Study of screening, transport pathway, and vasodilation mechanisms on angiotensin-I converting enzyme inhibitory peptide from Ulva prolifera proteins
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Abstract: In this study, Ulva prolifera protein was used for preparing angiotensin-I converting enzyme (ACE)-inhibitory peptide via virtual gastrointestinal digestion and in silico screening. Some parameters of the obtained peptide, such as inhibition kinetics, docking mechanism, stability, transport pathway, were explored by Lineweaver-Burk plots, molecular docking, in vitro stimulate gastrointestinal (GI) digestion and Caco-2 cells monolayer model, respectively. Then, a novel anti-ACE peptide LDF (IC50, (1.66 ± 0.34) μmol/L) was screened and synthesized by chemical synthesis. It was a no-competitive inhibitor and its anti-ACE inhibitory effect mainly attributable to four Conventional Hydrogen Bonds and Zn701 interactions. It could keep activity during simulated GI digestion in vitro and was transported by peptide transporter PepT1 and passive-mediated mode. Besides, it could activate Endothelial nitric oxide synthase (eNOS) activity to promote the production of NO and reduce Endothelin-1 (ET-1) secretion induced by Angiotensin II (Ang II) in Human Umbilical Vein Endothelial Cells (HUVECs). Meanwhile, it could promote mice splenocytes proliferation in a concentration-dependent manner. Our study indicated that this peptide was a potential ingredient functioning on vasodilation and enhancing immunity.
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Figure 2. Lineweaver-Burk plots of the synthetic peptide. 1/V and 1/S represents the reciprocal of reaction velocity and substrate concentration, respectively.
Figure 3. Stability of LDF against GI proteases. Full scan primary MS chromatogram for Control group (a), pepsin (b), pepsin-trypsin (c). d presents inhibition rate of above groups in the same concentration (P, pepsin; P-T, pepsin-trypsin; 5 μmol/L). The values are represented as the mean of the triplicate ± SD.
Figure 4. Effect of incubation time on the transport of LDF across the Caco-2 cell monolayers. a. Transport rate of LDF at different times. Samples were collected from the B side at 30 min, 60 min, and 90 min for HPLC analysis. Effect of Gly-Pro (PepT1 inhibitor), cytochalasin D (Tight junction disruptor) and wortmannin (Transcytosis inhibitor) on the transport of LDF across Caco-2 cell monolayers.Values represent the mean±standard deviation, and the bars with different lowercase letters were significantly different (p < 0.05, n = 3).
Figure 5. Docking simulation of candidates binding with ACE, a-LDF (IC50 = 1.66 μmol/L), b-Lisinopril (ID:1O86, IC50 = 1.1 ng/mL).
Figure 7. Effect of LDF on HUVECs. a. Effect of LDF on eNOS activity and NO secretion (extracellular). b. Effect of LDF on ET-1 secretion (Control, cells were cultured by medium for 18 h; LDF, cells were cultured by LDF for 18 h; Ang II, cells were cultured by medium for 12 h and then, adding Ang-II for another 6 h; Ang-II-LDF, cells were incubated by LDF for 12 h and then, adding Ang II for another 6 h; The final concentration of LDF and Ang II were 100 μmol/L and 100 nmol/L, respectively). c. Effect of LDF on the NO secretion (intracellular). Values (mean ± SD) that do not share a common lowercase letter within a column differ significantly (p < 0.05) (n = 3).
Table 1. Ulva prolifera protein sequences used in in silico analysis
Protein Amino acid residues Molecular weight/kDa A Pyruvate orthophosphate dikinase 899 96.43 0.1624 Adenine phosphoribosyl transferase 182 19.19 0.3908 Photosystem Ⅰ assembly protein Ycf4 185 24.42 0.4510 Ribosomal protein L14 (chloroplast) 123 13.52 0.4016 50S ribosomal protein L5 (chloroplast) 179 20.22 0.3722 30S ribosomal protein S12 (chloroplast) 74 13.60 0.3659 γ-carbonic anhydrase 1 173 23.57 0.3815 γ-carbonic anhydrase 2 226 17.99 0.3274 Plastid geranylgeranyl diphosphate synthase 330 35.69 0.2485 Plastid isopentenyl-diphosphate delta-isomerase Ⅰ 245 27.66 0.3265 Plastid 4-cytidine-5-diphospho-2-C-methyl-D-erythritol kinase 324 35.45 0.2500 Plastid 4-diphosphocytidyl-2C-methyl-D-erythritol synthase 269 29.24 0.3048 Plastid 1-deoxy-D-xylulose 5-phosphate reductoisomerase* 437 46.94 0.2517 Plastid 1-deoxy-D-xylulose 5-phosphate synthase 713 76.88 0.1865 Note: Parameter A: the frequency of bioactive fragments occurring in a protein sequence. *, represents the precursor protein for the final selected peptide. 
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Table 2. Pool of potential ACE inhibitory peptides
Peptide Peptide ranker WS Toxin HIA BBB -CE score LDF 0.839471 Good NO +0.7591 +0.8952 75.0459 WKL 0.827822 Good NO +0.6493 +0.8556 Fail FLK 0.805333 Good NO +0.7591 +0.8371 Fail FLKF 0.956004 Good NO +0.7591 +0.8371 Fail FLPR 0.925723 Good NO +0.6554 +0.8066 Fail DLGW 0.876197 Good NO +0.6493 +0.9109 Fail LSRF 0.817020 Good NO +0.7139 +0.9595 Fail LDLF 0.806570 Good NO +0.7591 +0.8952 Fail RYIF 0.847108 Good NO +0.7519 +0.9403 Fail DFL 0.889906 Good NO +0.7591 +0.8952 Fail LDFL 0.831537 Good NO +0.7591 +0.8952 Fail LYRF 0.920545 Good NO +0.7519 +0.9186 Fail Note: WS, water solubility; HIA+, high human intestinal absorptivity; BBB +, higher blood brain barrier permeability; -CE score, score of -C Docker energy (−kcal/mol).
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Table 3. Interactions between ACE and candidates
Candidates Bond position Distance/Å Type Number LDF A:LYS511:HZ3-LDF:O54 1.81686 Electrostatic interaction 5 A:ASP415:OD2-LDF:H4 2.08741 − − A:HIS353:NE2-LDF:O31 5.1492 − − A:HIS353:NE2-LDF:O54 4.02229 − − A:ZN701:ZN-LDF:O31 2.31603 − − A:HIS353:HE2-LDF:O33 2.68386 Conventional Hydrogen Bond 4 A:TYR520:HH-LDF:O53 2.287 − − A:ASP415:OD1-LDF:H2 2.52546 − − A:ASP415:OD1-LDF:H3 2.54477 − − A:HIS353:HE1-LDF:O33 2.62587 Carbon Hydrogen Bond 4 A:HIS353:HE1-LDF:O54 2.32017 − − A:VAL380:HA-LDF:O21 2.32324 − − A:HIS513:HE1-LDF:O54 2.3485 − − A:VAL380-LDF:C16 5.39525 Alkyl 1 Lisinopril (Lis) A:HIS353:NE2-Lis:O1 5.54654 Attractive Charge 3 A:LYS511:NZ-Lis:O22 5.33806 − − A:ZN701:ZN-Lis:O1 2.0673 − − A:GLN281:HE21-Lis:O22 2.45266 Conventional Hydrogen Bond 6 A:GLN281:HE22-Lis:O23 2.28218 − − A:HIS353:HE2-Lis:O17 2.46511 − − A:TYR520:HH-Lis:O23 2.02826 − − A:ALA354:O-Lis:H40 2.119 − − A:HIS383:NE2-Lis:H58 2.01119 − − A:HIS353:HE1-Lis:O17 2.54683 Carbon Hydrogen Bond 3 A:GLU162:OE2-Lis:H52 2.77882 − − A:GLU384:OE1-Lis:H59 2.29522 − − A:HIS383-Lis:N24 4.95965 Pi-Cation 1 Note: − reprents no data.
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