Quantitative StructureâActivity Relationship Modeling Coupled with

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Functional Structure/Activity Relationships

QSAR Modeling Coupled with Molecular Docking Analysis in Screening of ACE Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-enzyme Combination Hydrolysis Kai Lin, Lan-wei Zhang, Xue Han, Zhao-xu Meng, Yi-fan Wu, Jian-ming Zhang, and Da-you Cheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00313 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Journal of Agricultural and Food Chemistry

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QSAR Modeling Coupled with Molecular Docking Analysis in Screening of ACE

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Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-enzyme

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Combination Hydrolysis †

Kai Lin , Lanwei Zhang

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†, ‡,§

*

, Xue Han

†

†

, Zhaoxu Meng , Jianming Zhang ,

†

Yifan Wu , Dayou Cheng

5

†

**

†

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† School of Chemistry and Chemical Engineering, Harbin Institute of Technology,

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Harbin 150000, China

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‡ College of Food Science and Engineering, Ocean University of China, Qingdao

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266003, China

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§ Hua Ling Biotechnology Research Center, Lanzhou 730000, China

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*

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e-mail: [email protected] phone number: +86-451-86282901, Fax: +86-451-

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86282901

14

**

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e-mail: [email protected] phone number: +86-451-86282901, Fax: +86-451-86282901

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Kai Lin e-mail: [email protected]

Corresponding Author: Lan-wei Zhang

Co-corresponding author: Xue Han

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1

ACS Paragon Plus Environment


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Abstract: In this study, Qula casein derived from yak milk casein was hydrolyzed using

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a two-enzyme combination approach and high angiotensin I-converting enzyme (ACE)

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inhibitory activity peptides were screened by quantitative structure-activity relationship

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(QSAR) modeling integrate with molecular docking analysis. Hydrolysates (< 3 kDa)

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derived from combinations of thermolysin+alcalase and thermolysin+proteinase K

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demonstrated high ACE inhibitory activities. Peptide sequences in hydrolysates derived

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from these two combinations were identified by liquid chromatography-tandem mass

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spectrometry (LC-MS/MS). Based on the QSAR modeling prediction, a total of 16

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peptides were selected for molecular docking analysis. The docking study revealed that

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four of the peptides (KFPQY, MPFPKYP, MFPPQ and QWQVL) bound the active site

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of ACE. These four novel peptides were chemically synthesized and their IC50 was

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determined. Among these peptides, KFPQY showed the highest ACE inhibitory

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activity (IC50: 12.37±0.43 μM). Our study indicated that Qula casein present an

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excellent source to produce ACE inhibitory peptides.

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Key words: ACE inhibitory peptide, Molecular docking, Two-enzyme combination,

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QSAR modeling, Qula casein

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2


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1. Introduction Every year, cardiovascular diseases (CVDs) take the lives of 17.7 million

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individuals,

which

represents

31%

of

all

deaths

worldwide

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(http://www.who.int/cardiovascular_diseases/en/). Hypertension is one of the main

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contributory factor to CVDs 1. Angiotensin I-converting enzyme (ACE) plays an

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important role in the regulation of blood pressure through both the renin-angiotensin

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system (RAS) and kallikrein kinnin system (KKS). In the RAS, the ACE is responsible

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for converting deca-peptide angiotensin I into a potent vasoconstrictor octa-peptide

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angiotensin II. In addition, in the KKS, ACE inactivated the vasodilator, bradykinin 2.

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Therefore, inhibiting the activity of ACE is considered an effective therapeutic

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approach for the treatment of hypertension. In the clinic, hypertension is treated with

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synthetic ACE inhibitors, such as Captopril, Lisinopril and Enalapril. However, these

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synthetic ACE inhibitors may lead to several side effects, including dry cough,

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headache, and fever 3.

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Due to the reported side effects of synthetic drugs, the extraction of ACE inhibitory

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peptides from natural food sources to replace synthetic drugs has attracted widespread

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attention. The potential bioactive peptides are inactive within the sequence of parent

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proteins, but can be released during enzymatic digestion 4. In many studies, the use of

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various enzymes has been reported to hydrolyze different proteins, including silkworm

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pupa 5, smooth-hound viscera 6, buffalo milk 7, pinto bean 8, marine sponge (Stalotella

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aurantium) 9. Most of the research strategies involved hydrolyzing the protein with 3



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individual enzymes to obtain ACE inhibitory peptides. However, since different

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enzymes have their own specific enzyme cleavage sites, using a combination of

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enzymes to hydrolyze a protein may produce novel ACE inhibitory peptides compared

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to single enzymatic hydrolysis. In previous studies, a combination of enzymes has been

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used to hydrolyze various proteins for the production of ACE inhibitory peptides 10-13.

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However, multi-step purification approaches in conventional peptide discovery

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strategies are required to obtain novel ACE inhibitory peptide, and it involves a low

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yield and high associates costs

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quantitative structure-activity relationship modeling, has successfully been applied to

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predict the biological activities of peptides 15-17. In addition, molecular docking analysis

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has been employed to illuminate the spatial interaction between receptor proteins and

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donor peptides 9, 18-19. Compared with traditional experimental studies, in silico analysis

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is time-saving and more economical to quickly discover novel ACE inhibitory peptides

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20

14

. In recent years, in silico approach, such as

.

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Yak milk is the most common product in northwestern China, and includes

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Xinjiang, Gansu, and Tibet. For residents of these areas, yak milk is the key ingredient

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of their daily diets. However, due to the lack of transportation and storage, it is a

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challenge to collect and process fresh yak milk. Most yak milk is naturally acidified,

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leached, collected and dried to produce Qula. Qula is a type of crude cheese, and its

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main component is casein 21. Currently, Qula has only been used as a raw material for

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the production of different grades of industrial casein

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casein into valuable bioactive peptides has significant potential.

22

4


. Therefore, converting Qula

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To our knowledge, data on the use of QASR modeling coupled with molecular

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docking analysis to screen ACE inhibitory peptides from Qula casein hydrolysates

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obtained by two-enzyme combination hydrolysis is limited. The aim of this study was

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to provide a fast and efficient method for screening novel ACE inhibitory peptides

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derived from Qula casein hydrolysates using two-enzyme combination through

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integrated QSAR modeling with molecular docking approach. Potential ACE inhibitory

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peptides were synthesized and evaluated for their in vitro activities.

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2. Materials and methods

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2.1. Chemicals

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Qula casein was obtained from Hualing Casein Co. (Gansu province, China).

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Angiotensin I-converting enzyme from rabbit lung (EC 3.4.15.1; 2U/mg protein), N-

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Hippuril-L-histidy-L-leucine (HHL), thermolysin from Geobacillus stearothermophilu

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(EC 3.4.21.1), alcalase from Bacillus licheniformis (EC 3.4.21.62), trypsin from

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porcine pancreas (EC 3.4.21.4), proteinase K from Tritirachium album (EC 3.4.21.64),

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and papain from Carica papaya (EC 3.4.22.2) were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). All other reagents were of analytical reagent grade.

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2.2. Preparation of Qula casein

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Qula casein was prepared from Qula through isoelectric precipitation following a

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previously published method 21, with slight modifications. In brief, Qula was suspended

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in double-distilled water (8 % w/v) and the pH was adjusted to 8 using 1 M NaOH. The

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suspension was stirred with a magnetic stirrer for 30 min at 500 rpm and 45 °C, then

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high-speed sheared at 10,000 rpm for an additional 15 min. The obtained solution was 5



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passed through a 200-mesh filter cloth and the permeate was centrifuged at 11,000 g

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for 10 min to remove residual fat. To induce casein precipitation, the supernatant was

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acidified to pH 4.6 with 1 M HCl. Finally, the casein precipitate was washed three times

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with double-distilled water, freeze-dried, and stored at -20 °C until further analysis.

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2.3. Hydrolysis of Qula casein with two-enzyme combination

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Based on our previous studies23, five enzymes (thermolysin, alcalase, papain,

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proteinase K, and trypsin) were selected for two-enzyme combined hydrolysis of Qula

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casein. The hydrolysis time, pH, and temperature of each enzyme were presented in

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Table 1. Qula casein (4 % w/v) was dissolved in 100 mM Tris-HCl buffer and heated

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for 10 min at 90 °C under constant stirring. Next, this solution was adjusted to the

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optimum temperature and pH for each enzyme. For hydrolysis using the two-enzyme

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approach, Qula casein was first hydrolyzed by one enzyme, and inactivated prior to

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addition of the second enzyme. Enzymes were added to the solution at a 3:1 (U/mg)

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enzyme/protein ratio. The pH was maintained constant by adding 1 M NaOH. At the

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end of hydrolysis, the reaction mixture was incubated in boiling water for 10 min to

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inactivate the enzyme. The mixture was centrifuged for 20 min at 12,000 g at 4 °C and

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the supernatant was collected. The degree of hydrolysis (DH) in Qula casein

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hydrolysates was determined using the o-phthaldialdehyde approach as previously

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described 24.

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The Qula casein content was determined by the Kjeldahl method (N × 6.38)

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(AOAC 2000) 25, and the peptide concentration in each hydrolysate was determined by

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the bicinchoninic acid (BCA) method (Pierce, Rockford, IL, USA) following the 6


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manufacturer’s instructions.

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2.4. Ultra-filtration

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Qula casein hydrolysates were separated by ultra-filtration using a regenerated

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cellulose membrane with a 3 kDa molecular mass cut-off (Millipore Co., Billerica, MA,

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USA). Permeates were stored at -20 °C until further analysis.

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2.5. IC50 determination of Qula casein hydrolysates (< 3 kDa)

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The ACE inhibitory activities of Qula casein hydrolysates < 3 kDa and synthetic

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peptides were evaluated and expressed as IC50 values, which were determined using

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methods described in our previously published study 23.The IC50 values were defined

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as the concentration that inhibited 50 % of the ACE activity.

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2.6. LC-MS/MS analysis

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Identification of peptides in 3 kDa permeates was performed using a Q-Exactive

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mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a

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Thermo Scientific EASY-nLC 1000 System (Thermo Fisher Scientific, Waltham, MA,

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USA). Samples were loaded in a reverse phase trap column (2 cm × 100 μm, 5 μm-

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C18), which was connected to a reverse phase analytical column (75μm × 100 μm, 3

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μm-C18). Buffer A (0.1 % formic acid in LC/MS grade H2O) was used for equilibration

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of chromatographic columns, whereas buffer B (0.1 % formic acid in 84 % LC/MS

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grade acetonitrile) was utilized for sample separation. For LC-MS/MS analysis, the

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sample was injected into the trapping column at a flow rate of 300 nL/min using a

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gradient from 0 % B to 35 % B over 50 min, followed by a gradient from 35 % B to

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100 % B over 5 min, and was finally maintained at 100 % B over 5 min. The mass 7



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spectrometer (MS) was operated in positive-ion detection mode, and the most abundant

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precursor ions from the scanning range of 300-1800 m/z were selected to obtain MS

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data. Automatic gain control target was set to 3e6 and the first-order maximum injection

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time was 50 ms. The dynamic exclusion duration was 40 s. Survey scans were obtained

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at a resolution of 70,000 at m/z 200, whereas the resolution for higher-energy collisional

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dissociation (HCD) spectra was set to 17,500 at m/z 200. Other parameters were as

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follows: isolation window: 2 m/z, second-order maximum injection time: 60 ms,

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normalized collision energy: 27 eV, and underfill ratio: 0.1%. Raw data were analyzed

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by Mascot software (version 2.3.0, Matrix Science, London, England) using a UniProt

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Knowledgebase (http://www.uniprot.org/), containing the sequences of αs1-casein

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(L8I5S0), αs2-casein (L8I6J3), β-casein (L8I8G5), and κ-casein (L8IIT8).

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2.7. Prediction of ACE inhibitory activities of peptides by QSAR modeling

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ACE inhibitory activities of peptides that were identified in the present study were

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predicted using the QSAR modeling that was described in our previous study 23. Four

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ACE inhibitory peptide databases containing penta-peptides, hexa-peptides, hepta-

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peptides and octa-peptides were constructed. In brief, identified peptides were

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converted into X-matrix by means of z-5 scales 26. In the peptide descriptor variable,

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the amino acid at the C-terminus was designated as c1, and its properties were described

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as c1z1, c1z2, c1z3, c1z4, and c1z5. Similarly, the second position from the C-terminus

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was designated as c2, and its properties were described as c2z1, c2z2, c2z3, c2z4, and

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c2z5, etc. ACE inhibitory activities of identified peptides were predicted using SIMCA-

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P version 11.5 software (Umetrics, Umeå, Sweden) with partial least squares (PLS) 8


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regression. For each QSAR model, the top two ranked peptides with the highest

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predicted ACE inhibitory activities were selected for further analysis.

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2.8. Molecular docking analysis

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The structures of the predicted peptides with the highest predicted ACE inhibitory

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activities were constructed using Chem Office 2015 software (Cambridge Soft Co.,

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Boston, MA, USA). The structure was energy minimized using steepest descent and

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conjugate gradient techniques. The 3D structure of human ACE (1O8A.pdb) was

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derived from the Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do).

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Prior to the docking analysis, the structure of water molecules and the inhibitor

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Lisinopril were removed from the data set using Discovery Studio 2.5 software (San

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Diego, CA, USA), whereas atoms of the cofactors zinc and chloride were retained in

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the active site of the crystal structure of ACE. The molecular docking study of the

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peptides at the ACE binding site was performed using Autodock Tools (ADT, version:

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1.5.6). Docking runs were carried out using a radius of 80 Å, with coordinates x: 40.79,

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y: 33.61, and z: 43.38 18. The best ranked docking pose of peptides in the active site of

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ACE was obtained according to the binding energy value.

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2.9. Peptide synthesis

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Potential ACE inhibitory peptides as selected by QSAR modeling and molecular

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docking studies were chemically synthesized by Sangon Biotech. Co., Ltd. (Shanghai,

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China). The purity (> 98 %) and sequences of these peptides were verified by analytical

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HPLC-MS/MS analysis. ACE inhibitory activity was determined as described above.

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2.10. In silico estimation of anti-gastrointestinal digestion and toxicity of ACE 9



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inhibitory peptides

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In silico analysis of the potential survival of synthetic peptides in vivo was

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performed by using Peptidecutter software (http://web.expasy.org/peptide_cutter/).

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ACE inhibitory peptides were evaluated using enzymes that were present in the

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gastrointestinal tract, including pepsin (EC 3.4.23.1, pH 1.3 and pH > 2), trypsin (EC

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3.4.21.4), and chymotrypsin (EC 3.4.21.1). The toxicity of the peptides was predicted

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using the online tool ToxinPred (http://www.imtech.res.in/raghava/toxinpred/).

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2.11. Statistical analysis

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Data are expressed as the mean ± standard deviation (SD) and were analyzed by

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one-way ANOVA using SPSS17.0 software (SPSS Inc., Chicago, IL, USA). Group

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mean comparisons were conducted using Duncan multiple range test. A value of p

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