Two novel potent ACEI peptides isolated from Pinctada fucata meat hydrolysates using in silico analysis: identification, screening and inhibitory mechanisms

Jiao Li; Jilei Su; Min Chen; Jiao Chen; Wenping Ding; Yanqun Li; Hao Yin

doi:10.1039/d0ra10476k

Two novel potent ACEI peptides isolated from Pinctada fucata meat hydrolysates using in silico analysis: identification, screening and inhibitory mechanisms

RSC Adv. 2021 Mar 25;11(20):12172-12182. doi: 10.1039/d0ra10476k. eCollection 2021 Mar 23.

Authors

Jiao Li^{1

2

3

4}, Jilei Su^{1

2

3

4}, Min Chen^{1

2

3

4}, Jiao Chen^{5

6}, Wenping Ding^{1

2

3

4}, Yanqun Li^{1

2

3

4}, Hao Yin^{1

2

3}

Affiliations

¹ CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences Guangzhou 510301 China yinhao@scsio.ac.cn.
² Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences Guangzhou 510301 China.
³ Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) Guangzhou 511458 China.
⁴ College of Earth and Planetary Sciences, University of Chinese Academy of Sciences Beijing 100049 China.
⁵ Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine Nanjing 210028 China.
⁶ Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine Nanjing 210028 China.

Abstract

The aim of this study was to discover potent angiotensin-converting enzyme (ACE) inhibitory (ACEI) peptides from Pinctada fucata (P. fucata) for treating hypertension and to characterize them using in silico analysis. The P. fucata proteins were hydrolyzed by Alcalase®, a serine endopeptidase with broad selectivity, at various times (0, 2, 4, 6, 8, 10 h). The degree of hydrolysis (DH) and ACEI activity of the different hydrolysates were measured. Considering the molecular weight and ACEI activity, the 10 h hydrolysate was purified by a series of traditional separation methods, including ultrafiltration, gel G-25 chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC), with ACEI activity as a guide. The results showed two fractions, C17 and C18, eluted by means of semi-preparative RP-HPLC, and showed the highest ACEI activities of 80.33 ± 2.70% and 81.66 ± 0.29%, respectively, at 1 mg mL^-1. The two fractions were then identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and their MS/MS spectra data were subjected to de novo sequencing. Subsequently, the potential ACEI peptides were screened by in silico methods, namely, to analyze the average local confidence (ALC) value obtained from the sequencing software and the P-value from the Pepsite 2. In total, 13 potential ACEI peptide sequences were obtained and identified from the two fractions by LC-ESI-MS/MS, and two novel tetrapeptides, FRVW (607.3314 Da) and LPYY (555.2881 Da), were screened for synthesis according to the in silico analysis. The in vitro ACEI tests indicated that FRVW and LPYY had IC₅₀ values of 18.34 and 116.26 μM, respectively. The Lineweaver-Burk plot showed that FRVW was a noncompetitive inhibitor, and LPYY was shown to be a mixed-mode type inhibitor. A stability study against ACE indicated that both peptides were hydrolyzed by ACE to some extent, the higher ACEI activity following incubation with ACE indicating that they should be classified as pro-drug substrates. Molecular docking results showed that hydrophobic amino acids (HAAs) within peptides formed vital interactions including hydrogen bonds, electrostatic forces, van der Waals forces and Pi-Pi interactions with ACE residues, which stabilized the enzyme-peptide complex. Furthermore, the docking results accorded with the inhibition kinetic mode. Our study demonstrated that FRVW and LPYY isolated from P. fucata have potential applications as antihypertensive agents.