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Introduction
In recent years, bioactive peptides, as functional substances beneficial to human health, have attracted more and more attention, and the research on bioactive peptides has become one of
the hot spots of scientific research.
Bioactive peptides are produced by protein hydrolysis or fermentation, after which peptide bonds are destroyed, resulting in the production
of peptides and amino acids.
Therefore, bioactive peptides are small in size, have good permeability, and are more easily absorbed
by the human body.
At present, the various effects of bioactive peptides have been reported by more and more studies, including anticancer, blood pressure, antibacterial, cholesterol lowering, anti-diabetes, etc
.
However, at present, the research on bioactive peptides mostly focuses on food-derived peptides, and there are few
studies and reports on non-food-derived peptides.
Previous studies have shown that non-food-derived bioactive peptides have higher affinity and can effectively exert their bioactive functions
compared with food-derived bioactive peptides.
Lymphocytes are found in animal lymphoid organs and tissues, including tonsils, spleen, etc
.
They are the central regulatory cells of the immune system, and cell lines composed of lymphocytes play an important role in the immune system, and most of the functions are mediated by small molecule polypeptides such as lymphokines, so it is of great significance
to explore the peptide components and peptide functions in lymphocytes for the development of functional products related to bioactive peptides.
Taking mouse splenic lymphocytes as the research object, the authors isolated and identified the peptide components in lymphocytes before and after LPS stimulation, analyzed and compared the pre-stimulation and post-stimulation peptides with the previously reported peptides, and found 6 new bioactive peptides with potential DPP-IV inhibitory functions, two of which had good DPP-IV inhibitory activity
.
Results and discussion
Isolation and identification of lymphocyte polypeptides
After extracting lymphocytes before and after LPS stimulation from the mouse spleen, the purity of lymphocytes was determined by flow cytometry to reach 98.
3%, and the peptides were further separated, and the separated peptides were identified by nanoliter liquid phase-Q Activate quadrupole ultra-high resolution orbital trap mass spectrometer, and a total of 385 peptides were found after library search and analysis by PEAKS X software, of which 309 were from the LPS-stimulated experimental group and 149 were from the control group, with 73 The results showed that the number and type of polypeptides changed greatly after lymphocytes were stimulated by LPS
.
In the process of the immune response generated by the cells after external stimulation, immune cells are acting on target cells through immune factors to complete the key link in the immune response, and immune factors are actually different kinds of peptides
.
Therefore, the new polypeptides produced by lymphocytes stimulated by LPS are likely to have certain active functions
.
Control group; B.
Experimental group
.
Fig.
1 Comparison of intracellular polypeptide mass spectra and quantity of lymphocytes before and after LPS stimulation
.
Prediction of peptide biological activity
The discovered new peptides were analyzed for functional activity using Peptide Ranker software and BIOPEP, and six of the peptides scored higher than 0.
8, indicating high biological activity
.
Among the 6 peptides, DPP-IV inhibitors have the most inhibitory active fragments, and DPP-IV inhibitors are a drug used for the treatment of type II diabetes, indicating that these 6 peptides may have certain hypoglycemic function, so the DPP-IV inhibitory function of these 6 bioactive peptides is further verified
.
Table 1 Bioactive fragments retrieved by BIOPEP
DPP-IV inhibitory activity of six novel bioactive peptides
The pNA standard curve was established, and the DPP-IV inhibitor screening model was constructed, and the inhibition rate
of six novel peptides on DPP-IV was verified.
The results showed that the peptide SAPRHGSLGFLPRK (IC50: (23.
443 7±1.
04) mg/L) and the peptide SGVSLAALKKALAAAAGYDVEK (IC50: (84.
193 9±2.
87) mg/L) showed better DPP-IV inhibition potential
.
Fig.
2 DPP-IV inhibition rate curves of six peptides
The DPP-IV inhibition types of the peptide SAPRHGSLGFLPRK and the peptide SGVSLAALKKALAAAGYDVEK were further determined, and the inhibition type was fitted by the Lineweaver-Burk double reciprocal plotting method, and the inhibition type was evaluated and found that the inhibition type of the peptide SAPRHGSLGFLPRK was competitively inhibited, which inhibited the binding of the enzyme to the substrate by binding the active site of the enzyme.
The type of inhibition of the polypeptide SGVSLAALKKALAAAGYDVEK is non-competitive, which binds to the enzyme through other sites without affecting the binding
of the substrate to the enzyme.
The DPP-IV inhibitory activity of the polypeptide SAPRHGSLGFLPRK may be related to the large number of hydrophobic amino acids and alanine at the N-terminal P1 position, and the lysine residues on the C-terminus of the two peptides will also significantly increase their DPP-IV inhibitory activity
.
Among them, the peptide SAPRHGSLGFLPRK was found after LPS stimulation, indicating that lymphocytes may produce more functionally active peptides
after external stimulation.
Fig.
3 Lineweaver-Burk plot of different concentrations of polypeptide SAPRHGSLGFLPRK
Fig.
4 Lineweaver-Burk plot of SGVSLAALKKALAAAGYDVEK at different concentrations
Conclusion
In this study, a method for the isolation and identification of peptides in mouse lymphocytes was established, among which a total of 131 new peptides were found, including peptides before LPS stimulation (45 kinds) and LPS stimulated polypeptides (108 kinds), the amino acid length was in the range of 8~30 amino acids, and it was found that cells would produce more functionally active polypeptides after external stimulation.
At the same time, a model to explore the inhibitory efficacy of DPP-IV was used to find that two new peptides SAPRHGSLGFLPRK and peptide SGVSLAALKKALAAAGYDVEK had potential DPP-IV inhibition functions, and their IC50s were (23.
443 7±1.
04) mg/L and (84.
193 9±2.
87) mg/L, respectively, and the inhibition types were competitive and non-competitive
, respectively 。 In addition, it is necessary to further explore the interconnection between molecular structures through molecular docking, which will contribute to the development of
bioactive peptide-related health products and special medical foods.
Author Jane
About the first author
Luan Yuanyuan, female, currently holds a master's degree in food engineering from the College of Agriculture and Biology, Shanghai Jiao Tong University, focusing on the functional application of bioactive peptides and participating in the research and development
of major projects of enterprises.
He has published one SCI paper as the first author (received by Food Science and Human Wellness), published one core paper as the second Chinese author, and participated in more than 10 published patents, two of which have been authorized
.
About the corresponding author
Zhang Shaohui, male, researcher and master supervisor of the College of Agriculture and Biology, Shanghai Jiao Tong University, graduated from Kagoshima University in Japan in 1998 with a doctorate.
Since 2000, he has held technical and management positions such as R&D manager, chief researcher and production technology director at Bright Dairy, and as the technical head of the Asia Pacific region of Döhler Food & Beverage Ingredients Group in Germany
.
In 2007, he entered Shanghai Jiao Tong University as a researcher, committed to the preparation and application of bioactive peptides, with a total investment of more than 10 million yuan, innovatively discovered 100 kinds of bioactive peptides and applied for invention patents, and published more than
30 articles related to bioactive peptides 。 Identification and dipeptidyl peptidase IV (DPP-IV) inhibitory activity verification of peptides from mouse lymphocytes
Juan Wanga,b, Yujia Xiea, Yuanyuan Luana, Tingting Guoa, Shanshan Xiaob, Xingxing Zengb, Shaohui Zhanga,b, *
a School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
b Zhejiang Go Peptides Life Science and Healthcare Technology Co.
, Ltd.
, Wenzhou 325000, China
*Corresponding author:
E-mail: shaohuizhang@sjtu.
edu.
cn
The objective of this study was to isolate and identify the intracellular bioactive peptides from mouse lymphocytes before and after lipopolysaccharide (LPS) stimulation, to explore novel peptides and to research the bioactive function.
Mouse spleen lymphocytes were isolated and cultured with LPS stimulation (experimental group) or not (control group) to collect intracellular peptides.
Totally 385 peptides were analyzed by nanoliter liquid phase-Q Exactive quadrupole ultra-high resolution orbitrap mass spectrometer (Nano LC-Q Exactive Plus) and identified by PEAKS X software.
After compared with peptides reported, 131 novel peptides were discovered, which then were predicted bioactivity by Peptide Ranker and 6 peptides with high bioactivity were predicted function by BIOPEP-UMW database.
Prediction data showed that they may have dipeptidyl peptidase IV (DPP-IV) inhibitory activity.
Finally, two peptides showed better potent inhibition were verified with competitive and noncompetitive modes.
Reference:
WANG J, XIE Y J, LUAN Y Y, et al.
Identification and dipeptidyl peptidase IV (DPP-IV) inhibitory activity verification of peptides from mouse lymphocytes[J].
Food Science and Human Wellness, 2022, 11(6): 1515-1526.
DOI:10.
1016/j.
fshw.
2022.
06.
009.
