Bioactive Peptides Isolated from Casein Phosphopeptides Enhance

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Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers Yong Cao, Jian-yin Miao, Guo Liu, Zhen Luo, Zumeng Xia, Fei Liu, Mingfei Yao, Xiaoqiong Cao, Shengwei Sun, Yanyin Lin, Yaqi Lan, and Hang Xiao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05711 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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Journal of Agricultural and Food Chemistry

TITLE RUNNING HEADER Bioactive Peptides Enhancing Calcium and Magnesium Transport

Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers Yong Cao†*, Jianyin Miao†,‡, Guo Liu†, Zhen Luo§, Zumeng Xia§, Fei Liu¶, Mingfei Yao ‡, Xiaoqiong Cao ‡, Shengwei Sun†, Yanyin Lin†, Yaqi Lan#, †, Hang Xiao‡*

†College of Food Science, South China Agricultural University, Guangzhou 510642, People’s Republic of China ‡Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States §Infinitus (China) Company Ltd ¶Guangzhou Greencream Biotech Co., Ltd # Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA

Corresponding Authors: *Tel.: +86 020 85286234. Fax: +86 020 85286234. Email:[email protected] (Y. Cao) *Tel.: +1 413 545 2281; fax: +1 413 545 1262. Email: [email protected] (H. Xiao)

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ACS Paragon Plus Environment


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ABSTRACT：The ability of casein phosphopeptides (CPPs) to bind and transport

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minerals has been previously studied. However, the single bioactive peptides

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responsible for the effects of CPPs have not been identified. This study was to purify

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calcium-binding peptides from CPPs and to determine their effects on calcium and

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magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1

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to P5 were isolated and the amino acid sequences were determined using LC-MS/MS.

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Compared with the CPP-free control, all five monomeric peptides exhibited

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significant enhancing effects on the uptake of calcium and magnesium (P < 0.05).

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Interestingly, when calcium and magnesium were presented simultaneously with P5,

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magnesium was uptaken with priority over calcium in the Caco-2 cell monolayers.

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For example, at 180 min, the amount of transferred magnesium and calcium was 78.4

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±0.95 μg/well and 2.56 ±0.64 μg/well, respectively, showing a more than 30-fold

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difference in the amount of transport caused by P5. These results provide novel

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insight into the mineral transport activity of phosphopeptides obtained from casein.

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KEYWORDS: casein phosphopeptides, identification, calcium transport,

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magnesium transport, Caco-2 cell monolayers.

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INTRODUCTION Calcium and magnesium are crucial elements to maintaining human health,1

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particularly in aging individuals. Given the current and future size of global elderly

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populations, research on calcium and magnesium supplements has been an area with

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increasing interest.2, 3 Calcium accounts for about 2 % of adult human body weight. 4,

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5

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and osteoporosis. 6 Magnesium is also a necessary component for proper bone health

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and plays an important role in the physiological functioning of the brain, heart, and

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skeletal muscles. 3 However, approximately 60% of all Americans receive less than

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the recommended daily amount of magnesium. 3

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The effects of calcium deficiency on health include increased risks of bone fracture

Currently, calcium, magnesium, and other elemental mineral supplements are

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mainly composed of inorganic ionic forms. 7 However, numerous studies have

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focused on using organic supplements due to their increased bioavailability. 8-10 To

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this end, many organic supplements improve the absorptive ability of mineral

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elements and include oligophosphopeptide, 11 chicken eggshell matrix proteins, 12

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tilapia protein hydrolysate, 13 whey protein hydrolysates, 14 hen egg yolk phosvitin 15

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and desalted duck egg white peptides. 16

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Casein phosphopeptides (CPPs) are derived from trypsin-mediated casein cleavage

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and are considered mineral carriers with potential roles in improving elemental

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mineral absorption. 17 Several factors affect the activity and mechanisms of mineral

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uptake of CPPs. These include what phosphate residues are present on serine, 15 amino

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acid composition 18, 19 and molecular structures. 16 The effects of CPP-mediated

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calcium absorption have been in-depth studied by previous researchers and obtained

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some significant results on the activity and mechanisms. 20, 21 However, the effects of

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CPPs on regulating magnesium absorption are few reported. At the same time, though 3



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the peptide mixture in CPPs has the effects of mineral transport, the activity and

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mechanisms of single peptide monomer may be different. It is significant to study the

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pure mineral element transport active peptide that has been isolated from the CPPs.

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With this in mind, the work presented here sought to purify and identify specific

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CPP-derived peptides that exhibited excellent calcium transport capacity. Additionally,

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magnesium transport capacity and the difference in transport activity between calcium

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and magnesium was also investigated. Thus, this study allowed for the further

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exploration of the exact activity and mechanism of action of CPP-derived calcium and

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magnesium transport peptides.

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MATERIALS AND METHODS

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Materials. A commercial CPP mixture was provided by Guangzhou Greencream

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Biotech Co., Ltd (20% CPPs, Guangzhou, China) was used.

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Chemicals. Cell culture media, fetal bovine serum (FBS), antibiotic, Hank’s balanced

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salt solution (HBSS, without calcium and magnesium), and nonessential amino acids

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and all other chemicals were purchased from Sigma (St. Louis, MO, USA).

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Calcium-binding assay. The final concentration of 1.0 mg/mL of lyophilized sample

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dissolved in deionized water was mixed with 5 mM CaCl2 in 0.2 M sodium phosphate

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buffer (pH 8.0). The solution was stirred at 37 °C for 2 h and the pH was maintained

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at 8.0. The reaction mixture was centrifuged at 10,000×g at room temperature for 10

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min. The calcium content of the supernatant was determined using inductively

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coupled plasma mass spectrometry (ICP-MS) (NexION 300X ICP, MA, USA). 22

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Purification of active peptides. CPPs were first isolated by preparative

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high-performance liquid chromatography (LC-8, Shimadzu, Japan) with a 4


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reversed-phase (RP) column (20 mm × 450 mm) of C18 (10 μm, 300 Å, Macherey

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Nagel, France). Solvent A was 0.1% trifluoroacetic acid in double-distilled water, and

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solvent B was 100% acetonitrile. A linear gradient of solvent B at a flow rate of 10

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mL/min for 55 min was applied as follows: 10%–20% of solvent B for 10 min and

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20%-26% of solvent B for 45 min. Fractions were collected, concentrated, and tested

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using a calcium-binding assay. The active fractions were then further purified using

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HPLC in order to search for monomeric peptides that had an increased calcium

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transport ability. The active fraction solution collected in the first step was filtered

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through a 0.45-μm membrane filter and separated using a Diamosil C18 column (5

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μm, 4.6 mm × 250 mm) (Dikma Technologies, Beijing, China). The mobile phases

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were solvent A (double-distilled water with 0.1% trifluoroacetic acid) and solvent B

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(acetonitrile with 0.1% trifluoroacetic acid). Elution was performed using a gradient

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of 5-18% of solvent B (0-19.5 min) then 18-30% of solvent B (19.5-55 min)

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at a flow rate of 1 mL/min. The main peaks were collected, concentrated, and

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lyophilized. All elutions were monitored at 215 nm. Calcium transport activity of the

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eluted peaks was tested using the Caco-2 cell model.

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Transport studies in Caco-2 cell monolayer. Cell culture. Caco-2 cell lines (passage

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50−60) were the model for the intestinal epithelium that was used in the calcium

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transport experiments. Cells were cultured in complete Dulbecco’s modified essential

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medium (DMEM) with 10% fetal bovine serum (FBS), 1% antibiotic, and 1%

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nonessential amino acids as we described previously. 23 Cells were seeded at a density

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of 3 × 105 cells/mL on transwell culture plates (Corning Inc., MA, USA) with a

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polycarbonate membrane (24 mm diameter inserts, 0.4 μm pore size). The culture

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medium was added to both the basolateral (3.5 mL) and to the apical sides (2.5 mL).

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The medium was changed every other day for three weeks. 5



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Calcium transport studies. Before the calcium transport experiments began,

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transepithelial electrical resistance (TEER) was measured using a Millicell ERS-2

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epithelial voltammeter (World Precision Instruments, FL, USA). TEER values of the

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cell monolayers were about 260 Ω/cm2. 23 Culture medium was discarded and

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monolayers were immediately washed twice with Hank’s balanced salt solution

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(HBSS, without calcium and magnesium). Monolayers were then transferred to a new

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cluster plate containing 2 mL of HBSS buffer. Prior to the start of the calcium

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transport experiments, the apical side was also added to 2 mL of HBSS buffer and

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incubated for 30 min at 37 °C with 5% CO2. After 30 min of incubation, the CPPs

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(300 µg/well) and the HPLC-isolated peaks (P1 to P5, 100 µg/well each) were added

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to the apical sides in a calcium solution (300 µg/well). The samples and calcium

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solution were pre-mixed before the transport studies began. CPP-free controls had

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only the addition of a calcium solution (300 µg/well) and no CPP or HPLC-isolated

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peaks. At various time periods (20, 40, 60, 90, 120, 180, and 240 min), 1 mL of HBSS

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buffer was collected from the basolateral side to measure calcium ion concentration.

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One mL of fresh HBSS buffer was simultaneously added to the basolateral side to

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keep a constant volume.

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Resulting calcium concentrations at each time point were determined using

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ICP-MS (NexION 300X ICP, MA, USA). 22 The total quantity of calcium transported

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to the basolateral side of each well was calculated according to the following formula:

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B n  2  An  1 

n 1

 Ak k

(1)

1

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in which Bn represents the total quantity of transported calcium in 2-mL of HBSS

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buffer in the basolateral side of each well at each selected time point (20, 40, 60, 90,

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120, 180, and 240 min) (unit: μg/well). Two is a constant, representing 2-mL of HBSS

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buffer in the basolateral side of each well. An represents the calcium ion concentration 6


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of the HBSS buffer in the basolateral side of each well at different time points,

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measured by ICP-MS (unit: μg/mL). One is also a constant and represents the 1-mL of

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HBSS buffer collected from the basolateral side of each well in order to measure the

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calcium ion concentration. n is an independent variable. For the present study, it could

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be the number of 1, 2, 3, 4, 5, 6, or 7, representing time points 20, 40, 60, 90, 120, 180,

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and 240 min, respectively).

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All assays were carried out in triplicate and the results were expressed as mean values ±standard deviation (SD). Magnesium transport studies. CPPs (300 µg/well) and the HPLC-isolated peaks

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(P1 to P5, 100 µg/well each) were added to the apical sides using a magnesium

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solution (150 µg/well). The samples and magnesium solution were pre-mixed before

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the transport studies began. CPP-free controls had just the magnesium solution (150

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µg/well) added. All other conditions (e.g. time points) and analysis (e.g. Equation 1)

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in the magnesium transport studies were the same as those described in the calcium

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transport studies.

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Calcium and magnesium transport studies. CPPs (300 µg/well) and the

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HPLC-isolated peaks (P1 to P5, 100 µg/well each) were each added to the apical sides

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in a solution containing both calcium and magnesium (calcium: 300 µg/well,

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magnesium: 150 µg/well). The samples as well as the combined calcium and

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magnesium solution were pre-mixed before the transport studies began. CPP-free

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controls had just the combined calcium and magnesium solution (calcium: 300

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µg/well, magnesium: 150 µg/well) added. All other conditions and analysis in the

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combined calcium and magnesium transport studies were the same as those in the

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calcium transport studies.

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Amino acid sequence analysis of active peptides. The amino acid sequence of the 7



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active peptides that displaying calcium transport activity were analyzed using

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nanospray LC-MS/MS with an HPLC instrument coupled to an LTQ-Velos-Orbitrap

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mass spectrometer. The active peptides were solubilized in 0.1% TFA, then 1 μL

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solution was loaded on the nano-LC column. The data were acquired using

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LTQ-Velos-Orbitrap mass spectrometry. Peak lists were generated using Proteome

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Discoverer software. The resulting data were searched against Swiss-Prot database

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using Mascot 2.2. 24

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Statistical Analysis. All experiments were carried out at least three times and the data

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are expressed as means ± standard deviations (SD). The differences among samples

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were analyzed by ANOVA with a significance level of p< 0.05.

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RESULTS AND DISCUSSION

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Purification of fractions with calcium binding activity. Several different

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purification procedures have been reported for calcium transport peptides. For

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instance, a novel oligophosphopeptide with high calcium binding activity was

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obtained from carp egg hydrolysate. This was achieved by way of a three-step

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purification that consisted of ultrafiltration, size exclusion chromatography, and

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high-performance liquid chromatography. 11 In another instance, Chen et al. purified a

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calcium-binding peptide from tilapia scale protein hydrolysate by hydroxylapatite

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affinity, gel filtration and RP-HPLC. 8 In a third example, a histidine-containing

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peptide was initially fractionated by an ultrafiltration membrane, then purified using

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ion-exchange chromatography, gel filtration, and RP-HPLC. 25 Our study featured

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active fractions capable of calcium transport that were then separated through a

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two-step purification process.

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In the first step, CPP fractions with high calcium binding activity were collected 8


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between 20 and 55 min and were termed F1, F2, and F3 (Figure 1). The collected

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fractions between 20 and 55 min were further separated into different fractions

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(Figure 2). The numerous peaks in Figure 2 suggested that there were more than ten

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different calcium-binding peptides present in the sample. The five main peaks were

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designated as P1 to P5. They were subsequently collected and lyophilized prior to

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activity analysis of calcium and magnesium transport (Figure 2).

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Calcium transport studies in Caco-2 cell monolayer. Caco-2 cells have been

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previously used as an effective model of human intestinal epithelial cells in vitro.

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They have been successfully applied to simulative absorption studies involving drugs,

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26, 27

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activity (300 µg/well) and the HPLC-isolated peaks (100 µg/well each) were

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evaluated using Caco-2 cell monolayers. The resulting effects on calcium transport

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are shown in Figure 3. CPPs showed significant calcium transport activity across all

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Caco-2 monolayers when compared with CPP-free control across all values from the

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20 to 240 min time points (P

Bioactive Peptides Isolated from Casein Phosphopeptides Enhance

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