Subscriber access provided by Eastern Michigan University | Bruce T. Halle Library
Bioactive Constituents, Metabolites, and Functions
Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate Maolin Tu, Hanxiong Liu, Ruyi Zhang, Hui Chen, Fengjiao Mao, Shuzhen Cheng, Weihong Lu, and Ming Du J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00732 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
Journal of Agricultural and Food Chemistry
Analysis and evaluation of the inhibitory mechanism of a novel ACE-inhibitory peptide derived from casein hydrolysate
Maolin Tu a,b, Hanxiong Liu a, Ruyi Zhang a, Hui Chen a, Fengjiao Mao a, Shuzhen Cheng a,c, Weihong Lu b, Ming Du a,b,*
a
School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China
b
Department of Food Science and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China
c
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
Author contributions: Maolin Tu and Hanxiong Liu contributed equally to this study.
*Corresponding author Dr. Ming Du Tel: +86-411-86332275 Fax: +86-411-86323262 E-mail:
[email protected].
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
ABSTRACT
2
Casein hydrolysates exert various biological activities and the responsible
3
functional peptides are being identified from them continuously. In this study, the
4
tryptic casein hydrolysate was fractionated by ultra-filtration membrane (3 kDa), and
5
the peptides were identified by CE-TOF-MS/MS. Meanwhile, in silico methods were
6
used to analyze the toxicity, solubility, stability, and affinity between the peptides and
7
ACE. Finally, a new angiotensin I-converting enzyme-inhibitory (ACEI) peptide,
8
EKVNELSK, derived from the αs1-casein (fragment, 35–42), was screened. The IC50
9
value of the peptide is 5.998 mM, which was determined by an HPLC method. The
10
Lineweaver-Burk plot indicated that this peptide is a mixed-type inhibitor against
11
ACE. Moreover, Discovery Studio 2017 R2 software was adopted to perform
12
molecular docking to propose the potential mechanisms underlying the ACEI activity
13
of the peptide. These results indicated that EKVNELSK is a new ACEI peptide
14
identified from casein hydrolysate.
15 16
Keywords: Casein, ACE-inhibitory activity, Peptide, In silico, CE-TOF-MS/MS,
17
Molecular docking,
18
2
ACS Paragon Plus Environment
Page 2 of 27
Page 3 of 27
Journal of Agricultural and Food Chemistry
19
INTRODUCTION
20
Cardiovascular disease (CVD) has been recognized the leading cause of death in
21
the worldwide. It is expected that there will be more than 23.6 million deaths in 2030
22
caused by CVD 1. Hypertension (blood pressure > 140/90), as one of the independent
23
risk factors for CVD, with characteristics of frequent, chronic, and age-related
24
disorders
25
can be an effective way to prevent CVD 5. To control the blood-pressure, the
26
angiotensin I-converting enzyme (ACE, EC 3.4.15.1) must be taken into consideration,
27
which plays a significant role in blood pressure regulation through both the
28
renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) in vivo. In the
29
RAS, the potent vasopressor angiotensin II (Ang II) can be released by the ACE
30
cleaving the C-terminal dipeptide from angiotensin I (Ang I) 6. In the KKS, ACE can
31
also inactivate the bradykinin (BK) by removing the two C-terminal dipeptides 7.
32
Therefore, more attentions have been gradually paid to find novel ACEI substances
33
for the sake of lowering blood pressure. In the recent years, pharmaceutical drugs
34
targeting ACE are shown evident in lowering high blood pressure. However, some
35
side effects have appeared in clinical treatments 8. To find safer drugs, increasingly
36
scholars have paid attention to food-derived ACE inhibitors, especially the peptides
37
which owner the natural advantages of safe and easily to be metabolized in bodies.
38
ACEI peptides have been continuously identified from milk, soybean, egg and so on.
39
2-4
, has been paid increasingly attention. Blood-pressure lowering treatment
Casein is an excellent source of bioactive peptides and has been widely used in 9-10
40
food industry as a functional ingredient
41
been identified from enzymatic hydrolysates of casein, especially ACEI peptides. For
42
example, peptide QSLVYPFTGPI (from β-casein) and ARHPHPHLSFM (from
43
κ-casein) showed potent ACEI activity in vitro 11.
. To date, lots of bioactive peptides have
3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 27
44
To identify the bioactive peptides more effectively, some in silico methods have
45
been employed in recent studies. Quantitative structure-activity relationship (QSAR)
46
method, which has been widely applied to elaborate the relationships between the
47
peptides’ primary structures and the ACEI activity
48
been used to study the active mechanisms of the inhibitors against the receptors, such
49
as ACE
50
peptides by analyzing the physicochemical characteristics of the peptides. For
51
instance,
52
https://web.expasy.org/compute_pi/, has the function of predicting the molecular
53
weight (MW) and Iso-electric point (PI) of the potential bioactive peptides. ProtParam
54
tool can be used to predict instability index of peptides, available at
55
https://web.expasy.org/protparam/. The toxicity of the peptides can be predicted by
56
the online server ToxinPred, available at http://crdd.osdd.net/raghava/toxinpred/.
13
12
. Molecular docking also has
and thrombin Online servers have been used to screen the bioactive
the
online
server
Expasy-Compute
pI/Mw,
available
at
57
The aim of this study is to screen and identify a novel ACEI peptide from casein
58
hydrolysate and elaborate its active mechanisms. The peptides from casein
59
hydrolysates were determined by CE-Q-TOF-MS/MS. Toxicity, solubility, and
60
stability of the peptides were predicted by in silico methods. A novel ACEI peptide
61
was identified and subsequently synthesized. The IC50 value and the inhibition mode
62
of the peptide were investigated. Molecular docking analysis also were used to
63
elucidate the potential mechanism of ACEI activity of the peptide.
64 65
MATERIALS AND METHODS
66
Materials and chemicals
67 68
ACE (from rabbit lung), Hippuryl-histidyl-leucine (HHL), HPLC grade acetonitrile (ACN), and trifluoroacetic acid
(TFA) were purchased from
4
ACS Paragon Plus Environment
Page 5 of 27
Journal of Agricultural and Food Chemistry
69
Sigma-Aldrich Co. (St. Louis, MO, USA). Casein and trypsin were obtained from
70
Solarbio (Beijing, China). All other chemical reagents were of analytical grade.
71
Preparation of casein hydrolysates
72
Bovine casein solution (50 mg mL-1) was prepared by dissolving bovine casein
73
powder in deionized water. Then, 0.1 M NH4HCO3 buffer was used to adjust the pH
74
of the casein solution to 8.0. The hydrolysis reaction was started by the adding trypsin
75
with 2500 U g-1 of enzyme concentration. Other hydrolysis parameters were set as
76
120 min of hydrolysis time and 37 °C of temperature. Heating the samples in boiling
77
water (10 min) to quench the reaction before adjusting the pH to 7.0 (after the samples
78
cooled to below 25 °C). Subsequently, centrifuged the samples at 8000×g for 20 min
79
at 4 °C. Then, the supernatant was fractionated by Amicon Ultra ultrafiltration tube
80
(Millipore, [MWCO] = 3 kDa) according to the molecular weight and the fraction
81
with peptides’ molecular weight no more than 3 kDa was collected and freeze-dried
82
before storage at -80 °C.
83
Determination of ACEI activity
84
The assay of in vitro ACEI activities of the casein hydrolysate and the peptide
85
were conducted by an HPLC method as previously reported 14.
86
Identification of peptides by CE-Q-TOF-MS/MS
87
CE analysis was conducted on a CESI 8000 Plus System (AB SCIEX, Inc.,
88
Redwood City, CA, U.S.A.). The uncoated fused-silica capillary (91 cm of total
89
length, i.d. 30 µm) was coupled to Q-TOF-MS/MS (impact II, Bruker Daltonic GmbH,
90
Bremen, Germany) through electrospray ionization source (ESI). In the process of
91
capillary separation, 10% acetic acid solution and 100 mM ammonium acetate were
92
used as the background electrolyte (BGE) buffer and the conductive liquid,
93
respectively.
The
washing
and
injection
of
the
5
ACS Paragon Plus Environment
sample
were
operated
Journal of Agricultural and Food Chemistry
Page 6 of 27
94
hydrodynamically. Before loading each sample, the bare fused capillary was
95
conditioned by forwarding with 0.1 M NaOH for 3 min, followed by 3 min with 0.1
96
M HCl and 4 min with ultrapure water and 3 min with BGE for reversing and
97
followed by 4 min forward with BGE. Subsequently, the samples were loaded on
98
using under 20 psi pressure (1 min), then followed with BGE (30 s, 20 psi). The
99
separation was then conducted electrodynamically (1 h) by applying a voltage of +20
100
kV after injection. The CE-ESI-Q-TOF-MS was set as the following parameters:
101
150 °C of dry temperature, 3.0 L min-1 of dry gas speed. Moreover, the top 20 intense
102
ions were collected in the MS spectra over an m/z range of 50–2200. Finally, Data
103
Analysis 4.0 software (Bruker Daltonic GmbH, Bremen, Germany) was used to
104
process the MS data. MASCOT searching engine (Matrix Science, London, UK) was
105
adopted to identify the sequence of the peptides in the sample.
106
Screening of the potential ACEI peptide
107
Some properties of the peptides are crucially important for the further studies of
108
the peptides especially drug-oriented, such as the toxicity, solubility, and stability.
109
Therefore, those factors have been taken into consideration for screening the potential
110
ACEI peptides. The ToxinPred server was used to analyze toxicity of the peptides,
111
available at http://crdd.osdd.net/raghava/toxinpred/. The solubility of the peptide was
112
evaluated
113
www.innovagen.com/proteomics-tools. The instability index of the peptide was
114
predicted by using ProtParam Tool (http://web.expasy.org/protparam/). In additional,
115
the affinities of the peptides against the ACE were compared by evaluating the
116
molecular docking results, which were conducted on the Discovery Studio R2
117
software (Neotrident Technology Ltd., Beijing, China).
118
Chemical synthesis of peptides
by
the
online
Innovagen
6
ACS Paragon Plus Environment
server,
available
at
Page 7 of 27
Journal of Agricultural and Food Chemistry
119
Selected peptide was synthesized by a solid-phase method at Cellmano Biotech
120
Limited Corporation (Hefei, China), which was with purity of 98.55% and verified by
121
HPLC.
122
Assay of ACEI kinetics
123
The inhibition pattern of the peptide, EKVNELSK, was explored by using
124
Lineweaver-Burk plots of 1/V versus 1/HHL. The concentrations of the HHL were set
125
as 0.5, 1, 2 and 3.5 mM, meanwhile, the concentrations of the peptide were set as 0,
126
4.228 and 8.456 Mm. The Vmax and Km were calculated, respectively, as the Y and
127
X-axis intercepts of the primary plot.
128
Molecular docking analysis
129
Discovery Studio 2017 R2 software has been used to further evaluate the active
130
mechanism of the ACEI peptide through molecular docking technology according to
131
the reported methods
132
PDB ID: 1O8A) was downloaded from RCSB Protein Data Bank. The 3D-structures
133
of the peptide was produced by DS 2017 R2, and then minimized it by given the
134
CHARMm force field. Meanwhile, ACE molecular was treated by the procedures of
135
cleaning, preparing, removing waters and adding hydrogen. Finally, partial flexibility
136
program CDOCKER was chosen to perform the docking program with special
137
binding sites (coordinates x: 36.189, y: 43.643 and z: 55.175) and receptor radius (16
138
Å). The results of molecular docking were evaluated based on the -CDOCKER
139
energy scores, interaction site, and interaction force types with ACE.
140
Statistical analysis
141
15
, with some modifications. ACE crystal structure (Human,
All experiments were conducted in triplicate and the data were presented as the
142
mean ± SD in this study. Origin 8.0 (OriginLab Corporation, Northampton, MA, USA)
143
software was adopted to process the data. 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 27
144 145
RESULTS AND DISCUSSION
146
ACEI activity and peptide composition of casein hydrolysate
147
In this study, the ACEI activity of the casein hydrolysate with the molecular
148
weight no more than 3 kDa, has been determined by an HPLC method. As shown in
149
Fig.1, the ACEI activity of the mixture became significantly from the concentration of
150
0.05 mg mL-1, and the IC50 value of the casein hydrolysate was 0.4424 mg mL-1. To
151
analyze the component of the active mixture, CE-ESI-Q-TOF-MS/MS was used to
152
identify the peptides (Table 1). There is a total of 32 peptides were identified from the
153
casein mixture and 14, 11, 5, and 2 peptides were obtained from αs1-, αs2-, β- and
154
κ-CN, respectively. Although the mixture has been fractionated by ultrafiltration tube,
155
it’s not hard to find that there are also two peptides with molecular weight more than
156
3 kDa, which may attribute to the fact that the ultrafiltration tube can’t separate the
157
mixture completely. As casein hydrolysate mixture with potent ACEI activity, the
158
identified peptides can be considered as the potential ACE inhibitors.
159
Screening of the potential active ACEI peptides
160
In the present casein hydrolysate, the ACEI peptides have reported were
161
explored
162
(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) and the published literatures.
163
As labelled in Table1, there are six peptides (FFVAPFPEVFGK 16, ALNEINQFYQK
164
17
165
activity, with IC50 values of 18 µM, 264 µM, 600 µM, 11.9 µM, 60 µM, 15 µM,
166
respectively. Therefore, those six peptides must be partly of the active factors, which
167
made the casein mixture exhibit high ACEI activity.
168
, AMKPWIQPK
by
18
searching
, FALPQYLK
19
the
, NMAINPSK 17, AVPYPQR
BIOPEP
20
) showed ACEI
The analysis of the human ACE-lisinopril complex has laid a solid foundation 8
ACS Paragon Plus Environment
Page 9 of 27
Journal of Agricultural and Food Chemistry
169
for the related studies of ACE inhibitors. There is a central active site groove that
170
extends for about 30 Å in the structure of ACE, which is the active site buried with
171
lisinopril
172
peptides with large spatial structures are less likely to be bioactive. Moreover, from a
173
cost point of view, the peptides with fewer amino acids are more meaningful for
174
exploring the drug-derived bioactive peptides. Therefore, the present study was aimed
175
to identify a novel ACEI peptide with the number of amino acids less than ten.
176
Twelve peptides with the characteristics of the novel and the sequences consist of less
177
than ten amino acids (Table 2). Toxicity is one of the fundamental factors should be
178
taken into consideration for the various products of foods and drugs. Consequently,
179
the identified ACEI peptides in this study were assessed for potential toxicity in silico
180
using ToxinPred 22. As shown in Table 2, all of the peptides showed no toxicity.
21
. Consequently, due to the limited space of the ACE active site, the
181
Solubility is also an important characteristic of the peptides, which will influence
182
the absorption, distribution, and elimination of the peptides in the human body 23. The
183
aqueous solubility of the potential ACEI peptides was evaluated by the online tool
184
Innovagen, one peptide (VLPVPQK) showed poor water solubility (Table 2).
185
Meanwhile, the stability has also been used as an evaluation index to screen
186
potential ACEI peptide, which was accessed by the online tool ProtParam. In this
187
program, if the instability index is smaller than 40, which is predicted as stable; when
188
the instability index value above 40, it is predicted as may be unstable. As shown in
189
Table 2, only 5 (EGIHAQQK, EKVNELSK, NRLNFLK, NRLNFLKK, FFSDKIAK)
190
out of 12 peptides were predicted as stable. Subsequently, molecular docking analysis
191
was adopted to evaluate the affinity of the peptides against ACE. The peptide
192
EKVNELSK against ACE acquired highest -CDOCKER ENERGY (kcal mol-1) score
193
(185.54), which means this peptide has the highest affinity with ACE (Table 3). 9
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
194
Hence, the peptide EKVNELSK is with higher probability to have the function of
195
inhibiting the ACE activity. To confirm the ACEI activity of the peptide,
196
EKVNELSK was synthesized for further study.
197
IC50 determination of the ACEI peptide activity
198
The HPLC method was adopted to evaluate the IC50 value of the peptide
199
EKVNELSK. Fig. 2 showed the ACE inhibitory activity of the peptide at various
200
concentrations. The regression equation, Y= (1.113E-2) X2+4.57673X+22.14678
201
(R2=0.98312), was used to evaluate the IC50 value of the peptide EKVNELSK. The
202
results showed that the peptide was a novel ACE inhibitor with an IC50 value of 5.998
203
mM. Although it is seemly that the peptide EKVNELSK showed week in vitro ACE
204
inhibitory activity. However, as the hypertension pathophysiology is very complicated
205
that related to a series of system activities 24, it is hard to judge the in vivo effect of the
206
peptide. Some studies showed that the relationship between in vitro ACE inhibition
207
and antihypertensive activity is not apparent
208
peptide KVLPVPQ with an IC50 value of 1000 µM, which is much higher than
209
YPFPGPIPN (IC50 of 15 µM). But, the peptide KVLPVPQ showed significant blood
210
pressure-lowering effect in vivo, which is 4.5 times that of YPFPGPIPN 25. Therefore,
211
the novel ACEI peptide, EKVNELSK, can be regarded as one of the blood
212
pressure-lowering inhibitor candidates and its effect in vivo of this peptide should be
213
further studied.
214
Inhibition mode of the peptide EKVNELSK
24
. Such as, β-casein derived ACEI
215
The ACE inhibition pattern of the peptide EKVNELSK was evaluated by using
216
the Lineweaver-Burk plot. As shown in Fig. 3, when the peptide concentration
217
increased, the Vm values decreased, which indicated that the existing of the peptide
218
EKVNELSK probably hindered the substrate bind to ACE active site. Meanwhile, the 10
ACS Paragon Plus Environment
Page 10 of 27
Page 11 of 27
Journal of Agricultural and Food Chemistry
219
higher of the peptide concentrations, the higher of Km values in the reactions, which
220
implied that the higher concentration of substrate is essential for the ACE catalytic
221
reaction. Moreover, the peptide EKVNELSK is a mixed-type ACE inhibitor. Similarly,
222
the peptide MLLCS has been identified from the hydrolysates of Styela plicata
223
peptides WVYY and WYT derived from the hemp seed proteins
224
mixed inhibition pattern against ACE. The mixed-type mode of ACE inhibition of the
225
peptide EKVNELSK indicated that the peptide binds to both the active and nonactive
226
sites of ACE, which consequently reduces the catalytic activity of ACE 27.
227
Interaction of the peptide and ACE
27
26
,
also showed the
228
The docking study of the peptide (EKVNELSK) at the ACE active site was
229
performed by Discovery Studio 2017 R2 software. As shown in Table 3, the value of
230
peptide EKVNELSK from -CDOCKER_Energy indicated that the peptide
231
EKVNELSK has the highest strength of the affinity against ACE in the five potential
232
ACEI peptides. The most stabilized poses of the peptide EKVNELSK against ACE
233
were obtained with the values of electrostatic energy (Eele), Van der Waals energy
234
(Evdw), and potential binding energy (Epot) of -675.211, -14.8202 and -631.721 kcal
235
mol-1, respectively. The hydrogen bonds formed between the peptide and ACE played
236
a vital role in inhibiting the ACE activity by stabilizing the structure of the
237
non-catalytic enzyme-peptide complex 15, 27. Table 4 and Fig. 4 showed that totally of
238
15 H-bonds have formed between ACE and peptide EKVNELSK, which contains
239
nine (LYS118, ASP121, GLU123, SER219, ASP358, TYR360, VAL399, ARG402,
240
ARG522) kinds of ACE residues. The residues GLU123 and ARG522 also formed
241
H-bonds with the ACEI peptides (WG and PRY), as reported by Fu et al.
242
Meanwhile, three kinds of electrostatic also formed between EKVNELSK and ACE at
243
the ACE residues of LYS118, GLU411, and ARG522. Moreover, the electrostatic 11
ACS Paragon Plus Environment
15
.
Journal of Agricultural and Food Chemistry
244
interaction of ZN701 and hydrophobic interactions of ALA89, ALA125, PRO519,
245
ARG124, ILE88) with ACE also contributed to the stabilization of the
246
EKVNELSK-ACE complex. Therefore, peptide EKVNELSK is a novel and effective
247
casein-derived ACE inhibitor.
248 249
ACKNOWLEDGEMENTS
250
We are grateful to Dr. Fang Luo of the NeoTrident Technology Ltd. (Beijing,
251
China) for the molecular docking instruction and Qin Zhang of the Dalian Polytechnic
252
University for helpful discussion.
253 254 255 256
FUNDING This study was financially supported by the National Natural Science Foundation of China (31371805).
257 258 259
Notes All authors declare that they have no conflicts of interest.
260 261
12
ACS Paragon Plus Environment
Page 12 of 27
Page 13 of 27
Journal of Agricultural and Food Chemistry
262
REFERENCES
263
(1) Deshwal, S.; Di, S. M.; Di, L. F.; Kaludercic, N. Emerging role of monoamine
264
oxidase as a therapeutic target for cardiovascular disease. Curr. Opin. Pharmacol.
265
2017, 33, 64−69.
266
(2) Micha, R.; Peñalvo, J. L.; Cudhea, F.; Imamura, F.; Rehm, C. D.; Mozaffarian, D.
267
Association between dietary factors and mortality from heart disease, stroke, and type
268
2 diabetes in the United States. J. Am. Med. Assoc. 2017, 317, 912−924.
269
(3) Cannon, C. P. Cardiovascular disease and modifiable cardiometabolic risk factors.
270
Clin. Cornerstone. 2008, 9, 24−41.
271
(4) Gu, Q.; Dillon, C. F.; Burt, V. L.; Gillum, R. F. Association of hypertension
272
treatment and control with all-cause and cardiovascular disease mortality among US
273
adults with hypertension. Am. J. Hypertens. 2010, 23, 38−45.
274
(5) Ettehad, D.; Emdin, C. A.; Kiran, A.; Anderson, S. G.; Callender, T.; Emberson, J.;
275
Chalmers, J.; Rodgers, A.; Rahimi, K. Blood pressure lowering for prevention of
276
cardiovascular disease and death: a systematic review and meta-analysis. Lancet.
277
2016, 387, 957−967.
278
(6) Ahmad, I.; Yanuar, A.; Mulia, K.; Mun'Im, A. Review of angiotensin-converting
279
enzyme inhibitory assay: rapid method in drug discovery of herbal plants.
280
Pharmacogn. Rev. 2017, 11, 1−7.
281
(7) Negraes, P. D.; Trujillo, C. A.; Pillat, M. M.; Teng, Y. D.; Ulrich, H. Roles of
282
kinins in the nervous system. Cell Transplant. 2015, 24, 613-623
283
(8) Rai, A. K.; Sanjukta, S.; Jeyaram, K. Production of angiotensin I converting
284
enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in
285
reducing hypertension. Crit. Rev. Food Sci. Nutr. 2015, 57, 2789−2800.
286
(9) Capriotti, A. L.; Cavaliere, C.; Piovesana, S.; Samperi, R.; Laganà, A. Recent 13
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
287
trends in the analysis of bioactive peptides in milk and dairy products. Anal. Bioana.
288
Chem. 2016, 408, 2677−2685.
289
(10) Field, K. L.; Kimball, B. A.; Mennella, J. A.; Beauchamp, G. K.; Bachmanov, A.
290
A. Avoidance of hydrolyzed casein by mice. Physiol. Behav. 2008, 93, 189−199.
291
(11) Ibrahim, H. R.; Ahmed, A. S.; Miyata, T. Novel angiotensin-converting enzyme
292
inhibitory peptides from caseins and whey proteins of goat milk. J. Adv. Res. 2017, 8,
293
63−71.
294
(12) Wu, J.; Aluko, R. E.; Nakai, S. Structural requirements of angiotensin
295
I‐ converting enzyme inhibitory peptides: quantitative structure‐ activity relationship
296
modeling of peptides containing 4−10 amino acid residues. QSAR Comb. Sci. 2010,
297
25, 873−880.
298
(13) Wang, Y.; Jiang, Y.; Yin, Y.; Liu, J.; Ding, L.; Liu, J.; Zhang, T. Identification and
299
inhibitory mechanism of angiotensin I-converting enzyme inhibitory peptides derived
300
from bovine hemoglobin. Protein J. 2017, 36, 1−8.
301
(14) Tu, M.; Wang, C.; Chen, C.; Zhang, R.; Liu, H.; Lu, W.; Jiang, L.; Du, M.
302
Identification of a novel ACE-inhibitory peptide from casein and evaluation of the
303
inhibitory mechanisms. Food Chem. 2018, 256, 98−104.
304
(15) Fu, Y.; Alashi, A. M.; Young, J. F.; Therkildsen, M.; Aluko, R. E. Enzyme
305
inhibition kinetics and molecular interactions of patatin peptides with angiotensin
306
I-converting enzyme and renin. Int. J. Biol. Macromol. 2017, 101, 207−213.
307
(16) Tauzin, J.; Miclo, L.; Gaillard, J. L. Angiotensin‐ I‐ converting enzyme
308
inhibitory peptides from tryptic hydrolysate of bovine αS2-casein. FEBS lett. 2002,
309
531, 369−374.
310
(17) Murray, B.; FitzGerald, R. Angiotensin converting enzyme inhibitory peptides
311
derived from food proteins: biochemistry, bioactivity and production. Curr. Pharm. 14
ACS Paragon Plus Environment
Page 14 of 27
Page 15 of 27
Journal of Agricultural and Food Chemistry
312
Des. 2007, 13, 773−791.
313
(18) Mora, L.; Escudero, E.; Aristoy, M. C.; Toldrá, F. A peptidomic approach to study
314
the contribution of added casein proteins to the peptide profile in spanish
315
dry-fermented sausages. Int. J. Food Microbiol. 2015, 212, 41−48.
316
(19) López-Expósito, I.; Quirós, A.; Amigo, L.; Recio, I. Casein hydrolysates as a
317
source of antimicrobial, antioxidant and antihypertensive peptides. Dairy Sci. Technol.
318
2007, 87, 241−249.
319
(20) Maruyama, S.; Miyoshi, S.; Tanaka, H. Angiotensin I-converting enzyme
320
inhibitors derived from Ficus carica. Agric. Biol. Chem. 1989, 53, 2763−2767.
321
(21) Natesh, R.; Schwager, S. L.; Sturrock, E. D.; Acharya, K. R. Crystal structure of
322
the human angiotensin-converting enzyme-lisinopril complex. Nature. 2003, 421,
323
551−554.
324
(22) Gupta, S.; Kapoor, P.; Chaudhary, K.; Gautam, A.; Kumar, R.; Raghava, G. P. In
325
silico approach for predicting toxicity of peptides and proteins. Plos One. 2013, 8,
326
e73957.
327
(23) Balakin, K. V.; Savchuk, N. P.; Tetko, I. V. In silico approaches to prediction of
328
aqueous and DMSO solubility of drug-like compounds: trends, problems and
329
solutions. Curr. Med. Chem. 2006, 13, 223−241.
330
(24) Wu, J.; Liao, W.; Udenigwe, C. C. Revisiting the mechanisms of ACE inhibitory
331
peptides from food proteins. Trends Food Sci. Technol. 2017, 69, 214−219.
332
(25) Fitzgerald, R. J.; Murray BAWalsh, D. J. Hypotensive peptides from milk
333
proteins. J. Nutr. 2004, 134, 980S−988S.
334
(26) Ko, S. C.; Kang, M. C.; Lee, J. K.; Byun, H. G.; Kim, S. K.; Lee, S. C.; Jeon, B.
335
T.; Park, P. J.; Jung, W. K.; Jeon, Y. J. Effect of angiotensin I-converting enzyme
336
(ACE) inhibitory peptide purified from enzymatic hydrolysates of Styela plicata. Eur. 15
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
337
Food Res. Technol. 2011, 233, 915−922.
338
(27) Girgih, A. T.; He, R.; Aluko, R. E. Kinetics and molecular docking studies of the
339
inhibitions of angiotensin converting enzyme and renin activities by hemp seed
340
(Cannabis sativa L.) peptides. J. Agric. Food Chem. 2014, 62, 4135−4144.
341
16
ACS Paragon Plus Environment
Page 16 of 27
Page 17 of 27
Journal of Agricultural and Food Chemistry
Figure Captions Fig. 1. Total ion current (TIC) chromatogram and ACEI activity of bovine casein hydrolysate (molecular weight no more than 3 kDa). Hydrolysis was digested by trypsin at 37 °C for 2 h, with 50 mg mL-1 of protein and 2500 U g-1 of the enzyme as the substrate. Fig. 2. The ACEI activity of the peptide EKVNELSK. The ACEI activity of the peptide was determined by the HPLC method. The IC50 value was determined by the regression equation: Y= (1.113E-2) X2+4.57673X+22.14678 (R2=0.98312). Fig. 3. Lineweaver-Burk plot of ACE inhibition by the peptide EKVNELSK. The ACE activities were measured in the absence or presence of the peptide EKVNELSK (■, control; ●, 4.228 mM; ▲, 8.456 mM). 1/[S] and 1/[V] represent the reciprocal substrate concentration and velocity, respectively. Fig. 4. The molecular docking simulation of the peptide EKVNELSK with ACE (PDB: 1O8A). (A), (B) and (C) represent the general overview, local overview, and 2D-diagram of docking pose of peptide EKVNELSK, respectively.
17
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 18 of 27
Table 1 Peptides Profile of Casein Hydrolysate Identified by CE-ESI-Q-TOF-MS/MS. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Protein αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN αs1-CN
αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN αs2-CN β-CN
m/z meas. 416.1951 455.7382 1070.1953 473.7646 1159.0664 692.8659 669.3396 1118.6130 880.4720 753.8928 460.7463 790.9140 624.9996 634.3531 684.3493 549.8074 490.2843 623.8211 509.7609 817.4429 437.7254 452.7708 344.8805 451.2648 624.3294 415.7288
z 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2 2 3 3 2 2
Mr. calc. 830.3770 909.4668 3206.5859 945.5131 2315.1296 1383.7227 1336.6735 2234.2283 1758.9377 1505.7773 919.4804 1579.8206 1870.9788 1266.6972 1366.6881 1097.6055 978.5538 1245.6353 1017.5090 1632.8835 873.4378 903.5290 1031.6240 1350.7732 1246.6517 829.4446
Scores 53.99 25.78 63.49 47.88 76.75 37.63 63.12 28.98 94.33 67.13 21.25 39.49 40.56 67.12 60.79 32.44 30.38 87.48 49.19 69.10 38.44 27.29 23.17 34.90 51.98 28.95
Start K K K K K R K K K R K K R K K K K K K K K K K K K
AA Sequence EDVPSER EGIHAQQK EGIHAQQKEPMIGVNQELAYFYPELFR EKVNELSK EPMIGVNQELAYFYPELFR FFVAPFPEVFGK HIQKEDVPSER HPIKHQGLPQEVLNENLLR HQGLPQEVLNENLLR LHSMKEGIHAQQK PFPEVFGK VPQLEIVPNSAEER YKVPQLEIVPNSAEER YLGYLEQLLR ALNEINQFYQK AMKPWIQPK FALPQYLK ITVDDKHYQK LTEEEKNR LTEEEKNRLNFLK NMAINPSK NRLNFLK NRLNFLKK RNAVPITPTLNR TKLTEEEKNR AVPYPQR
18
ACS Paragon Plus Environment
End Y E Q D Q E Y F F E L L L F T T A L K E K I E L D
Length 7 8 27 8 19 12 11 19 15 13 8 14 16 10 11 9 8 10 8 13 8 7 8 12 10 7
ACE activity
ACE inhibitor
ACE inhibitor ACE inhibitor ACE inhibitor
ACE inhibitor
ACE inhibitor
Page 19 of 27
Journal of Agricultural and Food Chemistry
27 28 29 30 31
β-CN β-CN β-CN β-CN κ-CN
507.2634 436.7798 437.2458 390.7523 478.2653
2 2 2 2 2
1012.5164 871.5491 872.4789 779.4905 954.5175
40.11 33.53 33.89 33.29 35.83
K R K K R
32
κ-CN
1052.5454
3
3153.6063
20.63
R
HKEMPFPK INKKIEK VKEAMAPK VLPVPQK FFSDKIAK SPAQILQWQVLSNTVPAKSCQAQPTTM AR
19
ACS Paragon Plus Environment
Y F H A Y
8 7 8 7 8
H
29
Journal of Agricultural and Food Chemistry
Page 20 of 27
Table 2 Toxicity, Solubility and Stability of the Potential ACEI Peptides. Number of
Solubility in
residues
water
EDVPSER
7
2
EGIHAQQK
3
No.
Peptide sequence
ToxinPed
Instability index
1
Good
Non-Toxin
unstable (118.63)
8
Good
Non-Toxin
stable (37.64)
EKVNELSK
8
Good
Non-Toxin
stable (-1.86)
4
PFPEVFGK
8
Good
Non-Toxin
unstable (68.01)
5
LTEEEKNR
8
Good
Non-Toxin
unstable (114.33)
6
NRLNFLK
7
Good
Non-Toxin
stable (-25.03)
7
NRLNFLKK
8
Good
Non-Toxin
stable (-20.65)
8
HKEMPFPK
8
Good
Non-Toxin
unstable (141.43)
9
INKKIEK
7
Good
Non-Toxin
unstable (93.04)
10
VKEAMAPK
8
Good
Non-Toxin
unstable (44.65)
11
VLPVPQK
7
Poor
Non-Toxin
unstable (118.63)
12
FFSDKIAK
8
Good
Non-Toxin
stable (-12.48)
20
ACS Paragon Plus Environment
Page 21 of 27
Journal of Agricultural and Food Chemistry
Table 3 The -CDOCKER_Energy Scores of the Potential ACEI Peptides Obtained from Molecular Docking. -CDOCKER_ENERGY
No.
Peptides
1
EGIHAQQK
177.172
2
EKVNELSK
185.540
3
NRLNFLK
154.963
4
NRLNFLKK
159.759
5
FFSDKIAK
169.805
(kcal mol-1)
21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 27
Table 4 Hydrogen Bonds, Electrostatic and Hydrophobic Interactions Observed in the Best Peptide Poses Based on the ACE-peptide Complex. Interaction Residues
Distance
Types
EKVNELSK:H132 - A:ASP121:OD2
2.28054
Hydrogen Bond;Electrostatic
EKVNELSK:H133 - A:GLU123:OE2
2.353
Hydrogen Bond;Electrostatic
EKVNELSK:H134 - A:GLU123:OE2
2.16624
Hydrogen Bond;Electrostatic
A:LYS118:NZ - EKVNELSK:O137
4.3831
Electrostatic
A:ARG522:NH1 - EKVNELSK:O15
5.48899
Electrostatic
A:ZN701:ZN - EKVNELSK:O15
2.30831
Electrostatic
EKVNELSK:N1 - A:GLU411:OE2
4.41662
Electrostatic
A:LYS118:HZ1 - EKVNELSK:O111
2.07162
Hydrogen Bond
A:LYS118:HZ3 - EKVNELSK:O114
2.12179
Hydrogen Bond
A:TYR360:HH - EKVNELSK:O64
2.77065
Hydrogen Bond
A:ARG522:HH12 - EKVNELSK:O17
3.02249
Hydrogen Bond
A:ARG522:HH12 - EKVNELSK:O39
2.97451
Hydrogen Bond
EKVNELSK:H66 - A:ARG402:O
2.16968
Hydrogen Bond
EKVNELSK:H67 - A:VAL399:O
3.08084
Hydrogen Bond
EKVNELSK:H86 - A:GLU123:OE1
2.89964
Hydrogen Bond
EKVNELSK:H116 - A:GLU123:OE2
2.05597
Hydrogen Bond
EKVNELSK:H32 - A:ASP358:O
2.658
Hydrogen Bond
EKVNELSK:H88 - A:GLU123:OE1
2.56598
Hydrogen Bond
EKVNELSK:H130 - A:SER219:O
2.79581
Hydrogen Bond
A:ALA89 - EKVNELSK:C98
4.43141
Hydrophobic
A:ALA125 - EKVNELSK:C98
4.26213
Hydrophobic
EKVNELSK:C46 - A:PRO519
4.97062
Hydrophobic
EKVNELSK:C94 - A:ARG124
4.93511
Hydrophobic
EKVNELSK:C98 - A:ILE88
4.68749
Hydrophobic
EKVNELSK:C98 - A:ARG124
4.24887
Hydrophobic
22
ACS Paragon Plus Environment
Page 23 of 27
Journal of Agricultural and Food Chemistry
Fig. 1.
23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Fig. 2.
24
ACS Paragon Plus Environment
Page 24 of 27
Page 25 of 27
Journal of Agricultural and Food Chemistry
Fig. 3.
25
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Fig. 4.
A)
B)
C)
26
ACS Paragon Plus Environment
Page 26 of 27
Page 27 of 27
Journal of Agricultural and Food Chemistry
Graphic for table of contents
27
ACS Paragon Plus Environment