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Article
An Exploration of the Calcium-Binding Mode of Egg
White Peptide, Asp-His-Thr-Lys-Glu, and in vitro Calcium
Absorption Studies of Peptide-Calcium Complex
Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, and Songyi Lin
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03705 • Publication Date (Web): 24 Oct 2017
Downloaded from http://pubs.acs.org on October 25, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical
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Page 1 of 33
Journal of Agricultural and Food Chemistry
1
An Exploration of the Calcium-binding Mode of Egg White Peptide,
2
Asp-His-Thr-Lys-Glu, and in vitro Calcium Absorption Studies of
3
Peptide-Calcium Complex
4
Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, Songyi Lin*
5
6
7
National Engineering Research Center of Seafood, School of Food Science and
8
Technology, Dalian Polytechnic University, Dalian 116034, P. R. China
9
10
11
*
12
Professor Songyi Lin
13
No. 1 Qinggongyuan, Ganjingzi District
14
National Engineering Research Center of Seafood, School of Food Science and
15
Technology, Dalian Polytechnic University, Dalian, P.R. China, 116034
16
Tel.: +86 18840821971
17
Fax: +86 0411 86318655
18
E-mail address: linsongyi730@163.com
Corresponding author:
19
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ABSTRACT:
21
The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates
22
and calcium ions was elucidated upon its structural and thermodynamics
23
characteristics. The present study demonstrated that the DHTKE peptide could
24
spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding
25
site corresponded to the carboxyl oxygen, amino nitrogen and imidazole nitrogen
26
atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex
27
on improving the calcium absorption was investigated in vitro using Caco-2 cells.
28
Results showed that the DHTKE-calcium complex could facilitate the calcium influx
29
into the cytosol and further improve calcium absorption across Caco-2 cell
30
monolayers by more than seven times when compared to calcium-free control. This
31
study facilitates the understanding about the binding mechanism between peptides
32
and calcium ions as well as suggests a potential application of egg white peptides as
33
nutraceuticals to improve calcium absorption.
34
35
KEYWORDS: egg white peptide, calcium binding, calcium absorption, Caco-2 cells
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
37
Calcium is known as an essential mineral for human body to maintain bone health.1,2
38
Bone loss, which results in metabolic bone diseases such as rickets and osteoporosis,3
39
has been proved to be mainly related to insufficient calcium absorption. It has been
40
reported that the amount of calcium absorbed by the body depends on the amount of
41
soluble calcium in the duodenum and proximal jejunum.4 The solubility of calcium
42
can be diminished due to the precipitation of calcium ions with antagonists such as
43
oxalate, phytates and cellulose in the intestines.5,6
44
Recently, there has been a great deal of interest in the enhancing effects of food
45
substances (particularly peptides) on calcium solubility and subsequent calcium
46
absorption. Binding calcium to peptides for preventing from calcium precipitation can
47
effectively increase the absorption of calcium in the body.7,8 Currently,
48
calcium-binding peptides have been found from various food sources, including hen
49
egg yolk,9 cow milk casein,10 whey,12,14 soy,15 wheat germ,16 tilapia fish,17,18 Alaska
50
pollock19,20 and shrimp processing byproducts.21 Casein phosphopeptides (CPPs) and
51
phosvitin phosphopeptides can promote the absorption of calcium by chelating
52
calcium with the phosphoserine residues. However, several peptides lacking
53
phosphoserine residues also enhance intestinal calcium uptake, by binding calcium to
54
the Asp and Glu residues.
55
Asp, Glu, Ser, His, and Lys are the most frequently reported calcium-binding
56
ligands. CPPs are originally found to have an ability to bind calcium ions and increase
57
calcium absorption, which is exactly attributed to the role of the acidic sequence
58
“Ser(P)-Ser(P)-Ser(P)-Glu-Glu”.22, 23, 24 Afterwards, Chen et al.18 reported that Asp,
59
Gly and Glu were the most abundant amino acids in the calcium-binding peptide from
60
tilapia scale protein. The calcium-binding peptides derived from soybean protein
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61
hydrolysates,15 bovine serum protein hydrolysates,25 and porcine blood plasma protein
62
hydrolysates,26 also possessed Asp and Glu residues. Furthermore, peptides containing
63
histidine residues have been reported to possess high affinity to calcium ions. Huang
64
et al.21 isolated a histidine-containing tri-peptide, Thr-Cys-His, from shrimp
65
processing byproducts to possess high calcium-binding capability, which might be
66
responsible for the presence of His residue. As a matter of fact, the calcium-chelating
67
activity of these amino acids could be ascribed to their specific groups, including
68
carboxyl groups of Asp and Glu, the δ-N in the imidazole ring of His, and the ε-amino
69
nitrogen of Lys. Most current researches have focused on the isolation and
70
identification of calcium-binding peptides, and the exploration of specific amino acids
71
and groups that contribute to calcium binding. More detailed studies are limited that
72
characterize the stoichiometry, binding affinity and thermodynamics of calcium
73
binding to purified peptides, or their biological properties through in vitro and in vivo
74
studies.
75
Our previous study isolated an egg white pentapeptide, Asp-His-Thr-Lys-Glu
76
(DHTKE),33 which possesses the amino acids related to calcium binding. This study
77
was designed to elucidate the binding mode between the DHTKE peptide and calcium
78
ions upon its structural and thermodynamics characteristics, and investigate the effect
79
of the DHTKE peptide on calcium absorption by human intestinal epithelial cells. The
80
possible calcium-binding sites were investigated using analytical techniques such as
81
ultraviolet absorption spectroscopy, Fourier transform infrared spectroscopy, and 1H
82
nuclear magnetic resonance (NMR) spectroscopy. Isothermal titration calorimetry was
83
applied to investigate the association constant and stoichiometry of the
84
peptide-calcium complex. A Caco-2 cell model was applied to assess calcium
85
absorption. This study could be conducive to better understand the binding
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mechanism between peptides and calcium ions, and suggests a potential application of
87
egg white peptides as nutraceuticals to improve calcium absorption.
88
MATERIALS AND METHODS
89
Materials. An egg white pentapeptide, Asp-His-Thr-Lys-Glu (DHTKE), was
90
synthesized in 98.85% purity by China Peptides Co., Ltd. (Shanghai, China). Eagle’s
91
minimal essential medium, penicillin-streptomycin-neomycin (PSN) antibiotic
92
mixture and trypsin-EDTA were obtained from Gibco (Burlington, Ontario). Fetal
93
bovine serum (FBS) was supplied by PAN-Biotech (Bavaria, Germany). Fluo-3 AM
94
was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin (from
95
porcine pancreas) and pepsin (from porcine gastric mucosa) were obtained from Bio
96
Basic Inc. (Toronto, Canada) and Sigma-Aldrich (St. Louis, MO), respectively.
97
Preparation of the DHTKE-calcium Complex. For fabrication of the
98
DHTKE-calcium complex, 3.6 mM of DHTKE peptide was mixed with 21.6 mM
99
CaCl2 in 20 mL Milli-Q water. The binding reaction was performed at pH 8.0 and
100
50 °C for 1 h under continuous stirring. Thereafter, absolute ethanol was added to the
101
mixture to a final concentration of 90%, resting for 60 min to precipitate the
102
complexes. After centrifugation at 12,000×g for 5 min, the precipitates was collected,
103
freeze-dried and labeled as DHTKE-calcium.
104
Ultraviolet-visible Absorption Spectroscopy. Ultraviolet-visible spectra were
105
measured to testify the occurrence of the chelation reaction between peptide and
106
calcium. 0.1 mg/mL of the DHTKE peptide solution was prepared and mixed with 0.2,
107
0.4 or 0.8 mM CaCl2 to obtain the DHTKE-calcium complex. The ultraviolet-visible
108
spectra of the DHTKE peptide and its calcium complex were measured within a
109
wavelength range between 190 and 800 nm by using a UV-Vis spectrophotometer
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(Perkin Elmer, Salem, MA).
111
Zeta Potential Determination. The zeta potential of the DHTKE peptide and
112
DHTKE-calcium complex was measured using a Zetasizer Nano ZS90 particle size
113
analyzer (Malvern Instruments Ltd., Malvern, UK), according to the method from the
114
previous studies of Sun et al.11 1 mg/mL of the DHTKE peptide or DHTKE-calcium
115
solution was added into an U-shaped cell. Subsequently, the cell temperature was
116
maintained at 25 °C for 5 s, and all measurements was performed at 25 °C and
117
repeated 12 times.
118
Fourier Transform Infrared Spectroscopy. Freeze-dried DHTKE peptide or
119
DHTKE-calcium powder (2 mg) was grinded evenly with 100 mg KBr under infrared
120
light. The mixed powder was then compressed into a thin disc. The spectra between
121
4000 and 400 cm-1 were performed through a Fourier transform infrared (FTIR)
122
spectrometer (Perkin Elmer, Salem, MA) at a resolution of 4 cm-1. A total of 32 scans
123
were recorded per sample in the FTIR spectra, and the analysis of FTIR spectra was
124
performed using an OMNIC 8.2 software (Thermo Fisher Scientific Inc.)
125
1
H Nuclear Magnetic Resonance (NMR) Spectroscopy. The 1H NMR spectra of
126
the DHTKE peptide and DHTKE-calcium complex were determined by a Bruker
127
AVANCE III 400 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany), using
128
a modification of the methodology of Lin et al.27 with some modifications. 5 mg
129
samples were dissolved in 600 µL of dimethyl-d6 sulfoxide solution containing 0.03%
130
(v/v) tetramethylsilane (TMS), transferred into 5 mm NMR tubes and subjected to
131
NMR analysis. A spectral width of 8012.8 Hz was used with a relaxation delay of 1.5
132
s and a total of 16 scans were recorded.
133
Isothermal Titration Calorimetry (ITC). ITC measurements were conducted at
134
25 ± 0.2 °C by an Affinity ITC calorimeter (TA Instruments Ltd., New Castle, DE).
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The DHTKE peptide and CaCl2 were dispersed in Tris/HCl buffer (pH 8.0), passing
136
through a 0.22-µm Millipore membrane. All samples were degassed before loading
137
into the sample cell and injection syringe. Reactions were conducted by titrating 40
138
consecutive CaCl2 solution (50 mM) into 5 mM of DHTKE peptide solution.
139
Controlled trials were conducted by injecting CaCl2 solution into Tris/HCl buffer.
140
Raw ITC data were fitted to an independent-site binding model using the Nano
141
Analyze software by which stoichiometry (n), binding constant (K), and enthalpy (∆H)
142
and entropy change (∆S) were calculated.
143
Calcium Absorption Studies. Cell culture. Caco-2 cells were supplied by Cell
144
Resource Center in Shanghai Institutes for Biological Sciences, the Chinese Academy
145
of Sciences (Shanghai, China). The cells were cultured in Eagle’s minimal essential
146
medium, supplemented with 20% FBS and PSN antibiotic mixture, and incubated at
147
37 °C in a humidified 5% CO2 incubator. The differentiation of Caco-2 cells was
148
achieved according to the well-established procedure29 consisting of successive
149
sub-cultivations.
150
Calcium imaging. Calcium influx into Caco-2 cells, which is expressed with
151
calcium imaging, was measured as previously described by Perego et al.8 with some
152
modifications. Caco-2 cells were seeded on 35-mm confocal dishes, and loaded with
153
2.5 µM Fluo-3 AM, in Krebs-Ringer HEPES (KRH) solution (140.0 mM NaCl, 5.0
154
mM KCl, 2.0 mM CaCl2, 0.55 mM MgCl2, 6.0 mM glucose and 10.0 mM HEPES,
155
pH 7.4). After incubation at 37 ºC in darkness for 30 min, cells were washed in KRH
156
solution and allowed to a further 30 min incubation for de-esterification of the
157
fluorescent probe. Fluorescence analysis was performed by a Leica SP8 confocal
158
laser-scanning microcopy (Leica Microsystems, Wetzlar, Germany) using a 40× (oil)
159
magnification. The cells, maintained in 2 mL of KRH solution, were excited at a
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wavelength of 488 nm. The emission was recorded at 510 nm. Baseline calcium
161
signal was monitored, and then 2 mmol/L of DHTKE-calcium complex or CaCl2 (100
162
µL) was added to the 35-mm confocal dishes, followed by the measurement of
163
cytoplasmic calcium signal. Finally, 100 µL of ATP (100 µmol/L) was added prior to
164
the end of the assay.
165
Establishment of Caco-2 cell monolayer model. Passage numbers between 30 and
166
50 were applied in the establishment of Caco-2 cell monolayer model for calcium
167
transport studies. Cells were seeded at a density of 1.5 × 105 cells/mL on 12-well
168
transwell culture plates (Corning Inc., NY) with a polycarbonate membrane (12 mm
169
diameter inserts, 0.4 µm pore size). The culture medium in the apical and basolateral
170
sides was replaced every other day during the first week and then replaced daily until
171
applied for calcium transport tests. Transepithelial electrical resistance (TEER) was
172
measured every other day using a Millicell-ERS system (Millipore, Billerica, MA,
173
USA) to assess the integrity of the cell monolayers.
174
Calcium transport studies. The monolayers with TEER values above 400 Ω × cm2
175
were applied for this experiment.30 The cell monolayers were gently rinsed twice with
176
Hank’s balanced salt solution (HBSS, without calcium and magnesium) and then
177
moved to a new plate containing 1.5 mL of HBSS buffer. Afterwards, different
178
concentrations of DHTKE-calcium (0-6 mM) or 2 mM CaCl2 in 0.5 mL HBSS buffer
179
(pH 7.4) were added to the apical side, and incubated at 37 °C for 2 h. 1.0 mL of
180
sample was extracted from the basolateral side at 30, 60, 90, and 120 min, and then
181
1.0 mL of HBSS buffer was added to the basolateral side in order to keep the volume
182
constant.
183
spectrophotometer (Hitachi Co., Tokyo, Japan). Calcium absorption was calculated
184
according to Cao et al.31 by using the following equation:
Calcium
contents
were
determined
by
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atomic
absorption
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Journal of Agricultural and Food Chemistry
n −1
185
Bn = 1.5 × An + 1.0 × ∑ Ak
(1)
k =1
186
where B n represents the transported calcium content in 1.5 mL of HBSS buffer in the
187
basolateral side of each well at 30, 60, 90 and 120 min; 1.5mL is a constant,
188
representing 1.5 mL of HBSS buffer in the basolateral side of each well; An represents
189
the calcium concentration of the HBSS buffer in the basolateral side of each well at
190
different time points; 1.0 is also a constant and represents the 1.0 mL of HBSS buffer
191
collected from the basolateral side of each well in order to measure the calcium
192
content; n is an independent variable. For the present study, it could be the number of
193
1, 2, 3 or 4, representing time points 30, 60, 90, and120 min, respectively.
194
Statistical Analysis. All experiments were conducted in triplicate, and data
195
analysis was made by employing SPSS 18.0 software (SPSS Inc., Chicago, IL). A
196
one-way ANOVA was used to evaluate the significant differences of data, with the
197
confidence level set at P<0.05.
198
RESULTS AND DISCUSSION
199
Formation of DHTKE-calcium Complex. Ultraviolet-visible spectra analysis. The
200
UV-vis spectra of the DHTKE peptide and DHTKE-calcium complex are depicted in
201
Figure 1. The maximum absorption band was observed at about 197 nm, which was
202
regarded as the characteristics of the amide bond in the peptide.32 However, the
203
absorption intensity of the DHTKE-calcium complex is slightly lower than that of the
204
DHTKE peptide in the near ultraviolet region. As the calcium ion concentration
205
increased, the absorbance of the maximal absorption band decreased from 2.74 to
206
2.66, indicating a hypochromic effect in the UV-vis absorption spectrum. The
207
intensity changes of the band demonstrated that the chromophore groups (-C=O,
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-COOH) and auxochrome groups (-OH, -NH2) produced polarizing changes when
209
calcium ions bound to the organic ligands.13
[Figure 1]
210
211
Zeta potential analysis. Zeta potential is a physicochemical indicator that can
212
reflect the surface charge state of particles. The partial ionization of various amino
213
acid residues produces the surface charge of protein particles.34 Figure 2 exhibits the
214
zeta potential values of the DHTKE peptide and DHTKE-calcium complex. The
215
absolute value of the zeta potential of the DHTKE-calcium complex decreased
216
significantly (P < 0.05) when compared to those of the DHTKE peptide. Moreover,
217
the zeta potential ranged from positive charge (5.25 mV) to negative charge (-2.64
218
mV) when the calcium ions were added to the DHTKE peptide (Fig. 2B). These data
219
indicated that the calcium ions could be chelated with positively charged moieties,
220
such as amino groups.
221
[Figure 2]
222
Binding Sites of Cacium on the DHTKE Peptide. FTIR spectroscopy. The FTIR
223
spectra of the DHTKE peptide and DHTKE-calcium complex are depicted in Figure 3.
224
The absorption band at 3,400-3,200 cm−1, corresponding to the N-H and O-H bonds,
225
is assigned to water of hydration. Because the nitrogen atoms could form coordination
226
bonds with calcium ions by offering their electron pairs,35 the N-Ca bond replaced the
227
N-OH hydrogen bonds; this was attributed to the chemical shift from 3,277 cm-1 to
228
3,412 cm-1 following calcium fortification of the DHTKE peptide. Bands due to the
229
vibration of amide I (C = O) and amide II (N-H and C-N) were observed at 1,672 and
230
1,533 cm−1 in the spectra of the DHTKE peptide, which were shifted to 1,651 and
231
1,561 cm−1 after binding with calcium ion, respectively. The results implied the
232
contribution of the amide carbonyl (C = O) and the N-H of the amide bond in the
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233
binding between the DHTKE peptide and calcium ions. Moreover, the peak (1,431
234
cm−1) for the −COO− group was shifted to a higher frequency (1,453 cm−1) in the
235
FTIR spectrum of DHTKE-calcium complex, which indicated that the −COO− group
236
in the DHTKE peptide also played a crucial role in the chelation of calcium ions.
237
These findings were analogous to other metal-binding peptides,11,16,28 and suggested
238
that the calcium-binding sites referred principally to the carboxyl and amino groups
239
and to a smaller extent to the peptide bonds.
[Figure 3]
240
241
1
H-NMR spectroscopy. The 1H NMR spectra can also analyze the interaction
242
between calcium ions and ligand groups in the DHTKE peptide via reflecting the
243
distribution of the electron cloud around a hydrogen nucleus through the changes of
244
chemical shift.32 As depicted in Figure 4, the broad peak at 5.09 ppm, arose from the
245
N-H bonds, shifted to a lower magnetic field of 5.33 ppm after the DHTKE peptide
246
binds to calcium ions. Another broad peak at 12.78 ppm corresponded to the -COO−
247
carboxylate groups. Upon the binding of calcium ions, the peak of -COO− groups
248
shifted to a higher magnetic field of 12.22 ppm. Simultaneously, changes of the
249
carboxylate and amino groups result in chemical shifts of the hydrogen atoms in the
250
–CH2 and –CH3 groups, corresponding to the peaks at 1-3 ppm in the high magnetic
251
field area. Furthermore, the signal at 14.19 ppm is attributed to the hydrogen atom of
252
the imidazole nitrogen group in the DHTKE peptide, which is not present in the 1H
253
NMR spectra of the DHTKE-calcium complex. These changes were caused by the
254
chelation of the DHTKE peptide to calcium ions affecting the electron density around
255
the protons of the peptide, and suggested that calcium ions might bind to carboxyl
256
oxygen, amino nitrogen and imidazole nitrogen atoms of the DHTKE peptide.
257
The above-mentioned amino acid groups have been demonstrated to be calcium
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36
258
binding sites and promote calcium-binding capacity. Bao et al.
259
calcium-chelating capacity of soy protein hydrolysates was linearly correlative to the
260
content of carboxyl group, and that the sites for calcium binding are most likely to be
261
carboxyl groups of Glu and Asp. Similarly, Fourier transform infrared spectra
262
demonstrated that carboxyl oxygen and amino nitrogen atoms of Asp and Glu on
263
wheat germ protein hydrolysates were involved in the peptide-calcium chelation.16
264
The role of imidazolyl group on calcium-binding capacity of peptides was also
265
reported.
266
calcium binding by peptides derived from tilapia protein hydrolysates and shrimp
267
processing byproducts. Therefore, in the present study, the calcium-binding site of the
268
DHTKE peptide might correspond to the carboxyl oxygen and amino nitrogen atoms
269
of Glu and Asp together with imidazole group of His.
270
17,21
reported that the
Results stated that the imidazolyl group of His was conducive to
[Figure 4]
271
Cacium Binding Studies by Isothermal Titration Calorimetry (ITC). ITC has
272
been widely applied to study the thermodynamics of biomolecular interactions.37
273
Figure 5 exhibits a representative calorimetric titrations of CaCl2 into the DHTKE
274
peptide. The integrated heat data obtained in an ITC titration were fit to an
275
independent-site binding model. Results revealed that the titration of CaCl2 into the
276
peptide generated exothermic binding isotherms and the interaction gave rise to
277
negative ∆H and ∆G values, which means that the binding reaction occurs
278
spontaneously. Furthermore, a positive ∆S values (56.20 J/mol·K) might be
279
associated with conformational changes of peptide side-chains or the solvation
280
effect.38,39 One DHTKE molecule could bind one calcium ion (n = 0.906 ± 0.058) and
281
the binding constant was equal to 1.000×103 M-1, which demonstrated the peptide
282
possessed one functional calcium-binding site.
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[Figure 5]
283
284
Calcium Absorption Studies. Effect of DHTKE-calcium complex on calcium
285
influx into Caco-2 cells. Caco-2 cells, possessing enterocyte-like biochemical and
286
morphological characteristics, have been previously used as an effective model of
287
human intestinal epithelial cells in vitro.5,8,40,41 In the present study, the possible
288
influence of the DHTKE-calcium complex on calcium uptake by Caco-2 cells was
289
firstly assessed by observing the calcium influx into the cytosol after exposure to the
290
DHTKE-calcium complex, which is expressed with the fluorescence intensity.5 A
291
prerequisite for this experiment is that the cells need to keep full viability. In the
292
present study, cell viability was evaluated by stimulation with ATP prior to the end of
293
the assay and observing the increase of cytoplasmic calcium ions, which is
294
well-known
295
(1,4,5)-triphosphate.42,48 It can be seen that the treatment of ATP was followed by the
296
expected calcium ions rise (Figure 6A), indicating that cells were fairly viable and
297
normally responsive to effective stimuli. Moreover, Figure 6(A) shows changes in
298
intracellular calcium ions in Caco-2 cells which were treated with the
299
DHTKE-calcium complex or CaCl2. Results showed that the addition of both the
300
DHTKE-calcium complex and CaCl2 lead to an increase in intracellular calcium ions
301
when compared with untreated cells. Moreover, the DHTKE-calcium complex
302
appeared to be more effective than than CaCl2. These findings demonstrated calcium
303
ions bound to the DHTKE peptide were comparable, or even beneficial, to free
304
calcium ions. Similar results were observed in published studies of casein
305
phosphopeptide-calcium complexes8 and soybean protein hydrolysate-calcium
306
complexes,41 which may interact with the cell membrane to transport calcium ions to
307
the cytoplasm.
to
be
triggered
by
ATP-induced
production
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Calcium transport studies in Caco-2 cell monolayer model. Caco-2 monolayer
309
model has been successfully applied to simulative absorption studies involving
310
proteins,46 peptides,47 amino acids,45 and minerals.43,44 To well establish the Caco-2
311
cell monolayers with enterocyte morphology to mimic in vitro calcium absorption,
312
TEER values between apical and basolateral side of the monolayers were measured,
313
as illustrated in Figure 6B. The TEER values increased as the incubation time was
314
prolonged, and exceeded 400 Ω × cm2 at day 12. This indicated the formation of tight
315
junctions of the Caco-2 cell monolayers and, thus, the model could be used for the
316
calcium absorption experiment.
317
After the formation of Caco-2 cell monolayers, different concentrations of
318
DHTKE-calcium (0-6 mM) were added to the apical side and calcium contents on the
319
basolateral compartment were determined. 2 mM CaCl2 was used as control. As
320
shown in Figure 6C, all investigated DHTKE-calcium and CaCl2 showed greater
321
calcium transport activity across Caco-2 cell monolayers when compared to
322
calcium-free control (P < 0.05). Whether the DHTKE-calcium complex or CaCl2, the
323
contents of transported calcium markedly increased (P < 0.05) as the incubation time
324
was prolonged. At 120 min of incubation, 2 mM of DHTKE-calcium complex
325
showed significant increases (P < 0.05) in calcium transport when compared to 2 mM
326
CaCl2. Moreover, calcium absorption exhibited a dose-dependent increase with regard
327
to the DHTKE-calcium complex. In particular, 6 mM of DHTKE-calcium complex
328
showed the highest calcium transport capacity (P < 0.05), with increases in calcium
329
transport by approximately 7.7-, 8.0-, 8.9-, and 7.4-fold when compared to
330
calcium-free control values at 30, 60, 90, and 120 min, respectively. These results
331
demonstrated that the DHTKE-calcium complex could improve the absorption of
332
intestinal calcium. Interestingly, the casein phosphopeptides and desalted duck egg
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white peptides, identified by Cao et al.31 and Hou et al.40, respectively, can improve
334
calcium uptake, and more remarkable, contain Asp, Glu or His in their structure,
335
similar to the DHTKE peptide investigated in our study.
336
Calcium transported across the intestinal epithelium by peptides might involve one
337
or more of three different routes: transcellular pathway, paracellular pathway, and
338
transcytosis.6 For instance, several different theories have been proposed on how
339
casein phosphopeptides (CPPs) influenced calcium absorption. Ferraretto et al.48
340
found that CPPs improved calcium absorption directly through interaction with the
341
plasma membrane but did not affect calcium ion channels. On the contrary, Perego et
342
al. 24 demonstrated that CPPs increased the absorption of calcium via interacting with
343
a calcium channel in the plasma membrane. Additionally, the mechanism of calcium
344
absorption by desalted duck egg white peptides (DPs) was illuminated by Hou et al.
345
(2015), which indicated that the paracellular pathway might play a minor role in
346
calcium transport by DPs. On the contrary, DPs promoted calcium absorption
347
primarily via interaction with the plasma membrane to open a special calcium channel.
348
In our study, the DHTKE-calcium complex showed significant calcium transport
349
activity. However, the possible mechanism of action by which this occurs will require
350
further investigation.
351
[Figure 6]
352
In summary, a specific peptide (DHTKE) from egg white hydrolysates was
353
synthesized, and examined for its calcium-binding mode as well as stimulating effect
354
on calcium absorption in Caco-2 cells. Results indicated that the DHTKE peptide
355
could spontaneously bind calcium with a 1:1 stoichiometry, and that the
356
calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen and
357
imidazole nitrogen atoms of the DHTKE peptide. Moreover, the DHTKE-calcium
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complex could facilitate the calcium influx into the cytosol and further improve
359
calcium absorption across Caco-2 cell monolayers by more than seven times when
360
compared to calcium-free control values. This study facilitates the understanding
361
about the binding mechanism between peptides and calcium ions as well as suggests a
362
potential application of egg white peptides as nutraceuticals to improve calcium
363
absorption. Further studies are required to clarify the possible mechanism underlying
364
calcium absorption by the DHTKE-calcium complex.
365
366
ABBREVIATIONS
367
Casein phosphopeptides (CPPs)
368
Ultraviolet-visible (UV-vis)
369
Fourier transform infrared (FTIR)
370
1
371
Isothermal titration calorimetry (ITC)
372
Fetal bovine serum (FBS)
373
Penicillin-streptomycin-neomycin (PSN)
374
Krebs-Ringer HEPES (KRH)
375
Transepithelial electrical resistance (TEER)
376
Hank’s balanced salt solution (HBSS)
377
AUTHOR INFORMATION
378
Corresponding Author
379
*Tel.:
380
linsongyi730@163.com
H nuclear magnetic resonance (NMR)
+86
18840821971;
fax:
+86
0411
86318655;
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E-mail
address:
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381
Notes
382
The authors declare no competing financial interest.
383
ACKNOWLEDGMENTS
384
This work was financially supported by the National Key Research and Development
385
Program of China (2017YFD0400504).
386
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Figure captions
Figure 1
Ultraviolet-visible spectra of the DHTKE peptide with different CaCl2
concentrations within a wavelength range between 190 and 800 nm.
Figure 2
Zeta potential profile of the DHTKE peptide and DHTKE-calcium
complex. The zeta potential values of the DHTKE peptide and
DHTKE-calcium complex are showed in illustration.
Figure 3
Fourier transform infrared spectra of the DHTKE peptide and
DHTKE-calcium complex within a wavenumber region between 4000
and 400 cm-1.
Figure 4
1
H NMR spectrum of the DHTKE peptide and DHTKE-calcium
complex. The DHTKE peptide or DHTKE-calcium complex were
dissolved in dimethyl-d6 sulfoxide solution containing 0.03% (v/v)
tetramethylsilane (TMS), subjected to NMR analysis. The blue line
means
the
DHTKE
peptide,
and
the
red
one
means
the
DHTKE-calcium complex.
Figure 5
The thermodynamics of binding interactions between the DHTKE
peptide and calcium ions as determined by ITC. The upper panel
exhibits a representative calorimetric titration curve. CaCl2 (50 mM)
was titrated into 5 mM of DHTKE peptide solution at 25 °C. The lower
panel shows the integrated areas corresponding to each titration, plotted
as a function of Ca2+/peptide molar ratio. The solid line represents the
best curve fit obtained by using an independent binding site model.
Figure 6
Calcium absorption studies. (A) Effect of the DHTKE-calcium
complex on calcium influx into Caco-2 cells. Caco-2 cells were seeded
on 35-mm confocal dishes, and loaded with 2.5 µM Fluo-3 AM. After
incubation at 37 ºC in darkness for 30 min, cells were performed with
fluorescence analysis by a Leica SP8 confocal laser-scanning
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microcopy using a 40× (oil) magnification. Baseline calcium signal was
monitored prior to the addition of the DHTKE-calcium complex or
CaCl2. Finally, 100 µmol/L of ATP was added prior to the end of the
assay. (B) Establishment of Caco-2 cell monolayer model. Cells were
seeded on 12-well transwell culture plates with a polycarbonate
membrane. Transepithelial electrical resistance (TEER) was determined
every other day by a Millicell-ERS system to assess the integrity of the
Caco-2 cell monolayers. (C) Calcium transport studies. The monolayers
were moved to a new plate containing 1.5 mL of HBSS buffer. 2 mM
CaCl2 or different concentrations of DHTKE-calcium (0-6 mM) in 0.5
mL HBSS buffer (pH 7.4) were added to the apical side, and incubated
at 37 °C for 2 h. 1.0 mL of sample was extracted from the basolateral
side at 30, 60, 90, and 120 min, and calcium contents were determined
by an atomic absorption spectrophotometer. Different letters indicate
significant difference among the groups at the given time point (P <
0.05).
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538
Figure 1
539
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Abstract graphic
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