Phytoferritin Association Induced by EGCG Inhibits ... - Springer Link

Plant Foods Hum Nutr (2014) 69:386–391 DOI 10.1007/s11130-014-0451-8

ORIGINAL PAPER

Phytoferritin Association Induced by EGCG Inhibits Protein Degradation by Proteases Aidong Wang & Kai Zhou & Xin Qi & Guanghua Zhao

Published online: 11 November 2014 # Springer Science+Business Media New York 2014

Abstract Phytoferritin is a promising resource of non-heme iron supplementation, but it is not stable against degradation by proteases in the gastrointestinal tract. Therefore, how to improve the stability of ferritin in the presence of proteases is a challenge. Since (−)-epigallocatechin-3-gallate (EGCG) is rich in phenolic-hydroxyl groups, it could interact with ferritin through hydrogen bonds, thereby preventing protein from degradation. To confirm this idea, we focus on the interaction between EGCG and phytoferritin, and the consequence of such interaction. Results demonstrated that EGCG did interact with ferritin, and such interaction induced the change in the tertiary/quaternary structure of protein but not in its secondary structure. Furthermore, stopped-flow and dynamic light scattering (DLS) results showed that EGCG could trigger ferritin association. Consequently, such protein association markedly inhibited protein digestion by pepsin at pH 4.0 and by trypsin at pH 7.5. These findings raise the possibility to improve the stability of phytoferritin in the presence of proteases. Keywords (−)-Epigallocatechin-3-gallate . Recombinant soybean seed H-2 ferritin . Association . Protection . Degradation Abbreviations Electronic supplementary material The online version of this article (doi:10.1007/s11130-014-0451-8) contains supplementary material, which is available to authorized users. A. Wang : K. Zhou : G. Zhao (*) CAU & ACC Joint-Laboratory of Space Food College of Food Science and Nutritional Engineering Key Laboratory of Functional Dairy Ministry of Education, China Agricultural University, Beijing 100083, China e-mail: [email protected] X. Qi National institute of Metrology, Beijing 100013, China

BSA CD DLS EGCG IDA rH-2 EP

albumin from bovine serum circular dichroism dynamic light scattering (−)-epigallocatechin-3-gallate iron deficiency anemia recombinant soybean seed H-2 ferritin extension peptide

Introduction Dietary iron deficiency is the most common nutritional disorder in the world. Iron deficiency anemia (IDA) mainly affects vegetarians, women and young children in developing countries. Nutritional iron can be classified as heme iron and nonheme iron. While heme iron is absorbed as porphyrin complex unaffected by polyphenols in dietary materials, non-heme iron absorption from plant foodstuffs is likely to be interfered. Therefore, exploring new non-heme iron sources, which are unaffected by polyphenols in dietary materials, is vitally important. Ferritin is a superfamily of iron storage protein, which represents a new class of non-heme iron sources. All ferritins are composed of 24 subunits that arranged in a 4–3-2 symmetry to form a hollow protein shell that can store up to ~4500 Fe3+ atoms. Phytoferritin from legume seeds is rich in iron because legume seeds store ~90 % of its iron in ferritin [1]. Therefore, phytoferritin, especially from legumes, has been considered as a novel alternative dietary iron source due to three major advantages [2]: the protection of protein shell from interaction with other dietary factors [4], the safer form of iron stored as ferric cores rather than FeSO4, and the possible intact absorption by receptor-mediated endocytosis [3]. However, ferritin is not stable enough against degradation by proteases existing in the gastrointestinal tract [5, 6].

Plant Foods Hum Nutr (2014) 69:386–391

Therefore, it is important to find edible compounds which have the ability to prevent phytoferritin from degradation by the protease(s). (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant and active catechin in green tea [7]. Many of the biological properties of green tea such as reducing the risk of cancer, diabetes and cardiovascular disease have been attributed to its antioxidant and free radical-scavenging activity [8–10]. EGCG and phytoferritin co-exist in plant foodstuffs, thus the interaction between the two components by hydrogen bonds might occur because EGCG is rich in hydroxyl groups as shown in Fig. 1c. Consistent with this idea, recent studies have shown that EGCG interacts with several kinds of proteins including protein disulfide isomerase isoform A3 [11], human erythrocyte membrane proteins [12], and salivary proline-rich protein IB5 [13]. The above considerations raise two questions: (1) Is there an interaction between EGCG and phytoferritin? (2) If so, can such interaction inhibit phytoferritin from degradation by proteases? We will answer these two important questions in this study.

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light-scattering instrument (Viscotek Europe Ltd.) as recently reported [18]. Simulated gastric fluid (SGF), simulated intestinal fluid (SIF) digestion stability experiments and iron release measurement were carried out as recently described [16]. (Detailed information about fluorescence titration experiments, CD spectra, SLS and DLS measurements, SIF and SGF digestion, and iron release measurement are presented in Online Resource 1) Transmission electron micrographs were obtained at 80 kV using a Hitachi S-5500 scanning electron microscope. Liquid samples were diluted with 50 mM Tris–HCl buffer (pH 8.0) prior to being placed on carbon-coated copper grids, and excess solution was removed with filter paper. Resulting samples were stained using 2 % uranyl acetate for 2 min [19]. The data were analyzed using Origin 8.0 software (Micro Cal Inc.) and the structural formula was processed by ChemDraw 7.0. All experiments were carried out in triplicate.

Results and Discussion Materials and Methods Materials EGCG with a purity of 98 % was gained from J&K Chemical (Beijing, China). Recombinant soybean seed H-2 ferritin (rH-2) and holo-rH-2 were prepared as previously described [14, 15]. Methods Fluorescence titration experiments were performed using the Cary Eclipse spectrophotometer (Varian, Palo Alto, USA) as previously described [16]. CD spectra were recorded with a Chirascan Plus spectrometer (Applied Photophysics, Leatherhead, UK) as previously described with some modifications [16]. The SLS measurements were performed with a pneumatic drive Hi-Tech SFA-20 M apparatus in conjunction with a Cary Eclipse spectrofluorimeter (Varian, Palo Alto, USA) as previously described [17]. The DLS measurements were performed at 25 °C using a Viscotek model 802 dynamic

Fig. 1 a Native-PAGE analysis of rH-2 ferritin; b SDS-PAGE analysis of rH-2 ferritin c Chemical structure of (−)-epigallocatechin-3-gallate (EGCG)

Interaction of rH-2 with EGCG Non-denaturing gel electrophoresis (native PAGE) revealed purified recombinant soybean seed H-2 ferritin (rH-2) as a single complex, estimated to be about 560 kDa (Fig. 1a). SDS-polyacrylamide gel electrophoresis analysis indicated that rH-2 complex contains only H-2 subunit (28.0 kDa) (Fig. 1b). These two values are in good agreement with previous reports [3, 20]. Transmission electron microscope (TEM) analysis revealed apo-rH-2 molecules were well dispersed with an outside diameter of 12 nm (Fig. 1c), which is consistent with our previous studies [21]. Subsequently, a combination of fluorescence and CD spectra was used to characterize whether EGCG interact with rH-2. Since the intrinsic fluorescence emission from tryptophan residue in proteins is sensitive to the microenvironment surrounding the fluorophore residue [22, 23], firstly fluorescence spectroscopy is used to study the interaction between apo-rH-2 and EGCG, and results are shown in Fig. 2. In the absence of EGCG, apo-rH-2 exhibited a strong fluorescence emission band at 324 nm on excitation of 280 nm, indicating that most of the observed fluorescence was contributed by the sole Trp residue at the position of 243 [20], whereas EGCG had no fluorescence under the experimental condition. With the addition of EGCG as a series of increasing concentrations, the fluorescence intensity of apo-rH-2 decreased significantly, accompanied by obvious blue shift of maximum emission wavelength, demonstrating that there is an interaction between rH-2 and EGCG. In order to gain further information about the interaction, circular dichroism (CD) experiments were carried out in far-

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Fig. 2 Fluorescence intensity of apo-rH-2 ferritin treated with different concentrations of EGCG. Conditions: 0.5 μM apor-H-2, in 50 mM Tris–HCl, 150 mM NaCl, pH 8.0, 25 °C

UV region, and results were shown in Online Resource 2. Changes in far UV-CD are related to the alteration in the overall secondary structure of the protein [23]. RH-2 ferritin has two negative ellipticities in the far-UV spectrum, at 208 and 222 nm, being in good agreement with the literature reports for native SSF secondary structure [16], with a content Fig. 3 a Scatter light intensity of rH-2 ferritin aggregation induced by EGCG as a function of time. The curve represented an average of three experimental measurements. Conditions: 0.9 μM apo-rH-2, in 50 mM Tris– HCl, 150 mM NaCl, pH 8.0, 25 °C b Relative mass distributions of different particle sizes of apo-rH-2 in the presence and absence of EGCG c and d Transmission electron micrographs of apo-rH-2 in the absence c and presence d of EGCG. Conditions: 0.175 mM EGCG; 0.05 μM apo-rH-2 ferritin in 50 mM Tris–HCl, 150 mM NaCl, pH 8.0, 25 °C; samples were negatively stained by 2 % uranyl acetate

of 78 % α-helix, 10 % β-trun and 14 % random coil estimated by using CDNN secondary structure analysis. Upon the addition of EGCG to rH-2 ferritin, the CD spectrum of the protein was almost unchanged, indicating that the interaction between EGCG and rH-2 hardly changes the secondary structure of the protein.


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6 % in that of H-4, and 12 % in that of H-5), and that the EP is on the outer surface of ferritin where it can easily interact with EGCG, this might be an important reason why the interaction of the association of rH-2 induced by EGCG occurs. The phenolic hydroxyl of EGCG could form hydrogen bonds with the amide carbonyl of the peptide backbone. In addition, hydrophobic interaction between EGCG and rH-2 ferrintin could also be responsible for the association of rH-2 induced by EGCG.

Phytoferritin Association Induced by EGCG The above results indicate that there is an interaction between EGCG and rH-2 ferrtin. This raises a question of whether such interaction may induce protein association. To answer this question, the light scattering intensity was measured upon rapid mixing apo-rH-2 with EGCG solution by stopped-flow as recently reported [2], and results are given in Fig. 3a. It was found that the light scattering intensity of rH-2 rapidly increased when EGCG was shot against rH-2 ferritin by stopped-flow. With increasing EGCG concentration, the light scattering intensity increased to a greater extent. All kinetic curves are hyperbolic, and the light scattering intensity reached its maximum within 75 s when EGCG was shot against rH-2 at a mass ratio of rH-2 to EGCG (1 : 1.64). These results manifested that EGCG induced protein-protein association in a dose-dependent manner. Moreover, the light scattering intensity did not decrease with time, indicating that the association of rH-2 caused by EGCG was irreversible, and that resulting protein aggregate was stable. The present phenomenon is different from phytoferritin association induced by low pH or ferric ions, but similar with that triggered by tannic acid [2, 18, 16].

Effect of EGCG on the Stability of rH-2 in Simulated Digestive Fluid The above mentioned results demonstrate that EGCG induces the association of rH-2 ferritin and the formed aggregates are quite stable under the present experimental conditions, so it is of great interest to know whether such association can help ferritin stand against protein degradation by proteases. To answer this question, simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion of rH-2 ferritin in the presence of EGCG was performed and the results are shown in Fig. 4a and b, respectively. In SGF digestion (Fig. 4a), compared with the control sample (lane 2) in which the subunit of 28.0 kDa was degraded into two

Dynamic light scattering (DSL) was used to further characterize rH-2 association induced by EGCG. Samples tested in this experiment were allowed to stand for 10 min at room temperature prior to DLS measurements at pH 8.0. One population with size value of 7.0 nm was evident in the relative mass distribution of rH-2 ferritin sample (Fig. 3b), spectrum a, which is rich in the monomer of rH-2 ferritin. Furthermore, after the addition of EGCG to rH-2, the relative distribution was notably altered toward larger aggregates with increasing EGCG concentration. These results again demonstrated that the cross-linking of rH-2 and EGCG led to the aggregation of rH-2, being in accordance with the above stopped-flow results as shown in Fig. 3a. To confirm the above observation, TEM analysis was also employed to further investigate the association of rH-2 induced by EGCG. As shown visibly in Fig. 3c, most rH-2 ferritins without the treatment of EGCG were mono-dispersed, while the rH-2 ferritin exposed to EGCG became aggregated (Fig. 3d). This observation confirmed the conclusion that EGCG induced the association of rH-2. It was reported that proteins containing proline residues easily formed a haze when combined with polyphenolic compounds in beer, while the synthetic polypeptides and proteins lacking such proline residues did not [24]. Consistent with this idea, recent study revealed that EGCG induced the aggregation of the proline-rich protein IB5 in salivary [13]. Thus, it appears that phenolic-hydroxyl-rich compounds like EGCG could have high affinity for proline-rich proteins [25, 26]. Considering that the extension peptide (EP) of phytoferritin is also rich in proline, which accounts for ~10 % of total amino acid residues in the EP of H-2 subunit (15 % in that of H-1,

Fig. 4 a SDS-PAGE analysis of the stability of holo-rH-2 at different EGCG concentrations by SGF digestion stability assay at pH 4.0. Lane M, protein markers; Lane 1, holo-rH-2 alone; Lane 2, holo-rH-2+pepsin; Lane 3, pepsin alone; Lane 4–9, holo-rH-2+pepsin+EGCG, a ratio of ferritin to EGCG ranging from 1:10, 1:6, 1:3, 3:1, 6:1, to 10:1, respectively b SDS-PAGE analysis of the stability of holo-rH-2 at different EGCG concentrations by SIF digestion stability assay at pH 7.5. Lane M, protein markers; Lane 1, holo-rH-2 alone; Lane 2, holo-rH-2+trypsin; Lane 3, trypsin alone; Lane 4–9, holo-rH-2+trypsin+EGCG, a ratio of ferritin to EGCG ranging from 1:10, 1:6, 1:3, 3:1, 6:1, to 10:1, respectively

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main bands in smaller molecular weight at pH 4.0, treatment with EGCG at different concentrations inhibited the degradation of rH-2 to different extents, and such inhibition increased with increasing a ratio of EGCG to rH-2. The pH value of the stomach of infants and young children is around 4.0 [27], therefore EGCG might serve as a natural food compound to prevent ferritin from degradation by pepsin in their stomachs. Once escaping from stomach, the whole ferritin could be absorbed by a receptor-mediated endocytic process in the intestine, although the receptor is unclear [1, 3]. In SIF digestion (Fig. 4a), similar with the results above, the degradation of rH-2 ferritin were significantly inhibited by EGCG to different extents. The inhibitory effect was remarkable when the ratio of EGCG to rH-2 ferritin is not less than 3:1. Thus, it is possible that ferritin molecules can be protected by EGCG against trypsin degradation in the intestine. However, whether a complex formed by EGCG and ferritin can be taken up by Caco-2 cells in a similar mechanism to ferritin alone remains unknown at this moment. To answer this question it is necessary more investigation in future. On the other hand, we found that the interaction between EGCG and rH-2 resulted in iron release from rH-2 ferritin as shown in Online Resources 3. The rate of iron release from ferritin (0.5 μM) by EGCG (0.4 mM) is about 0.32 μM/min which is 1.8 times faster than that by ascorbic acid (0.18 μM/ min) under the same experimental condition. A faster iron release by EGCG than ascorbic acid could be ascribed to its stronger activity of chelating iron due to more phenolichydroxyl groups on its aromatic rings as recently reported [16]. Such iron release is unexpected before reaching the gut since it inhibits the absorption of iron by intact ferritin. However, it has to be mentioned that the absolute rate of iron release from ferritin induced by EGCG is still very slow. For example, the total iron concentration in holo-rH-2 is approximately 200 μM, and only 20 μM of iron (which accounts for ~10 % of the total) was released upon treatment of holo ferritin with 0.4 mM of EGCG for 60 min (Online resources 3). Therefore, the interaction of EGCG with ferritin has a small effect on iron release from protein.

Conclusion The present study reveals, for the first time, that EGCG, the most abundant and active polyphenol in green tea, can bind to and induce ferritin association. Such association greatly inhibits the degradation of ferritin by pepsin at pH 4.0 as well as by trypsin at pH 7.5. Phytoferritin can be considered as a promising dietary iron supplement since it is less sensitive to chelators existing in foodstuffs, but it is not stable enough against the degradation by proteases existing in the gastrointestinal tract. The present results represent the possibility that


the association induced by EGCG could be effective to protect ferritin from degradation in both stomach and intestine. Additionally, the effect of EGCG as a protector of ferritin in the gastrointestinal tract on iron release is very limited. Acknowledgments This work was supported by the National Natural Science Foundation of China (31271826, 31471693). Conflict of interests The authors declare that they have no conflict of interest.

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