Purification and characterization of a novel immunomodulatory hexapeptide from alcalase hydrolysate of ultramicro-pretreated silkworm (Bombyx mori) pupa protein

Journal of Asia-Pacific Entomology 22 (2019) 633–637

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Purification and characterization of a novel immunomodulatory hexapeptide from alcalase hydrolysate of ultramicro-pretreated silkworm (Bombyx mori) pupa protein

T

Zhiyong Lia, Shan Zhaoa, Xiangdong Xina, Bei Zhanga, Attaribo Thomasa, Asakiya Charlesa, ⁎ Kwang Sik Leeb, Byung Rae Jinb, Zhongzheng Guia,c, a b c

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea Sericultural Research Institute, ChineseAcademy of Agricultural Science, Zhenjiang, Jiangsu 212018, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Bombyx mori Pupa protein Immunomodulatory peptide Purification

Silkworm (Bombyx mori) pupa protein is one potential source of insect protein for use in food. Immunomodulatory peptides are specific protein fragments that can positively influence human health. Here, we purified a novel immunomodulatory hexapeptide from the alcalase hydrolysate of ultramicro-pretreated silkworm pupa proteinusing Sephadex gel filtration chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide sequence was determined by liquid chromatography–electrospray ionization– tandem mass spectrometry (LC-ESI-MS/MS). The results showed that the molecular mass of the purified peptide was 656.17 Da, and the amino acid sequence was Pro-Asn-Pro-Asn-Thr-Asn (PNPNTN). Splenocyte proliferation was 87.35% in the presence of 100 μg/ml of purified peptide. The splenocyte proliferation could be promoted upto 248.4% at 100 μg/ml of PNPNTN after induction by Concanavalin A (Con A). PNPNTN was stable in the presence of the gastrointestinal proteases pepsin and trypsin and at temperatures up to120°C. Taken together, these results show that this novel immunomodulatory hexapeptide from silkworm pupae has potential therapeutic value as an immunomodulatory component of functional food.

Introduction The silkworm (Bombyx mori) pupa is a potential source of insect protein and is traditionally used as food and medicine in Asian countries, such as China, Japan, Korea, India and Thailand(Zhou and Han, 2006). The silkworm pupa is made of48–60% crude protein and contains 18 amino acids, including high levels of hydrophobic amino acids such as valine, methionine and phenylalanine (Tomotake et al., 2010). Silkworm pupa hydrolysates generated by various enzymatic treatments exhibit numerous health-related properties, such as antioxidant activity (Yang et al., 2013), anti-obesity activity(Lee et al., 2012), antitumor activity (Hu et al., 2005), and anti-bacterial activity (Hara and Yamakawa, 1996). However, the abundant, beneficial properties of the silkworm pupa protein have not been fully utilized. Most silkworm pupae are used only as fertilizer and livestock feed or are even regarded as industrial waste (Hu et al., 2017). Therefore, it is important to assess the nutritional value and the potential medicinal value of silkworm pupa as food supplements.

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Immunomodulatory peptides are specific protein fragments that positively affect body functions and may ultimately influence health (Conlon et al., 2014).Such peptides can enhance immune cell functions (Fitizgerald and Murray, 2010), natural killer (NK) cell activity, antibody synthesis and cytokine regulation (Hartmann and Meisel, 2007; Horiguchi et al., 2005). Splenocytes consist of a variety of cell populations, such as T and B lymphocytes, dendritic cells and macrophages, that have different immune functions and are often used to test for immune responses. The development of immunomodulators from natural sources for diet supplementation in both animals and humans is an active area of research (Fitzgerald and Murray, 2010). To date, numerous immunomodulatory peptides have been purified from different protein sources, such as fish (Duarte et al., 2006), sheep bone (Yoshikawa et al., 1993), bovine milk (Gill et al., 2000), silkworm pupa (Hara and Yamakawa, 1996), Musca larvae(Chen et al., 2015), frog skin (Conlon et al., 2014), alga (Chlorella vulgaris) (Humberto et al., 2009), and Alaska pollock (Hou et al., 2012). However, most immunomodulatory pharmaceuticals are not suitable for long-term or

Corresponding author at: School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China. E-mail address: [email protected] (Z. Gui).

https://doi.org/10.1016/j.aspen.2019.04.005 Received 9 October 2018; Received in revised form 10 December 2018; Accepted 16 April 2019 Available online 17 April 2019 1226-8615/ © 2019 Korean Society of Applied Entomology. Published by Elsevier B.V. All rights reserved.

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Determination of the peptide sequence by ion trap (IT)-MS

preventive use. Thus, there is ongoing interest to identify new immunomodulators to enhance non specific host defense mechanisms. Ultramicro grinding is an advanced technology that generates changes in the structure and surface area of bulk materials and that thereby stimulates various beneficial protein characteristics. This technique is used to improve quality of food, chemicals, pharmaceuticals and other types of products (Zhao et al., 2010). Some studies have reported that ultramicro powder exhibited higher fluidity, solubility, electric conductivity and water holding capacity (Zhou et al., 2017). Other studies have shown that enzymatic hydrolysis is an effective method for releasing immunomodulatory peptides from food proteins (Mercier et al., 2004). In this study, we separate an immunomodulatory peptide from silkworm pupa protein using ultramicro-pretreatment and alcalase hydrolysis. The immunomodulatory peptide was purified by gel filtration chromatography using Sephadex-G100 and Sephadex-G15 resins and reverse-phase high-performance liquid chromatography (RP-HPLC). The sequence of the purified peptide was analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESIMS/MS). Additionally, the purified peptide was characterized for its stability in the presence of gastrointestinal enzymes and at high temperatures and for its capacity to stimulate Con A- and lipopolysaccharide (LPS)-induced splenocyte proliferation.

The molecular mass and amino acid sequence of the purified peptide were determined by ESI-IT-MS (Thermo LXQ Linear Ion Trap Mass Spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) with capillary temperature at 300 °C and spray voltage of 4.5 kV. Mass spectra were acquired over a mass range of 50–1506 m/z. The peptide molecular mass was determined by charge state (M + H)+ analysis of the mass spectrum, and the peptide was sequenced by MS/MS. Determination of peptide immunomodulatory activity Six-week-old ICR mice (animal license no. 20160010) were sacrificed by cervical dislocation, and their spleens were removed, minced, and washed through sterilized 200 mesh copper with 5 mL RPMI-1640 medium to obtain a single-cell suspension. Lymphocyte separation medium (Solarbio, China) was added, and the mixture was centrifuged at 400 ×g for 15 min at 4 °C. Erythrocytes in the pellet were lysed with Tris-NH4Cl solution (0.14 mol/l NH4Cl, 20 mmol/l Tris) for 2 min at 37 °C. The lysed solution was centrifuged as described above, and the pellet was washed twice with RPMI-1640 medium. The cell concentration was adjusted to 5 × 106 cells/ml with RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Splenocyte proliferation was determined using the MTT colorimetric assay (Wu et al., 2012). The stimulation index (SI) was calculated using the following equation: SI = (absorbance of peptide-treated cells − absorbance of blank wells) / (absorbance of peptide-treated wells).

Materials and methods Materials and reagents Silkworm pupae were provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (Zhenjiang, China). Female Kunming mice (6–8 weeks old) were purchased from the Laboratory Animal Center of Jiangsu University (Jiangsu, China). All experimental procedures were carried out in accordance with the guidelines for the Centre of Experimental Animal, Jiangsu, China. RPMI1640 cell culture medium, trypan blue, Con A, LPS, proteomics grade of pepsin and trypsin, and 3[4,5-dimethylthiazoyl-2-yl]2,5-diphenyltetrazoliumbromide (MTT) (purity, 98%) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Alcalase 2.4 L was suppliedby Sangon Biotechnology Co. (Shanghai, China). Sephadex G-100 and Sephadex G-15 columns were purchased from Sangon Biotechnology Co. (Shanghai, China). All other chemicals and reagents were of analytical grade.

Evaluation of Con A- or LPS-induced splenocyte proliferation Splenocyte proliferation induced by Con A or LPS was assayed according to the method of Wu et al. (2012). The spleen cell suspension containing 5 × 106 cells/ml was seeded into a 96-well plate at a final volume of 100 μl/well. Three different concentrations (25.0, 50.0, and 100.0 μg/ml) of the purified peptide were added to triplicate wells. Wells also received 100 μl of either Con A at a final concentration of 5 μg/ml or LPS at a final concentration of 10 μg/ml. For the blank control group, the sample solution was replaced with 100 μl of RPMI1640 medium. After incubation at 37 °C in 5% CO2 for 48 h, 20 μl MTT (5 mg/ml) was added to each well and incubated for an additional 4 h, after which the samples were centrifuged at 400 ×g at 4 °C for 5 min. After removing the supernatant, 150 μl of DMSO was added to each well. The plates were gently agitated to dissolve the purple crystals. After incubating for 15-min in a dark room, the absorbance was measured at 570 nm, using a microplate reader (Bio-TEK EL310, USA).

Preparation of ultramicro-pretreated silkworm pupa protein hydrolysate Silkworm pupa protein was isolated from defatted silkworm pupae according to the method of Jia et al. (2015). The protein solution was pretreated with ultramicro pulverization according to our previous work (Zhou et al., 2017), using superfine grinding in a ball mill to process protein at a concentration of 8.3% (w/v), pH 8.1, and with a processing time of 61 s. Hydrolysis was performed with 6000 U/g of alcalase at a constant temperature of 50 °C and pH 9.0 for 50 min.

Stability of the purified peptide at different temperatures The thermostability of the immunomodulatory peptide was assessed according to the method of Tidarat et al. (2015). Four plates of the purified peptide (25 μg/ml) were incubated at 37, 80, 100, and 121 °C, for 30 min prior to being added to the splenocytes. The splenocyte proliferation-inducing activity of heat-treated peptide was assessed as described above.

Peptide purification by gel filtration chromatography and RP-HPLC Hydrolysates were passed through a Sephadex-G100 column (2.5 × 70 cm) that was equilibrated and eluted with distilled water at a flow rate of 1.0 ml/min. Fractions were collected at 3-min intervals, and each fraction was assayed for splenocyte proliferation-inducing activity. The fraction with the highest activity was purified by gel filtration on a Sephadex-G15 column under the same conditions as described for the G-100 column, followed by filtration through a 0.45-μm filter (Millipore, Billerica, MA, USA) and a final round of purification by RPHPLC on an octadecyl-silica (C18) column using a mobile phase of acetonitrile in water a column temperature of 30 °C and an inflow rate of 1.0 ml/l for 20 μl of sample.

Stability of the purified peptides in the presence of gastrointestinal enzymes The stability of the immunomodulatory peptide in the presence of gastrointestinal proteases was evaluated in vitro according to a previously described method (Jia et al., 2015). A 1% (w/w) pepsin solution was prepared in 0.1 mM KCl-HCl buffer at pH 2.0, while a 1% (w/w) trypsin solution was prepared in 50 mM sodium phosphate buffer at pH 7.0. The purified peptide was successively treated with pepsin (pH 2.0, enzyme-substrate ratio of 20 U/mg) and trypsin (pH 7.0, 634

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enzyme-substrate ratio of 20 U/mg peptide and incubated at 37 °C for 4 h. The peptide-induced splenocyte proliferation response was measured as described above.

the original fraction 1.

Statistical analyses

The amino acid sequence of the purified component was determined by IT-MS/MS (Fig. 3). The results indicated a range of molecular mass for the purified peptide between 401.07 and 656.11 Da (Fig. 3A). The molecular mass of the primary peptide, which was deduced from the m/ z value of (M + H)+ by subtracting one mass unit for the attached proton, was 656.11 Da. Based on this value and the IT-MS/MS spectrum (Fig. 3B), the component was determined to be a hexapeptide with the sequence Pro-Asn-Pro-Asn-Thr-Asn (PNPNTN). Splenocyte proliferation in the presence of 100 μg/ml PNPNTN was 92.9% (P < .05).

Amino acid sequencing of the immunomodulatory peptide

All data are expressed as the mean ± standard deviation of triplicate determinations. Data were analyzed using SPSS v.22 software (SPSS Inc., Chicago, IL, USA), and differences between means were evaluated using one-way analysis of variance and Fisher's least significant difference test. A Pvalue < 0.05 was considered statistically significant. Results

Effects of the purified peptide on Con A- or LPS-induced splenocyte proliferation

Preparation of ultramicro-pretreated silkworm pupa protein hydrolysate

The effects of PNTNTN on splenocyte proliferation induced by Con A or LPS were investigated. The stimulatory effects of this peptide upon proliferation were higher with increasing dosage (Table 1). The proliferation of splenocytes induced by Con A was promoted by 248.4% at 100 μg/ml of PNPPNTN, which was a statistically significant increased compared to the Con A control group (P < .01). The stimulatory effects of PNPNTN upon lymphocyte proliferation induced by LPS were nearly identical to those see with Con A and were also significant (P < .01) at all test concentrations. These results suggested that PNPNTN could dose-dependently promote murine T- and B-cell proliferation induced by specific mitogens.

Prior to alcalase hydrolysis, the silkworm pupa protein was subjected to superfine grinding in a ball mill (i.e., ultramicro pretreatment) to enhance its immunoregulatory activity. Silkworm pupa protein hydrolysate with or without ultramicro pretreatment both induced splenocyte proliferation in a dose-dependent manner. However, splenocyte proliferation was stimulated to a greater extent by hydrolysates with ultramicro pretreatment, with 76.53% proliferation achieved with 100 μg/ml of pretreated protein. Thus, the ultramicro pretreatment process potentiates the immunomodulatory activity of the hydrolysate (Fig. 1). Purification of an immunomodulatory peptide from the pupa protein

Stability of the purified peptide in different conditions

As shown in Fig. 2A, two components (fraction 1 and 2) were separated from ultramicro-pretreated silkworm pupa protein hydrolysate by chromatography using Sephadex-G100. The yields of fraction 1 and fraction 2 were 68.1 mg/g total protein and 91.1 mg/g total protein, respectively. Splenocyte proliferation was enhanced in the presence of each fraction; the maximum proliferation of 87.35% (P < .05) was observed with 100 μg/ml of fraction 1 (Fig. 2A). Therefore, this fraction was further purified by chromatography using Sephadex-G15, which yielded a single peak (Fig. 2B). The peak-containing fraction was collected and further purified by RP-HPLC on a C18 column with 0.5 ml/l trifluoroacetic acid in 90% acetonitrile. The C18 chromatogram showed a single large peak (Fig. 2C), indicating that the fraction was sufficiently pure for amino acid sequencing. Splenocyte proliferation was 16.4 times greater when treated with the C18-purified component than with

The stability of the purified peptide PNPNTN in different temperatures and in simulated gastric and intestinal fluids was evaluated. The splenocyte proliferation-inducing activity of PNPNTN was stable in temperatures ranging from 37 °C to 121 °C (Table 2). Gastrointestinal proteases had no effect on the immunomodulatory activity of PNPNTN, as evident from the equivalent splenocyte proliferation observed with peptides that were or were not treated with enzyme (Table 3). Thus, PNPNTN was stable, possibly due to its short length, at a wide range of temperature and in the presence of the gastrointestinal proteases pepsin and trypsin (Ghassem et al., 2011; Jia et al., 2015). Discussion Currently, ultramicro pretreatment by superfine grinding is widely used to enhance protein properties. Zhao et al. (2010) reported that Astragalus membranaceous protein powder obtained with superfine grinding exhibited higher water-holding capacity, fluidity, and solubility. Our previous results showed that silkworm pupa protein treatment by superfine grinding increased the solubility, emulsibility and foaming of the protein by 402%, 187% and141%, respectively (Li et al., 2018). In this study, the ultramicro-pretreated technique was applied to silkworm pupa protein to improve the immunomodulatory activity of the hydrolysate. The splenocyte proliferation-inducing activity of the hydrolysates increased with substrate concentration for both the nonpretreated and ultramicro-pretreated hydrolysate groups. However, the proliferation was significantly higher with ultramicro-pretreated hydrolysate than with non-pretreated hydrolysate (Fig. 1). Subsequently, a novel immunomodulatory peptide was purified by Sephadex chromatography and RP-HPLC and was identified as a hexapeptide with the sequence Pro-Asn-Pro-Asn-Thr-Asn (PNPNTN) and a molecular mass of 656.1094 Da. The proliferative effect of PNPNTN was greatly influenced by its physicochemical characteristics, such as its positive charge, hydrophobicity and length. The molecular mass of peptides with immunoactivity are usually < 1000 Da, and these peptides usually contain hydrophobic amino acids (Jacquot et al., 2010). Specific amino acids or

Fig. 1. Effects of silkworm pupa protein with ultramicro-pretreated hydrolysates on splenocyte proliferation rate. Values are presented as the mean ± standard deviations from three replications (n = 3). 635

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Fig. 2. Purification profiles of immunomodulating peptide from silkworm pupa protein with ultramicro-pretreated hydrolysates. (a) Sephadex G-100 gel chromatography of the silkworm immunomodulating peptide fraction from ultrafiltration. The inserted table shows the splenocyte proliferation rate of each fraction at different concentrations. (b) Sephadex G-15 gel chromatography of fraction 1. (c) RP-HPLC with C18 column and 90% acetonitrile containing 0.5 ml/l TFA from Sephadex G-15 gel. Values are presented as the mean ± standard deviations from three replications (n = 3).

Table 1 Effects of the purified peptide on Con A- or LPS-induced mouse splenocyte proliferation rates. Preparation

Dose

Con A (A570)

LPS(A570)

Control (Con A) (μg/ml) Control (LPS) (μg/ml) PNTNTN (μg/ml)

5.0 10.0 25.0 50.0 100.0

18.99 – 86.04 87.62 89.58

– 44.59 88.89 90.18 90.87

a b

± 0.017 ± 0.015 ± 0.018a ± 0.018a

± ± ± ±

0.021 0.047b 0.080b 0.114b

P < .01 when compared with the Con A control group. P < .01 when compared with the LPS control group. Table 2 Splenocyte proliferation rates of PNTNTN after treatment by different temperature. Temperature

Splenocyte proliferation rate (%)

37 °C 80 °C 100 °C 121 °C

86.69 86.51 87.02 87.06

± ± ± ±

0.033 0.021 0.012 0.021

Splenocyte proliferation rate was tested at a concentration of 50 μg/ml. Values are presented as mean ± standard deviations from three replications (n = 3). Fig. 3. Molecular mass and amino acid sequence of immunomodulatory peptide from the purified fraction. (a) LC-ESI-MS spectrum of purified fraction. (b) LCESI-MS/MS spectrum of m/z 656.11, identified as the sequence as Pro-Asn-ProAsn-Thr-Asn (PNPNTN).

frequencies comparable with those induced by LPS. Another rimmunomodulatory peptide (Arg-Pro-Gln-Gln-Pro-Tyr-Pro-Gln-Pro-Gln-ProGln) from wheat protein hydrolysate has been reported to stimulate splenic T cells and NK cells by inducing the production of interferon-γ (Cornell et al., 1994). Hou et al. (2012) separated three peptides, namely, Asn-Gly-Met-Thr-Tyr, Asn-Gly-Leu-Ala-Pro and Trp-Thr, from Alaska Pollock frame that stimulated high levels of splenic lymphocyte proliferation. Zhou et al. (2014) reported that the peptide Gln-Glu-ProVal-Leu (QEPVL) from fermented milk can significantly activate

short chain length can greatly influence the immunoregulatory function of the peptide (Takahashi et al., 1994). Julius et al. (1998) purified a proline-rich peptide from sheep colostrum that stimulated resting mouse B cells and supported their progression through the cell cycle at 636

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Table 3 Splenocyte proliferation rate of PNTNTN after treatment by gastrointestinal proteases. Treatment

Splenocyte proliferation rate (%)

Control Gastric fluida Intestinal fluidb

85.05 ± 0.021 84.61 ± 0.116 85.72 ± 0.118

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Splenocyte proliferation rate was tested at a concentration of 50 μg/ml. Values are presented as mean ± standard deviations from three replications (n = 3). a The purified peptides were hydrolyzed by pepsin for 4 h. b The purified peptides were hydrolyzed by pepsin for 4 h and followed by trypsin hydrolysis for 4 h.

lymphocytes both in vitro and in vivo, can increase the lymphocyte proliferation rate and cyclic AMP levels, and can also inhibit LPS-induced inflammation by regulating nitric oxide release and the production of the cytokines IL-4, IL-10, IFN-γ, and TNF-α in vivo. In this study, the purified peptide could significantly stimulate the proliferation of T-lymphocytes and/or B-lymphocytes in the presence of ConA or LPS. Several research groups have reported that some compounds potentiate the proliferative ability of ConA, PHA, and LPS on immunocytes, suggesting that they have important effects in immune function (Manosroi et al., 2005; Gupta et al., 2006). T cells are involved in cell-mediated immunity and can be stimulated by ConA, where as LPS can stimulate the production of B cells, which are primarily responsible for humoral immunity (Wang et al., 2013). To probe the potential in vivo utility of PNPNTN for stimulation of immune cell proliferation, purified PNPNTN was incubated under temperature, pH, and enzymatic conditions that simulated those in the gastrointestinal tract. Our results showed that the purified peptide was stable against high temperatures and gastrointestinal proteases, similar to the peptides described in other reports (Kjellev et al., 2007; Jia et al., 2015).The stability of PNPNTN could be because short-chain peptides are less susceptible to degradation by gastro intestinal proteases (Ghassem et al., 2011). In summary, these results provide a scientific basis for the preparation of immunomodulatory peptides from silkworm pupa protein and indicate that PNPNTN has potential value as an ingredient in functional food devised to improve immune cell activity. Acknowledgements Financial support for this research was provided by the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201403064). References Chen, L.Q., Zhang, J., Sun, H.X., 2015. Immunological adjuvant effect of the peptide fraction from the larvae of Musca domestica. BMC Complement. Altern. Med. 15, 427. https://doi.org/10.1186/s12906-015-0951-6. Conlon, J.M., Mechkarska, M., Lukic, M.L., Flatt, P.R., 2014. Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, antiviral, immunomodulatory, and anti-diabetic agents. Peptides 57 (7), 67–77. Cornell, H.J., Skerritt, J.H., Puy, R., Javadpour, M., 1994. Studies of in vitro γ-interferon production in coeliac disease as a response to gliadin peptides. Biochim. Biophys. Acta 1226 (2), 126–130. Duarte, J., Vinderola, G., Ritz, B., Perdigón, G., Matar, C., 2006. Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation. Immunobiology 211 (5), 341–350. Fitzgerald, R.J., Murray, B.A., 2010. Bioactive peptides and lactic fermentations. Int. J. Dairy Technol. 59 (2), 118–125. Ghassem, M., Arihara, K., Babji, A.S., Said, M., Ibrahim, S., 2011. Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC-ESI-TOF MS/MS. Food Chem. 129, 1770–1777. Gill, H.S., Doull, F., Rutherfurd, K.J., Cross, M.L., 2000. Immunoregulatory peptides in

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