Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp

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Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp (Hypophthalmichthys molitrix Val.) Proteins Ying Zhang,† Ran Chen,† Xiling Chen,† Zhu Zeng,† Huiqin Ma,‡ and Shangwu Chen*,†,§ †

Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China ‡ College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, People’s Republic of China § Beijing Laboratory for Food Quality and Safety, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China S Supporting Information *

ABSTRACT: The dipeptidyl peptidase IV (DPP-IV)-inhibitory bioactivity of silver carp protein (SCP) hydrolysates were investigated, and their containing efficacious DPP-IV-inhibitory peptides were explored by in silico hydrolysis analysis, peptide separation combined with liquid chromatography−tandem mass spectrometry (LC−MS/MS) identification, and chemical synthesis. SCP hydrolysates generated by six proteases all showed efficient DPP-IV-inhibitory activities, and Neutrase-generated hydrolysates had the greatest DPP-IV inhibition (IC50 of 1.12 mg/mL). In silico Neutrase hydrolysis revealed hundreds of fragments released from myosin, actin, and collagen of SCPs, which include different Pro-motif peptides but only three reported peptidic DPP-IV inhibitors with moderate or weak bioactivity. In addition, three new DPP-IV-inhibitory peptides were identified using LC−MS/MS; in particular, LPIIDI and APGPAGP showed high DPP-IV-inhibitory activity with IC50 of 105.44 and 229.14 μM, respectively, and behaved in competitive/non-competitive mixed-type DPP-IV inhibition mode. The results indicate that the SCP-derived DPP-IV-inhibitory peptides could be potential functional ingredients in the diabetic diet. KEYWORDS: dipeptidyl peptidase IV, silver carp protein, peptide inhibitor, in silico analysis, Pro-motif peptide, LC−MS/MS identification

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INTRODUCTION Diabetes is one of the most prevalent and fastest growing chronic metabolic disorders worldwide, with type 2 diabetes accounting for 90−95% of the cases; the number of people affected by type 2 diabetes is anticipated to reach 366 million by 2030.1,2 Peptidic incretin glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have insulin-tropic and β-cell-proliferative effects, which are considered to be new therapy for the management of type 2 diabetes.1,3 However, GIP and GLP-1 are vulnerable to the cleavage of peptidase dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), resulting in their rather short half-life.1 DPP-IV inhibitors can reduce the DPP-IV activity and increase the lifetime of incretin; many natural-derived dietary proteins, such as milk,4,5 tuna,6 eggs,7 rice,8 and amaranth9 proteins, have been reported to be good sources to produce peptidic DPP-IV inhibitors, which could be released by enzymatic hydrolysis from protein chains. Silver carp (Hypophthalmichthys molitrix Val.) is one of the four major species of freshwater fish in China, with an estimated annual harvest of 3 850 873 tons in 2013.10 Silver carp is rich in proteins but with extremely muddy flavor and many bones, seriously limiting its market values.11,12 To make full use of this abundant protein resource, silver carp proteins (SCPs) could be hydrolyzed with proteolytic enzymes to provide more marketable and value-added fish protein hydrolysate products, which might be conferred nutritional and functional properties beneficial to human health.11,13 Silver © XXXX American Chemical Society

carp protein hydrolysates (SCPHs) generated by protease hydrolysis have been reported to yield peptides with antioxidant bioactivity but lack more functional property research data, which could contribute to accelerating its market application and promotion.11,12 To the best of our knowledge, the DPP-IV-inhibitory potency of SCPHs have not been reported and the containing SCP-derived effective DPP-IVinhibitory sequences also remain unclear and need investigation. Therefore, this work investigated the in vitro DPP-IVinhibitory activities of different SCPHs generated by six commercial proteases and aimed to select the most appropriate protease for preparation of SCPHs enriched in DPP-IVinhibitory peptides, which was further explored and analyzed by virtue of in silico hydrolysis of myosin, actin, and collagen of SCPs. New DPP-IV-inhibitory peptides from SCPHs were also isolated and identified by successive separation techniques combined with nano liquid chromatography−tandem mass spectrometry (LC−MS/MS), and their kinetic characteristics were assayed with synthetic sequences. Received: November 13, 2015 Revised: January 7, 2016 Accepted: January 12, 2016

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DOI: 10.1021/acs.jafc.5b05429 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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measured at 405 nm. Diprotin A was used as a reference inhibitor. The percent DPP-IV inhibition was defined as the percentage of DPP-IV activity inhibited by a given sample. The half maximal inhibitory concentration (IC50) value of the tested sample was determined by the plot of percent DPP-IV inhibition (%) against log10(sample concentration), and Lineweaver−Burk plots were used to visualize the DPP-IV inhibition modes with substrate concentrations ranging from 0.1 to 0.5 mM (assay concentration) in the absence and presence of the inhibitors.14,15 In Silico Neutrase Hydrolysis of SCPs. The three SCP sequences of myosin (A8R0Q7), actin (Q9DF40), and collagen (A0A077B3P8) were obtained from the UniProtKB database16 and conducted for in silico Neutrase hydrolysis to predict the release of peptide sequences. This was performed by the Proteolytic Cleavage tool of Vector NTI Advance software (Thermo Fisher Scientific, Waltham, MA) with peptide bond cleaving at the N terminal of hydrophobic amino acid Leu, Val, Phe, or Ala, as a result of the proteolytic characteristic of commercially available Neutrase from Bacillus subtilis.17 The potential DPP-IV-inhibitory sequences from SCPs were analyzed by comparing the released peptide fragments to previously reported DPP-IV peptidic inhibitors summarized in the BIOPEP database.18 Membrane Ultrafiltration. Membrane molecular-weight cutoff separation was applied for Neutrase-generated SCPH (NSCPH) fractionation. The NSCPHs were first filtered through 0.22 μm syringe membrane filters (Jinteng Corporation, Tianjin, China), and then the filtrate was sequentially ultrafiltered using an Amicon Ultra-15 centrifugal filter device (Millipore Corporation, Bedford, MA) with molecular-weight cutoffs of 10, 5, and 3 kDa. The different-sized ultrafiltration fractions were collected and freeze-dried for further use. Thin-Layer Chromatography (TLC) Fractionation. The fraction displaying the highest DPP-IV-inhibitory activity from ultrafiltration (labeled SF-0, 500 μL at 5 mg/mL peptides) was further separated by TLC on an 100 × 100 × 0.2−0.25 mm ready-use silica gel 60 TLC plate (Haiyang Corporation, Qingdao, China) with chloroform/methanol/25% ammonia (2:2:1, v/v/v) as the developing solvent according to Zhang et al.14 The TLC-developed peptide fractions were scraped according to the retardation factor (Rf) and the ninhydrin-colored spots. Peptides adsorbed on silica powders were recovered by twice extraction with double-distilled H2O (ddH2O). The supernatant of each fraction was adjusted to the initial volume of SF-0 for the DPP-IV inhibition assay. RP-HPLC Analysis and Fractionation. The peptide compositions of SCPHs were analyzed by RP-HPLC using a LC3050 HPLC system (Chuangxin Tongheng Science and Technology Co., Ltd., Beijing, China) according to Zhang et al.14 Briefly, samples were diluted to 0.5% (w/v) in ddH2O, filtered through a 0.22 μm syringe filter, and then injected into a analytical C18 column with 250 × 4.6 mm inner diameter, 5 μm, 100 Å (Chuangxin Tongheng Science and Technology Co., Ltd., Beijing, China). Samples were eluted with 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) as follows: 5% B, followed by a linear gradient from 5 to 80% B from 5 to 40 min, at a total flow rate of 1 mL/min at 30 °C. Eluate was monitored at 215 nm with an ultraviolet−visible (UV−vis) detector. The RP-HPLC separation of the highest DPP-IV-inhibitory fraction from TLC was performed using the same HPLC system as above with a linear increase of 0.1% TFA in acetonitrile (solvent B) from 10 to 50% B from 5 to 45 min in 0.1% TFA in water (solvent A). The peptide peaks with different retention time (tR) were collected, and the separation was repeated multiple times to obtain enough samples for analysis. After removal of acetonitrile by evaporation, each subfraction was adjusted to the initial volume as SF-0 for the DPP-IV inhibition assay. Identification of Peptide Sequences by LC−MS/MS. The peptide subfraction with the highest DPP-IV-inhibitory activity from RP-HPLC separation was subjected to sequence identification by LC− electrospray ionization (ESI)−MS/MS using a nanoAcquity nano HPLC system (Waters, Milford, MA) coupled to a Q-Exactive highresolution mass spectrometer (Thermo Scientific, Waltham, MA) according to the method by Zhang et al.14 The obtained MS/MS data were handled to confirm the amino acid sequence of peptides by

MATERIALS AND METHODS

Materials. Silver carp (H. molitrix Val.), with a mean fresh weight of 1800 g/fish, were purchased from a local market in Beijing, China, and transported live to the laboratory. Papain (from papaya, 8 × 105 units/g of protein) was purchased from Pangbo Biological Engineering Co., Ltd. (Nanning, China). Alcalase 2.4 L (from Bacillus licheniformis, 89 931 units/g of protein) and Flavourzyme (from Aspergillus oryzae, 11 243 units/g of protein) were supplied by Novozymes China, Inc. (Beijing, China). Neutrase (from Bacillus amyloliquefaciens, 35 143 units/g of protein) was supplied by Xindeli Biological Engineering Co., Ltd. (Taian, China). Trypsin (from porcine pancreas, with trypsin ≥ 250 NF units/mg and chymotrypsin ≤ 75 NF units/mg) was from Amresco LLC (Solon, OH). DPP-IV (from porcine kidney, ≥10 units/mg of protein; 1 unit will produce 1.0 μM p-nitroaniline from Gly-Pro-p-nitroanilide per minute in 100 mM Tris−HCl at pH 8.0 and 37 °C), Gly-Pro-pnitroanilide, and pepsin (from porcine gastric mucosa, 18 007 units/g of protein) were from Sigma-Aldrich (St. Louis, MO). Diprotin A (≥95% purity) were prepared by Qiangyao Biological Technology Co., Ltd. (Shanghai, China), and o-phthaldialdehyde (OPA) was from Jinlong Chemical Reagent Co., Ltd. (Beijing, China). Acetonitrile and trifluoroacetic acid (TFA) used for reverse-phase high-performance liquid chromatography (RP-HPLC) were HPLC-grade and from Fisher Scientific (Fair Lawn, NJ). All of the other chemicals were of analytical grade. Preparation of Hydrolysates. Freshly prepared silver carp dorsal muscle mince in distilled water (5%, w/w, protein basis) was boiled for 10 min to inactivate endogenous enzymes, then cooled, and homogenized at 13 000 rpm and 4 °C for 2 min using a homogenizer (FW2000, Fluko Co., Ltd., Shanghai, China). The homogenates were preincubated to the temperature for each proteinase hydrolysis. All reactions were conducted for 6 h at the optimal, static pH and temperature conditions according to manufacturers for each enzyme: trypsin, pH 8.0 and 37 °C; Neutrase, pH 7.5 and 45 °C; Alcalase 2.4 L, pH 8.0 and 60 °C; papain, pH 6.5 and 55 °C at an enzyme/substrate ratio of 3% (w/w, protein base); pepsin, pH 2.0 and 37 °C; and Flavourzyme, pH 7.0 and 37 °C at an enzyme/substrate ratio of 6%. The hydrolysates were sampled at 0.5, 1, 3, and 6 h, heated to 100 °C for 10 min to deactivate enzyme, and then cooled to 4 °C with an ice− water bath. Hydrolysates were centrifuged (TGL-20 M, Pingfan Instrument Co., Ltd., Changsha, China) at 1800 × g and 4 °C for 20 min. The supernatants were adjusted to pH 8.0, lyophilized (LGJ-12, Songyuan Huaxing Technology Development Co., Ltd., Beijing, China), and stored at −20 °C as SCPHs for further analysis. Measurement of the Degree of Hydrolysis (DH). The OPA method was used to measure the DH of SCPHs according to Zhang et al.5 A total of 150 μL of the suitably diluted SCPH sample (or standard) and 3 mL of the OPA reagent were mixed and reacted for 3 min, and then the absorbance was measured at 340 nm. The DH was calculated as

DH (%) =

(N − N0) 1 × 100 C htot

where N and N0 are the concentrations of amino nitrogen in the SCPHs and parent SCPs, respectively (mmol/mL), and N/N0 was calculated according to the linear regression equation N/N0 = d(aA340(SCPHs/SCPs) + b) obtained by the standard L-phenylalanine (0−6 mM), with d being the dilution factor of the sample and a and b being the constants of the linear equation, C is the SCP concentration (g/mL), and htot is the total number of peptide bonds per protein equivalent (htot of SCPs was 8.6 mmol/g). DPP-IV-Inhibitory Activity Assay. DPP-IV-inhibitory activity was measured according to the method by Zhang et al.14 in a 96-well microplate system. Briefly, 25 μL of peptide sample (diluted in 100 mM Tris−HCl buffer at pH 8.0) was mixed with 25 μL of Gly-Pro-pnitroanilide (1.6 mM) and preincubated at 37 °C for 10 min, and then 50 μL of DPP-IV (8 U/L) was added. The reaction was carried out at 37 °C for 60 min and stopped by adding 100 μL of 1 M sodium acetate buffer at pH 4.0. The absorbance of each reaction was B

DOI: 10.1021/acs.jafc.5b05429 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. (A) Degree of hydrolysis, (B) RP-HPLC profiles, and (C) DPP-IV-inhibitory activities of SCPHs produced using different proteases and (D) IC50 value of NSCPHs hydrolyzed for 3 h. DPP-IV inhibition values are measured under the assay concentration of 1.25 mg/mL SCPHs, and the SCPHs for RP-HPLC analysis are the hydrolysates hydrolyzed for 3 h for each enzyme. Bars indicate standard deviations. Values sharing different letters are significantly different (p < 0.05). searching against H. molitrix protein data from the National Center for Biotechnology Information (NCBI) database19 using Mascot 2.4 search engine (Matrix Science, London, U.K.). Peptide Synthesis. Peptides identified by nano LC−MS/MS were synthesized through the conventional 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis method by Leon Biological Technology Co., Ltd. (Nanjing, China) with a purity of ≥95%. Data Analysis. All experiments, except in silico hydrolysis, were carried out in triplicate, and the data were expressed as means with standard deviations (SDs). Tukey’s procedures of SPSS 17.0 (SPSS, Inc., Chicago, IL) were used to identify the significant differences (p < 0.05) between data.

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obviously different DH indicated considerable peptide composition variation of different SCPHs, which was corroborated by their distinct RP-HPLC peptide profiles (Figure 1B). Alcalase 2.4 L, Neutrase, papain, and Flavourzyme that generated SCPHs displayed similar and moderate hydrophilic peptide profiles, with the elution time mainly at 12−24 min, and the peak height distribution range was Alcalase 2.4 L > papain > Neutrase > Flavourzyme (Figure 1B). This suggested the different extents of releasing peptides from the parent SCPs by the four proteases, which also conformed to the DH value order of their generated SCPHs (Figure 1A). In addition, pepsin and trypsin that generated SCPHs contained more hydrophobic peptide peaks at tR of 16−22 min, especially for pepsin, with more narrower and higher peak distribution at tR of 18−22 min (Figure 1B), which might be due to the peptide-bond-cleaving tendency of pepsin at hydrophobic amino acid Phe, Met, Leu, or Tyr.20 Moreover, for trypsin and Flavourzyme, intact SCPs persisted, albeit with smaller peak areas (Figure 1B), suggesting the weaker hydrolysis ability of the two proteases for SCPs than the others, which was also in line with the lowest DH generated by them. The distinct peptide profiles and variable DH values of the six types of SCPHs corroborated their obviously different peptide compositions, which could result from the cleavage specificity of the

RESULTS AND DISCUSSION

DH and DPP-IV-Inhibitory Activity of SCPHs with Different Protease Hydrolysis. To compare the efficacy of the six commercial proteases at releasing DPP-IV-inhibitory peptides from SCPs, hydrolysis was carried out for 6 h at the optimal pH and temperature for each enzyme. As shown in Figure 1A, the DH of all SCPHs increased dramatically after protease hydrolysis: Alcalase 2.4 L resulted in the fastest proteolysis rate and the highest DH of 29.29 ± 0.02% after 6 h of hydrolysis, followed by papain, while Neutrase and trypsin generated an intermediate increase of DH, and the lowest DH were for Flavourzyme and pepsin, with values of 11.89 ± 0.11 and 11.35 ± 0.02%, respectively, after 6 h of hydrolysis. The C

DOI: 10.1021/acs.jafc.5b05429 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

inhibitors.23 Therefore, the above-mentioned three SCPs were selected for in silico Neutrase hydrolysis, and their major protein fragment or type forms included in the UniProtKB protein database are myosin heavy chain of fast skeletal type 3 (MHCFS-3), β-actin (β-AT), and collagen type I α1 (CGαI).16 Virtual Neutrase hydrolysis of the three SCPs released a total of 468, 80, and 253 fragments from MHCFS-3, β-AT, and CGαI, respectively (Supplement Table S1 of the Supporting Information). However, only 1, 1, and 6 fragments corresponding to respective sequences LP, AP, and APG (Table 1) matched previously reported DPP-IV-inhibitory peptides summarized in the bioactive peptide database of BIOPEP.18 However, the three sequences just had moderate or weak DPPIV-inhibitory potency, with IC50 values of 712.5, 7950, and 40 000 μM for LP, AP, and APG, respectively,8,24,25 sharply contrasting with the strong DPP-IV-inhibitory property of NSPCHs, which indicates unidentified efficient sequences among it. Previous reports have shown that the tripeptide sequence in the N terminal of the peptide inhibitor determined its binding to DPP-IV, and the sequences having Pro at position 2 are generally competitive inhibitors of DPP-IV as a result of their substrate-like structures.25,26 As well as LP and AP, VP and FP are also identified as DPP-IV-inhibitory peptides, with IC50 values of 93 and 3630 μM, respectively.8,27 Of those fragments released by in silico Neutrase hydrolysis, there were 4, 8, and 32 peptides with LP, AP, VP, or FP as the N-terminal dipeptide sequences from MHCFS-3, β-AT, and CG-αI, respectively, such as FPK from MHCFS-3, VPIYEGY from β-AT, and LPIIDI from CG-αI (Table 1). These 44 fragments with Pro motif/residue in the second position, especially the sequences with LP and VP at the N terminal, might be probably effective DPP-IV-inhibitory peptides in NSCPHs. In addition, more peptide fragments with other positional Pro motifs were obtained from CG-αI than MHCFS3 and β-AT (Table 1), which may also contribute to CG-αIderived DPP-IV inhibition for the bioactivity of NSCPHs. Overall, we might speculate that myosin is a weak protein source for generation of DPP-IV-inhibitory peptides, even as the premier protein in the silver carp white muscle, while collagen, albeit with a lower percentage in silver carp muscle, is a superior precursor of DPP-IV-inhibitory peptides as a result of its abundant Pro/Hyp content. DPP-IV-Inhibitory Peptide Distribution by Different Fractionations of NSCPHs. Ultrafiltration of NSCPHs into Four DPP-IV-Inhibitory Peptide Fractions. NSCPHs were preliminary separated using membrane filters into four different molecular weight peptide fractions, 10 kDa, taken as 40.15, 11.19, 33.82, and 14.84% of the original weight of NSCPHs, and the DPP-IV-inhibitory activity was found in all fractions, as shown in Table 2. In comparison to the parent NSCPHs, relative lower DPP-IV inhibition was observed for the 5−10 and >10 kDa fractions, while there was no significant difference between 5 kDa) also have noticeable inhibitory bioactivity. Similar phenomena have been reported in many other food proteins, such as milk casein,5 whey protein,4 and salmon skin gelatin21 hydrolysates. The apparent DPP-IVinhibitory activity in the higher molecular mass peptides (>5 kDa) might be attributed to their DPP-IV substrate-like structural features, which could act as substrate analogues to

proteases and possibly lead to apparent differences of SCPHs in DPP-IV-inhibitory activity. Figure 1C showed the DPP-IV-inhibitory activity of the SCPs and associated SCPHs. The intact SCPs showed quite low DPP-IV-inhibitory activity (inhibition value of Alcalase 2.4 L ≥ papsin ≫ Flavourzyme and trypsin (Figure 1C). This suggests the DPP-IV-inhibitory activity of SCPHs is determined by the peptide bond cleavage characteristics of the applied proteinases. The trypsin-genetared SCPHs displayed the weakest DPP-IV inhibition (Figure 1C), differing from the result of trypsin-treated bovine/goat milk casein hydrolysates as the highest inhibitor.5 Although the increased DH might mean more bioactive peptides releasing from proteins for an individual protease, the ultimate and summit of DPP-IV inhibition of hydrolysates was determined by the protein structure, especially the nature of released peptides in different hydrolysates; the DH and DPP-IV inhibition values were not coincidence necessarily. This was in accordance with previous reports for tuna cooking juice and salmon skin gelatin hydrolysates6,21 as well as our investigated bovine and goat casein hydrolysates.5 Neutrase was the most potent enzyme among six proteases for releasing DPP-IV-inhibitory peptides from SCPs, and its generated SCPHs showed quite limited variance in DPP-IVinhibitory activity after 3 h of hydrolysis (Figure 1C). This suggested that 3 h could be the optimal hydrolysis time for Neutrase to produce DPP-IV-inhibitory hydrolysates (NSCPHs), which displayed an IC50 value of 1.12 ± 0.04 mg/mL (Figure 1D). In addition, no significantly different (p ≥ 0.05) DPP-IV-inhibitory activity was observed for NSCPHs after simulated gastrointestinal digestion (data not shown), although with an apparent increment of the DH value from 18.04 ± 0.29 to 24.03 ± 0.13%, suggesting that NSCPHs have good function stability to pepsin and pancreatin proteolytic digestion. On the whole, these results initially indicate that NSCPHs could be potential functional ingredients with DPPIV-inhibitory properties for the management of type 2 diabetes. In Silico Neutrase Hydrolysis of SCPs To Predict Potential DPP-IV-Inhibitory Sequences. To understand NSCPHs as a new resource of food-derived DPP-IV-inhibitory functional peptides, the main protein components of SCPs were searched by in silico hydrolysis, to guide in depth the research on NSCPHs. Silver carp white muscle consists of various categories of proteins, of which myosin and actin are two principal proteins accounting for up to 20−35 and 10− 14% of the total SCPs, respectively, and another protein, collagen, contains 20−30% proportion of Pro/Hyp within its primary sequence, although with just an estimated ∼5% amount in the SCPs.22 The structure−bioactivity relationship of DPP-IV-inhibitory peptides has not yet been fully elucidated, while many peptides containing Pro as the first, second, third, or fourth N-terminal amino acid residue, preferably in the penultimate position, have been shown to act as good DPP-IV D

DOI: 10.1021/acs.jafc.5b05429 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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All three mature proteins were obtained from the UniProtKB database,16 and their ID number refers to the accession number of protein in this database. Peptide sequences refer to the released partial peptide fragments containing Pro residue by in silico Neutrase hydrolysis of three SCPs, and peptides in bold are DPP-IV inhibitors reported in the literature, while sequences underlined are peptides with Pro at the second position of the N terminus.

competitively interfere with the combination of the synthetic substrate and DPP-IV.4 In addition, although the