexapeptide (Glu-Cys-Phe-Ser-Thr-Ala) from wheat germ globulin, which enhanced the phagocytic function of RAW264.7 cells [28]. Mao et al. [29] isolated an oligopeptide from naked oat (Avena nuda L.), which significantly improved the innate and specific immunity of mice by enhancing cellular and humoral immunity, macrophage phagocytic capacity, and NK cell activity. However, reports on the extraction of immunoregulatory peptides from Stolephorus chinensis are lacking.

Stolephorus chinensis, commonly known as Stolephorus, is a small offshore fish belonging to family Engraulidae of order Clupeiformes, which is mostly distributed along the coast of the South Sea and East Sea of China [30]. Nutritional composition analysis showed that S. chinensis is rich in a variety of basic or hydrophobic amino acids, as well as various mineral elements, such as zinc, strontium, and calcium [31]. Despite this, current studies on Stolephorus are mainly related to the relationship between its distribution characteristics and the environment [32] or storage method [[33], [34], [35]], while reports on preparation of immunoregulatory peptides from Stolephorus chinensis are lacking. Hence in this study, we aimed to prepare immunoregulatory peptides from S. chinensis via process optimization, ultrafiltration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HLPC). The amino acid sequence of the immunoregulatory peptide was also determined using Edman degradation. Our findings provide experimental basis and theoretical guidance for improving the biological value of S. chinensis via the development of functional immunomodulatory food.

Section snippets

Materials and reagents

S. chinensis were purchased from a local seafood market in Zhoushan, China. The fish were washed and treated with 0.1 M NaOH at a ratio of 1:4 (w/v) for 6 h for defatting. Then, the defatted fish were rinsed with pure water until the pH was neutral and were ground to uniformity with a homogenizer. The minced fish were stored in plastic bags at -20 °C until used for enzymatic hydrolysis.

Pepsin and neutral protease were purchased from AOBOX Biotechnology Co., Ltd. (Beijing, China). Trypsin,

Preparation of immunoregulatory peptides from S. chinensis

Determination of the optimum enzymes. Enzymes cleave at specific positions on the polypeptide chain, and different proteases affect the composition and size of the resulting polypeptides differently, which consequently affect their biological activity [42,43]. Hence, selection of the appropriate protease is important. In this study, five different proteases (pepsin, neutral protease, alkaline protease, trypsin, and papain) were used to hydrolyze S. chinensis and the effect of the hydrolysates

Conclusions

The optimum conditions for preparing immunoregulatory peptides using S. chinensis as the substrate were as follows: enzymatic hydrolysis time, 4 h; pH, 1.0, enzyme amount, 3000 U/g, solid-liquid ratio, 1:3; temperature, 45 °C. After continuous separation and purification, an immunoregulatory peptide (Tyr-Val-Met-Arg-Phe, MW 715.4 Da) from S. chinensis (IPSC) was identified. We also showed that IPSC stimulated the differentiation of RAW 264.7 cells. Furthermore, the production of NO was

Funding

This work was supported by the National Natural Science Foundation of China (grant No. 41806153).

CRediT authorship contribution statement

Baogui Xu: Methodology, Data curation. Lei Ye: Methodology, Data curation. Yunping Tang: Supervision, Writing - review & editing, Funding acquisition. Jiawen Zheng: Visualization, Investigation. Xiaoxiao Tian: Visualization, Investigation. Yuxia Yang: Visualization, Investigation. Zuisu Yang: Conceptualization, Methodology, Writing - review & editing.

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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