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Shrimp (Pandalopsis dispar) waste hydrolysate as a source of novel β–secretase inhibitors

Shrimp (Pandalopsis dispar) waste hydrolysate as a source of novel β–secretase inhibitors In this study, purified peptides from shrimp waste hydrolysates (SWHs) were examined for their inhibitory effects against β–secretase. During consecutive purification using a Sephadex G–25 column chromatography and high performance liquid chromatography on a C18 column, a potent β–secretase inhibitory peptide Asp–Val–Leu–Phe– His (629 Da) was isolated and identified from SWH24 by Q–TOF MS/MS and the IC value was determined to be 92.70 μM. The β–secretase inhibition patterns of the purified peptides were found to be competitive. Among synthesized β–secretase inhibitory peptides, Leu–Phe–His had higher β–secretase inhibitory activity than the others. The result of this study suggests that the β–secretase inhibitory peptide derived from SWH24 could be potential candidates to develop nutraceuticals and pharmaceuticals. Keywords: Alzheimer’s disease, β–secretase inhibitory activity, Shrimp waste hydrolysates, Peptide Background drugs that can inhibit β–secretase and thereby reduce Aβ levels Successful public health policies and socioeconomic develop- as a therapeutic treatment for AD. High–throughput screen- ment have resulted in increasing number of elderly population ing of compound collections and natural product extracts, globally, which are accompanied by challenges to address vari- together with drug design building on structure–activity rela- ous health issues of an aging society. The World Health tionships, have led to the discovery and development of both Organization reported in 2012 that 35.6 million people world- peptide and non–peptide inhibitors of the enzyme. These in- wide are living with dementia or Alzheimer's disease (AD), and clude compounds such as the peptidic β–secretase inhibitor that this number will triple to 115.4 million by 2050 (World OM99–1, other aspartic protease inhibitors, an eight–residue Health Organization and Alzheimer’s Disease International, transition state inhibitor OM99–2 (Chen et al. 1995), and a 2012). Much of AD research has been focused on the amyloid more potent eight–residue transition state inhibitor OM00–3 cascade hypothesis, which states that amyloid beta (Aβ), a pro- (Turner et al. 2001). Chitosan derivatives from crab shell ex- teolytic derivative of the large trans–membrane protein amyl- hibited weak β–secretase inhibition (Byun et al. 2005), while oid precursor protein (APP), plays an early and crucial role in catechins from green tea, ellagic acid from pomegranate all cases of AD. Consequently, blocking the production of Aβ (Kwak et al. 2005), hispidin from mycelial cultures of Phellinus by specific inhibition of the β–secretase required for Aβ gener- linteus (Park et al. 2004), and several compounds isolated from ation is a major focus of research into AD therapy (Citron Sanguisorbae radix (Lee et al. 2011) have also all been studied 2004). β–secretase (EC 3.4.23.46), an aspartic peptidase also as β–secretase inhibitors. Moreover, several hydroxyl–con- knownasmemapsin2andBACE1,isthe firstproteasethat taining inhibitors have been reported (Cumming et al. 2004). processes APP in the pathway leading to Aβ production. Ex- Shrimp have a high market value but the remaining cessive levels of Aβ in the brain are closely related to AD patho- heads and shells, which account for half of the shrimp genesis, so much research has been focused on developing weight, are typically removed after processing (Cheung et al. 2012). Many studies have examined the potential use of these underutilized materials, termed shrimp * Correspondence: hgbyun@gwnu.ac.kr processing by–products, for functional properties that are Department of Marine Biotechnology, Gangneung–Wonju National University, Gangneung 210-702, Republic of Korea applicable to industries such as pharmaceutical, functional Full list of author information is available at the end of the article © 2016 Li-Chan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 2 of 7 food, and nutraceuticals (Dey and Dora 2014; Cheung and stored at −80 °C until use. The SWHs was provided Li–Chan 2010). from Li–Chan’s laboratory in UBC, Canada. Functional peptides can be produced from enzymatic hy- drolysis of various bio–resource proteins. Bioactive peptides Measurement of β–secretase inhibitory activity are usually 3–10 amino acid residue chains whose activity β–Secretase inhibitory activity was measured following is based on their amino acid composition and sequence Johnston et al. (2008), using a commercially available (Stachel et al. 2004), and whose functions include regulatory fluorogenic substrate, MCA–EVKMDAEFK–(DNP)– effects related to nutrient uptake, immune defense (Chen NH . This substrate corresponded to the wild–type et al. 1995), and antioxidant activity (Mendis et al. 2005). APP sequence, derivatised at its N–terminus with a Moreover, some peptides can influence higher brain func- fluorescent 7–methoxycoumarin–4–yl acetyl (MCA) tions, such as learning and memory, in humans and animals group, and on its C–terminal lysine residue with a (Mclay et al. 2001). However, there is a paucity of informa- 2,4–dinitrophenyl (DNP) group. In the intact peptide tion on bioactive peptides from food–derived products, the fluorescence of the MCA group was abolished by which may have potential to serve as β–secretase inhibitors. internal quenching from the DNP group. Upon cleav- The objective of this study was to isolate and characterize age by β–secretase activity the MCA fluorescence could β–secretase inhibitory peptides purified from shrimp waste be detected. Assays were performed in 96–well black hydrolysates, and to elucidate the active component pep- plates using a Spectrofluorometer (Molecular Devices). tide(s) and the mode of inhibition of β–secretase. β–secretase and β–secretase substrate I were incubated in a final volume of 200 μl in assay buffer (50 mM Methods sodium acetate, pH 4.5). The hydrolysis of β–secretase Materials substrate I was followed at 37 °C for 30 min, by Shrimp processing by–products (including shells, heads measuring the accompanying increase in fluorescence. and tails recovered from hand–peeling of cooked shrimp Readings (excitation 325 nm, emission 393 nm) were Pandalopsis dispar) in frozen form were donated by Albion taken every 60s. The inhibition ratio was obtained by Fisheries Ltd. (Vancouver, BC, Canada). Shrimp wastes the following equation: Inhibition (%) = [1– {(S–S0)/ hydrolysis was performed under experimental conditions (C–C0)} × 100], where C is the fluorescence of a con- according to Cheung and Li-Chan (2010). Protamex® trol (enzyme, assay buffer, and substrate) after 60 min (Bacillus amyloliquefaciens and Bacillus licheniformis, of incubation, C0 is the fluorescence of control at zero 1.5 AU/g), a product from Novozymes North America Inc. time, S is the fluorescence of tested samples (enzyme, (Salem, NC), was donated by Neova Technologies Inc. sample solution, and substrate) after 60 min of incuba- (Abbotsford, BC, Canada). β–secretase and MCA– tion, and S0 is the fluorescence of the tested sample at EVKMDAEFK–(DNP)–NH (β–secretase substrate I) zero time. All data are the means of triplicate was purchased from Sigma Chemical Co. (St. Louis, experiments. MO). All other reagents used in this study were reagent grade chemicals. Purification of β–secretase inhibitory peptide Preparation of shrimp waste hydrolysates(SWHs) The potent fraction as determined from β–secretase SWHs were prepared using Protamex enzyme for hydro- inhibitory activity assay was further purified by size lysis of the shrimp waste under varying conditions of exclusion chromatography on a Sephadex G–25 gel water:substrate ratio (1:1, 1:1.5, 1:2 or 1:2.5), percent en- filtration column (25 × 750 mm) equilibrated with dis- zyme (2, 4, 6 or 8 % w/w protein contents of shrimp tilled water. Separated fractions were monitored at processing by–products) and time of hydrolysis (1, 4, 8 215 nm, collected at a volume of 7.5 ml and mea- or 24 h) (Table 1). The lyophilized hydrolysates were sured for β–secretase inhibitory activity. The most ac- tive fraction was then injected into a preparative Table 1 The conditions for hydrolysis of shrimp processing by– reversed phase HPLC column (YMC, ODS C18, 10.0 × products 250 mm, 5 μm) and separated using a linear gradient of Sample Hydrolysis conditions acetonitrile (0–40 % v/v) containing 0.1 % trifluoroace- Protease W:S Enzyme(%) Time(h) pH tic acid (TFA) on an HPLC system (Agilent Technolo- gies, USA). The peak showing potent inhibitory activity SWH1 Protamex 1.5:1 4 1 8.4 was finally purified into a single peptide on a reversed SWH4 Protamex 2.5:1 6 4 8.3 phase HPLC analytical C18 column (4.6 × 250 mm, SWH8 Protamex 2:1 2 8 8.3 5 μm) using a linear gradient of acetonitrile (0–20 % v/ SWH24 Protamex 1:1 8 24 8.0 v) in 0.1 % TFA. Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 3 of 7 Amino acid sequence of purified peptide Statistical analysis To identify molecular weight and amino acid sequence Each experiment was performed at least three times and of the purified peptide, all MS/MS experiments were results were presented as the mean ± SD. Statistical com- performed on a Q–TOF tandem mass spectrometer parisons of the mean values were performed by analysis (Micromass Co., Manchester, UK) equipped with a of one–way ANOVA (SPSS 12, IBM, IL, Chicago, USA), nano–ESI source. The peptide solution was desalted followed by Duncan’s multiple–range test using SPSS using Capcell Pak C18 UG120 V (4.6 × 250 mm, (12) software. Differences were considered significant at 5 μm, Shiseido, Tokyo, Japan). The purified peptide dis- p < 0.05. solved in methanol/water (1:1, v/v) was infused into the ESI source and molecular weight was determined by 2+ Results and discussions doubly charged (M + 2H) state in the mass spectrum. β–secretase inhibitory activity of SWHs Following molecular weight determination, peptide was β–secretase inhibitory activity was measured using an automatically selected for fragmentation and sequence assay that we developed and validated using a commer- information was obtained by tandem MS analysis. cially available fluorogenic substrate. Figure 1a shows the kinetics of β–secretase inhibitory activity from Determination of β–secretase inhibition pattern SWHs and a control. A fluorescent signal (in relative For the Lineweaver-burk plot, the data were plotted as fluorescence units) was found over 0–60 min with hy- mean values of 1/v, the inverse of the increase in fluores- drolysates (or buffer) at 0.5 mg/ml and 10 mM β–secre- cence intensity per min (min/DRFU) of three independent tase substrate I. The fluorescence of β–secretase tests with different concentrations of fluorescent substrate. incubated in the absence of membrane protein was sub- The assay was performed in the presence of purified tracted at each time point. There appeared to be a linear peptide (final concentration of 0, 25, 50 and 100 μg/ml). increase in signal beginning after 5 min that continued for up to 1 h. Among hydrolysates, SWH24 was particu- larly potent. As seen in Fig. 1b, the lowest IC value Synthesis of β‑secretase inhibitory peptides was exhibited by SWH24 at 0.54 mg/ml. These discrep- The peptides were chemically synthesized in the peptide ancies may be attributed to differences in substrate spe- synthesis facility, PepTron Inc. (Daejeon, Korea). The cificity and conditions for optimal activity of the enzyme peptides were synthesized using the Fmoc–solid phase preparations, as well as to differing peptide sequences method with a peptide synthesizer (PeptrEX–R48, Peptron and structural factors affecting reactivity of the protein Inc., Daejeon, Korea). These synthetic peptides were substrates. The findings underline the importance of purified by RP–HPLC using a Capcell Pak C18 column selecting the appropriate combination of experimental (Shiseido, Japan). Elution was performed with a water– conditions to release bioactive peptide sequences acetonitrile linear gradient (0–80 % of acetonitrile) contain- (Cheung and Li–chan 2010). Protamex hydrolysates has ing 0.1 % (v/v) TFA. Elution was monitored at 220 nm on previously been reported to be effective in various bio- HPLC instrument (Prominence HPLC, Shimadzu, Tokyo, activity such as producing potent ACE inhibitory pep- Japan). tides from marine source (He et al. 2007). ab 3500 2.5 Control SWH 1 2.0 SWH 4 2500 SWH 8 SWH 24 1.5 1.0 1000 d 0.5 0.0 0 1000 2000 3000 4000 SWH1 SWH4 SWH8 SWH24 Time (s) Sample Fig. 1 Effect of shrimp waste hydrolysates (SWHs) on the inhibitory activity of β–secretase. a Relative fluorescence unit of SWHs under 10 mM β–secretase substrate I was incubated with hydrolysates (0.5 mg/ml) or buffer (control). b IC value (mg/ml) of SWHs. Letters indicate significantly (P < 0.05) different averages (ANOVA, Duncan’stest) Relative fluorescence units IC value (mg/ml) 50 Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 4 of 7 Purification of β–secretase inhibitory peptide of 1.0 ml/min (Fig. 2–IV). The IC value of this purified The use of Sephadex G–25 chromatography led to a peptide was 58.31 μg/ml. The β-secretase inhibitory ac- fraction with greatly improved β–secretase inhibitory ac- tivity of purified peptide was increased by 10.52-fold tivity (Fig. 2–I). First, fraction D from SWH24 had the compared to the SWH24 (0.54mg/ml), using the four highest β–secretase inhibitory activity, with an IC step purification procedure. value of 0.19 mg/ml. The lyophilized fraction D was fur- ther separated into eight sub–fractions by HPLC on an Identification of β–secretase inhibitory peptide ODS column with a linear gradient of acetonitrile (0– The amino acid sequence of the purified β–secretase 50 %) (Fig. 2–II). Finally, the purified fraction B2 was inhibitory peptide were identified using MS/MS. For found to have the highest β–secretase inhibitory activity SWH24, the sequence was found to be Asp–Val–Leu– (Fig. 2–III). The active fraction B2 was subjected to re– Phe–His (629 Da) for fraction B2, with an IC value of chromatography on the HPLC column using a isocratic 92.70 μM (Fig. 3). The amino acid sequence of this elution with 22.5 % acetonitrile for 30 min, at a flow rate peptide is critical in its β–secretase inhibitory activity. Fig. 2 Purification steps of β–secretase inhibitory peptide from SWH24 by Sephadex G–25 column chromatography and HPLC. I Sephadex G–25 Gel filtration chromatogram of hydrolysates prepared with SWH24 (b, lower layer). Separation was performed with 1.5 ml/min and collected at a fraction volume of 7.5 ml. The fractions isolated by Sephadex G–25 Gel column were separated (A ~ D) and β–secretase activity determined as upper panel (a). II,III,IV HPLC chromatogram of potent β–secretase inhibitory activity of separated fraction from previous step. Separation was performed with linear gradient of acetonitrile at a flow rate of 1.0 ml/min and Grom–sil 120 ODS–5 ST column (5 μm, 10 × 250 mm). Elution was monitored at 215 nm (b, lower layer). The fractions showing β–secretase inhibitory activity were determinded IC (mg/ml) as shown in upper layer (a) Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 5 of 7 520 Max. 503.4 cps. 514.25 O O O H H H N N N H N N OH H H HO O O D L H V F 194.13 629.30 95.07 415.19 184.14 77.06 176.12 -18.01 -87.99 54.0785.08 166.13 302.11 -187.05 -63.00 -144.05 100 150 200 250 300 350 400 450 500 550 600 650 Mass, Da Fig. 3 Identification of molecular weight and amino acid sequence of the purified peptide from SWH24 by HPLC. MS/MS experiments were performed on a Q–TOF tandem mass spectrometer equipped with a nano–ESI source Kimura et al. (2010) investigated the synthetic β–secretase aspartic acid groups (Bursavich and Rich 2002). Potent inhibitor, KMI–370 (IC value = 3.4 nM), whose activity transition state–mimetic β–secretase inhibitors have was greater than that of the purified peptide (IC value been reported by several groups, and the area has been =92.70 μM). Because its molecular weight was much reviewed recently (Hong et al. 2005). OM99–2, a syn- smaller than those of the others, it was considered suitable thesized peptidyl inhibitor of human brain β–secretase for absorption in the intestine. Lee et al. (2007) found that (Hong et al. 2005), was utilized to learn the interactions the amino acid sequence of a purified β–secretase inhibi- of the β–secretase active site. The inhibitor was bound tor peptide from Saccharomyces cerevisiae was Gly–Pro– in the substrate–binding cleft located between the Leu–Gly–Pro–Ile–Gly–Ser with N–terminal sequence analysis. The molecular weight of the purified β–secretase 1.0 inhibitor was estimated to be 697 Da by LC–MS, and its Control β–secretase inhibitory activity IC value was 2.59 μM. In g/ml spite of having the highest inhibition efficiency, they re- 0.8 50 g/ml ported that this octapeptide needs a reduced molecular 25 g/ml weight to overcome metabolic instability. The purified β– 0.6 secretase inhibitory peptide acted competitively with a substrate according to the Lineweaver–burk plots (Fig. 4). This strongly suggests that the purified peptide might have 0.4 an affinity for the active site of an enzyme where the sub- strate also binds; the substrate and inhibitor compete for 0.2 access to the enzyme's active site. Derivatives of these pep- tides are expected to be useful in the prevention of AD 0.0 through the development of novel peptidic inhibitors. -0.1 0.0 0.1 0.2 0.3 0.4 0.5 Availability of protein/ligand structures has opened up the 1/[S] possibility of structure–based design of β–secretase inhib- itors. Prototypical aspartic acid protease inhibitors are Fig. 4 Lineweaver–burk plots for determining inhibition pattern of the purified inhibitor against β–secretase. The intersection of the peptides of high molecular weight, and contain a sec- three lines on the vertical axis signified that the purified β–secretase ondary alcohol that acts as a transition-state mimetic inhibitor was a competitive inhibitor via the formation of hydrogen bonds with the catalytic Intensity, cps 1/[V] Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 6 of 7 N– and C–terminal lobes. Six of the eight OM99–2 report on the β–secretase inhibiting activity of marine or- residues (P4 ~ P'2) are bound in the active site of β– ganisms. These isolated and synthesized peptides from secretase in an extended structure and their respective SWH24 could be useful in the study of the mechanisms of binding sites (S4 ~ S'2) are well–designated by atomic Alzheimer’s disease. contacts with the inhibitor side. Conclusion β–secretase inhibitory activity of synthetic peptides In conclusion, the hydrolysate of shrimp waste protein The peptide Asp–Val–Leu–Phe–His was purified from generated by proteinases treatment followed by consecu- SWH24. Based on it, five synthetic peptides were tive purification of gel filtration and reversed-phase HPLC prepared in order to study their β–secretase inhibitory resulted in a novel β-secretase inhibitory peptide of activity relative to their amino acid sequences. They DVLFH. The purified peptide acted as a competitive in- were further purified using a reversed–phase HPLC. hibitor against β-secretase with an IC value of 92.70 μM The resulting IC values of the synthetic peptides are 50 and molecular weight of 629 Da. We were synthesized shown in Table 2. Among the synthetic peptides, the novel β-secretase inhibitory peptide base on amino acid IC value of Leu–Phe–His was 34.11 μM. Moreover, 50 sequences of DVLFH. Among the synthesized peptides, the IC values of the synthetic peptides were improved 50 LFH had higher β–secretase inhibitory activity the other over the original peptide isolated from SWH24 (Asp– synthesized peptides. Our present results proposed that Val–Leu–Phe–His, IC , 92.70 μM). Synthesized Leu– 50 the β–secretase inhibitory peptides derived shrimp waste Phe–His acted competitively according to the Linewea- protein could be used as nutraceutical ingredients and alz- ver–Burk plot (data not shown). In both peptides (Asp– heimer’s disease medicine. The manufacturing of hydroly- Val–Leu–Phe–His and Leu–Phe–His), leucine is likely to sates and peptides loaded with bioactive peptide-rich be the important residue for β–secretase inhibition. In the protein from shrimp by–products could be a new possibil- β–secretase inhibitory mechanism, leucine plays an im- ity for functional foods. portant role in the Swedish mutant APP, which has a mu- Competing interests tation at the P2–P1 positions from Lys–Met to Asn–Leu. The authors declare that they have no competing interests. Generally, β–secretase has eight (P1 ~ P4 and P1' ~ P4') residues that are important in the catalytic domain, deter- Authors’ contributions mined by its crystal structure. Inhibitory activities against HGB and ECYL conceived and designed the study. IWYC prepared the samples and assisted with data collection. HGB performed the β–secretase when the P2 position was changed to several experiments, analyzed the data, and drafted the manuscript. All authors other amino acids have been described (Hong et al. 2005). read and approved the final manuscript. In the case of hydrophilic amino acids (Asp, Asn, Glu, and Gln) in the P2 position, the inhibitory activities were weak Acknowledgement This study was supported by Gangneung–Wonju National University. (β–secretase inhibitory activity of 25–36 %). However, with hydrophobic amino acids like leucine in the P2 pos- Author details ition, significant inhibitory activity was present (β–secre- Food, Nutrition & Health Program, Faculty of Land & Food Systems, The University of British Columbia, 2205 East Mall, Vancouver, BC V6T 1Z4, tase inhibitory activity of >90 %). These results suggested Canada. Department of Marine Biotechnology, Gangneung–Wonju National that a hydrophobic interaction at the P2 site of β–secre- University, Gangneung 210-702, Republic of Korea. tase was more effective than a hydrophilic one, in spite of Received: 4 January 2016 Accepted: 28 January 2016 the hydrophilic property of the P2 site. Leucine was employed as the P2 moiety for the synthetic β–secretase inhibitor. The isolated and synthesized peptides may not References be directly considered as potential drug candidates, since Bursavich MG, Rich DH. Designing non–peptide peptidomimetics in the 21st century: inhibitors targeting conformational ensembles. J Med Chem. 2002; they have relatively groups. However, this is the first 45:541–58. Byun HG, Kim YT, Park PJ, Lin X, Kim SK. Chitooligosaccharides as a novel β– secretase inhibitor. Carbohyd Polym. 2005;61:198–202. Table 2 β–secretase inhibitory activity of synthesized peptides Chen J, Suetsuna K, Yamauchi F. Isolation and characterization of Synthesized Peptide IC value (μM) immunostimulative peptides from soybean. J Nutr Biochem. 1995;6:310–3. a Cheung IWY, Li–Chan ECY. Angiotensin–I–converting enzyme inhibitory activity Asp–Val–Leu–Phe–His 101.54 ± 11.54 and bitterness of enzymatically–produced hydrolysates of shrimp Asp–Val–Leu 41.93 ± 4.14 (Pandalopsis dispar) processing byproducts investigated by Taguchi design. Food Chem. 2010;122:1003–12. Asp–Val 67.46 ± 7.83 Cheung LKY, Cheung IWY, Li–Chan ECY. Effects of production factors and egg– Leu–Phe–His 34.11 ± 9.01 bearing period on the antioxidant activity of enzymatic hydrolysates from shrimp (Pandalopsis dispar) processing byproducts. J Agric Food Chem. 2012; Phe–His 104.76 ± 8.67 60:6823–31. a-c , Letters indicate significantly (P < 0.05) different averages (ANOVA, Citron M. Beta–secretase inhibition for the treatment of Alzheimer's disease– Duncan’s test) promise and challenge. Trends Pharmacol Sci. 2004;25:92–7. Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 7 of 7 Cumming JN, Iserloh U, Kennedy ME. Design and development of β–secretase inhibitors. Curr Opin Drug Disc Devel. 2004;7:536–56. Dey SS, Dora KC. Antioxidative activity of protein hydrolysate produced by alcalase hydrolysis from shrimp waste (Penaeus monodon and Penaeus indicus). J Food Sci Tech. 2014;51:449–57. He HL, Chen XL, Wu H, Sun CY, Zhang ZY, Zhou BC. High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin–I–converting enzyme inhibitory activity by capillary electrophoresis. Bioresource Technol. 2007;98:3499–505. Hong L, He X, Huang X, Chang W, Tang J. Structural features of human memapsin 2 (beta–secretase) and their biological and pathological implications. Acta Biochim Biophys Sin. 2005;36:787–92. Johnston JA, Liu WW, Coulson DTR, Todd S, Murphy S, Brennan S. Platelet β–secretase activity is increased in Alzheimer’ disease. Neurobiol Aging. 2008;29:661–8. Kimura R, Devi L, Ohno M. Partial reduction of BACE1 improves synaptic plasticity, recent and remote memories in Alzheimer’s disease transgenic mice. J Neurochem. 2010;113:248–61. Kwak HM, Jeon SY, Song BH, Kim JG, Lee JM, Lee KB, Jeong HH, Hur J M, Kang YH, Song KS. β–Secretase (BACE1) inhibitors from pomegranate (Punica granatum) husk. Arch Pharm Res. 2005;28:1328–32. Lee DH, Lee DH, Lee JS. Characterization of a new antidementia β–secretase inhibitory peptide from Saccharomyces cerevisiae. Enzyme Microb Tech. 2007; 42:83–8. Lee HJ, Seong YH, Bae KH, Kwon SH, Kwak HM, Nho SK, Kim KA, Hur JM, Lee KB, Kang YH, Song KS. β–secretase (BACE1) inhibitors from Sanguisorbae radix. Arch Pharm Res. 2011;28:799–803. McLay RN, Pan W, Kastin AJ. Effects of peptides on animal and human behavior. Peptides. 2001;22:2181–255. Mendis E, Rajapakse N, Kim SK. Antioxidant properties of a radicals scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate. 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Shrimp (Pandalopsis dispar) waste hydrolysate as a source of novel β–secretase inhibitors

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Springer Journals
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Copyright © 2016 by Li-Chan et al.
Subject
Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Animal Ecology
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Abstract

In this study, purified peptides from shrimp waste hydrolysates (SWHs) were examined for their inhibitory effects against β–secretase. During consecutive purification using a Sephadex G–25 column chromatography and high performance liquid chromatography on a C18 column, a potent β–secretase inhibitory peptide Asp–Val–Leu–Phe– His (629 Da) was isolated and identified from SWH24 by Q–TOF MS/MS and the IC value was determined to be 92.70 μM. The β–secretase inhibition patterns of the purified peptides were found to be competitive. Among synthesized β–secretase inhibitory peptides, Leu–Phe–His had higher β–secretase inhibitory activity than the others. The result of this study suggests that the β–secretase inhibitory peptide derived from SWH24 could be potential candidates to develop nutraceuticals and pharmaceuticals. Keywords: Alzheimer’s disease, β–secretase inhibitory activity, Shrimp waste hydrolysates, Peptide Background drugs that can inhibit β–secretase and thereby reduce Aβ levels Successful public health policies and socioeconomic develop- as a therapeutic treatment for AD. High–throughput screen- ment have resulted in increasing number of elderly population ing of compound collections and natural product extracts, globally, which are accompanied by challenges to address vari- together with drug design building on structure–activity rela- ous health issues of an aging society. The World Health tionships, have led to the discovery and development of both Organization reported in 2012 that 35.6 million people world- peptide and non–peptide inhibitors of the enzyme. These in- wide are living with dementia or Alzheimer's disease (AD), and clude compounds such as the peptidic β–secretase inhibitor that this number will triple to 115.4 million by 2050 (World OM99–1, other aspartic protease inhibitors, an eight–residue Health Organization and Alzheimer’s Disease International, transition state inhibitor OM99–2 (Chen et al. 1995), and a 2012). Much of AD research has been focused on the amyloid more potent eight–residue transition state inhibitor OM00–3 cascade hypothesis, which states that amyloid beta (Aβ), a pro- (Turner et al. 2001). Chitosan derivatives from crab shell ex- teolytic derivative of the large trans–membrane protein amyl- hibited weak β–secretase inhibition (Byun et al. 2005), while oid precursor protein (APP), plays an early and crucial role in catechins from green tea, ellagic acid from pomegranate all cases of AD. Consequently, blocking the production of Aβ (Kwak et al. 2005), hispidin from mycelial cultures of Phellinus by specific inhibition of the β–secretase required for Aβ gener- linteus (Park et al. 2004), and several compounds isolated from ation is a major focus of research into AD therapy (Citron Sanguisorbae radix (Lee et al. 2011) have also all been studied 2004). β–secretase (EC 3.4.23.46), an aspartic peptidase also as β–secretase inhibitors. Moreover, several hydroxyl–con- knownasmemapsin2andBACE1,isthe firstproteasethat taining inhibitors have been reported (Cumming et al. 2004). processes APP in the pathway leading to Aβ production. Ex- Shrimp have a high market value but the remaining cessive levels of Aβ in the brain are closely related to AD patho- heads and shells, which account for half of the shrimp genesis, so much research has been focused on developing weight, are typically removed after processing (Cheung et al. 2012). Many studies have examined the potential use of these underutilized materials, termed shrimp * Correspondence: hgbyun@gwnu.ac.kr processing by–products, for functional properties that are Department of Marine Biotechnology, Gangneung–Wonju National University, Gangneung 210-702, Republic of Korea applicable to industries such as pharmaceutical, functional Full list of author information is available at the end of the article © 2016 Li-Chan et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 2 of 7 food, and nutraceuticals (Dey and Dora 2014; Cheung and stored at −80 °C until use. The SWHs was provided Li–Chan 2010). from Li–Chan’s laboratory in UBC, Canada. Functional peptides can be produced from enzymatic hy- drolysis of various bio–resource proteins. Bioactive peptides Measurement of β–secretase inhibitory activity are usually 3–10 amino acid residue chains whose activity β–Secretase inhibitory activity was measured following is based on their amino acid composition and sequence Johnston et al. (2008), using a commercially available (Stachel et al. 2004), and whose functions include regulatory fluorogenic substrate, MCA–EVKMDAEFK–(DNP)– effects related to nutrient uptake, immune defense (Chen NH . This substrate corresponded to the wild–type et al. 1995), and antioxidant activity (Mendis et al. 2005). APP sequence, derivatised at its N–terminus with a Moreover, some peptides can influence higher brain func- fluorescent 7–methoxycoumarin–4–yl acetyl (MCA) tions, such as learning and memory, in humans and animals group, and on its C–terminal lysine residue with a (Mclay et al. 2001). However, there is a paucity of informa- 2,4–dinitrophenyl (DNP) group. In the intact peptide tion on bioactive peptides from food–derived products, the fluorescence of the MCA group was abolished by which may have potential to serve as β–secretase inhibitors. internal quenching from the DNP group. Upon cleav- The objective of this study was to isolate and characterize age by β–secretase activity the MCA fluorescence could β–secretase inhibitory peptides purified from shrimp waste be detected. Assays were performed in 96–well black hydrolysates, and to elucidate the active component pep- plates using a Spectrofluorometer (Molecular Devices). tide(s) and the mode of inhibition of β–secretase. β–secretase and β–secretase substrate I were incubated in a final volume of 200 μl in assay buffer (50 mM Methods sodium acetate, pH 4.5). The hydrolysis of β–secretase Materials substrate I was followed at 37 °C for 30 min, by Shrimp processing by–products (including shells, heads measuring the accompanying increase in fluorescence. and tails recovered from hand–peeling of cooked shrimp Readings (excitation 325 nm, emission 393 nm) were Pandalopsis dispar) in frozen form were donated by Albion taken every 60s. The inhibition ratio was obtained by Fisheries Ltd. (Vancouver, BC, Canada). Shrimp wastes the following equation: Inhibition (%) = [1– {(S–S0)/ hydrolysis was performed under experimental conditions (C–C0)} × 100], where C is the fluorescence of a con- according to Cheung and Li-Chan (2010). Protamex® trol (enzyme, assay buffer, and substrate) after 60 min (Bacillus amyloliquefaciens and Bacillus licheniformis, of incubation, C0 is the fluorescence of control at zero 1.5 AU/g), a product from Novozymes North America Inc. time, S is the fluorescence of tested samples (enzyme, (Salem, NC), was donated by Neova Technologies Inc. sample solution, and substrate) after 60 min of incuba- (Abbotsford, BC, Canada). β–secretase and MCA– tion, and S0 is the fluorescence of the tested sample at EVKMDAEFK–(DNP)–NH (β–secretase substrate I) zero time. All data are the means of triplicate was purchased from Sigma Chemical Co. (St. Louis, experiments. MO). All other reagents used in this study were reagent grade chemicals. Purification of β–secretase inhibitory peptide Preparation of shrimp waste hydrolysates(SWHs) The potent fraction as determined from β–secretase SWHs were prepared using Protamex enzyme for hydro- inhibitory activity assay was further purified by size lysis of the shrimp waste under varying conditions of exclusion chromatography on a Sephadex G–25 gel water:substrate ratio (1:1, 1:1.5, 1:2 or 1:2.5), percent en- filtration column (25 × 750 mm) equilibrated with dis- zyme (2, 4, 6 or 8 % w/w protein contents of shrimp tilled water. Separated fractions were monitored at processing by–products) and time of hydrolysis (1, 4, 8 215 nm, collected at a volume of 7.5 ml and mea- or 24 h) (Table 1). The lyophilized hydrolysates were sured for β–secretase inhibitory activity. The most ac- tive fraction was then injected into a preparative Table 1 The conditions for hydrolysis of shrimp processing by– reversed phase HPLC column (YMC, ODS C18, 10.0 × products 250 mm, 5 μm) and separated using a linear gradient of Sample Hydrolysis conditions acetonitrile (0–40 % v/v) containing 0.1 % trifluoroace- Protease W:S Enzyme(%) Time(h) pH tic acid (TFA) on an HPLC system (Agilent Technolo- gies, USA). The peak showing potent inhibitory activity SWH1 Protamex 1.5:1 4 1 8.4 was finally purified into a single peptide on a reversed SWH4 Protamex 2.5:1 6 4 8.3 phase HPLC analytical C18 column (4.6 × 250 mm, SWH8 Protamex 2:1 2 8 8.3 5 μm) using a linear gradient of acetonitrile (0–20 % v/ SWH24 Protamex 1:1 8 24 8.0 v) in 0.1 % TFA. Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 3 of 7 Amino acid sequence of purified peptide Statistical analysis To identify molecular weight and amino acid sequence Each experiment was performed at least three times and of the purified peptide, all MS/MS experiments were results were presented as the mean ± SD. Statistical com- performed on a Q–TOF tandem mass spectrometer parisons of the mean values were performed by analysis (Micromass Co., Manchester, UK) equipped with a of one–way ANOVA (SPSS 12, IBM, IL, Chicago, USA), nano–ESI source. The peptide solution was desalted followed by Duncan’s multiple–range test using SPSS using Capcell Pak C18 UG120 V (4.6 × 250 mm, (12) software. Differences were considered significant at 5 μm, Shiseido, Tokyo, Japan). The purified peptide dis- p < 0.05. solved in methanol/water (1:1, v/v) was infused into the ESI source and molecular weight was determined by 2+ Results and discussions doubly charged (M + 2H) state in the mass spectrum. β–secretase inhibitory activity of SWHs Following molecular weight determination, peptide was β–secretase inhibitory activity was measured using an automatically selected for fragmentation and sequence assay that we developed and validated using a commer- information was obtained by tandem MS analysis. cially available fluorogenic substrate. Figure 1a shows the kinetics of β–secretase inhibitory activity from Determination of β–secretase inhibition pattern SWHs and a control. A fluorescent signal (in relative For the Lineweaver-burk plot, the data were plotted as fluorescence units) was found over 0–60 min with hy- mean values of 1/v, the inverse of the increase in fluores- drolysates (or buffer) at 0.5 mg/ml and 10 mM β–secre- cence intensity per min (min/DRFU) of three independent tase substrate I. The fluorescence of β–secretase tests with different concentrations of fluorescent substrate. incubated in the absence of membrane protein was sub- The assay was performed in the presence of purified tracted at each time point. There appeared to be a linear peptide (final concentration of 0, 25, 50 and 100 μg/ml). increase in signal beginning after 5 min that continued for up to 1 h. Among hydrolysates, SWH24 was particu- larly potent. As seen in Fig. 1b, the lowest IC value Synthesis of β‑secretase inhibitory peptides was exhibited by SWH24 at 0.54 mg/ml. These discrep- The peptides were chemically synthesized in the peptide ancies may be attributed to differences in substrate spe- synthesis facility, PepTron Inc. (Daejeon, Korea). The cificity and conditions for optimal activity of the enzyme peptides were synthesized using the Fmoc–solid phase preparations, as well as to differing peptide sequences method with a peptide synthesizer (PeptrEX–R48, Peptron and structural factors affecting reactivity of the protein Inc., Daejeon, Korea). These synthetic peptides were substrates. The findings underline the importance of purified by RP–HPLC using a Capcell Pak C18 column selecting the appropriate combination of experimental (Shiseido, Japan). Elution was performed with a water– conditions to release bioactive peptide sequences acetonitrile linear gradient (0–80 % of acetonitrile) contain- (Cheung and Li–chan 2010). Protamex hydrolysates has ing 0.1 % (v/v) TFA. Elution was monitored at 220 nm on previously been reported to be effective in various bio- HPLC instrument (Prominence HPLC, Shimadzu, Tokyo, activity such as producing potent ACE inhibitory pep- Japan). tides from marine source (He et al. 2007). ab 3500 2.5 Control SWH 1 2.0 SWH 4 2500 SWH 8 SWH 24 1.5 1.0 1000 d 0.5 0.0 0 1000 2000 3000 4000 SWH1 SWH4 SWH8 SWH24 Time (s) Sample Fig. 1 Effect of shrimp waste hydrolysates (SWHs) on the inhibitory activity of β–secretase. a Relative fluorescence unit of SWHs under 10 mM β–secretase substrate I was incubated with hydrolysates (0.5 mg/ml) or buffer (control). b IC value (mg/ml) of SWHs. Letters indicate significantly (P < 0.05) different averages (ANOVA, Duncan’stest) Relative fluorescence units IC value (mg/ml) 50 Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 4 of 7 Purification of β–secretase inhibitory peptide of 1.0 ml/min (Fig. 2–IV). The IC value of this purified The use of Sephadex G–25 chromatography led to a peptide was 58.31 μg/ml. The β-secretase inhibitory ac- fraction with greatly improved β–secretase inhibitory ac- tivity of purified peptide was increased by 10.52-fold tivity (Fig. 2–I). First, fraction D from SWH24 had the compared to the SWH24 (0.54mg/ml), using the four highest β–secretase inhibitory activity, with an IC step purification procedure. value of 0.19 mg/ml. The lyophilized fraction D was fur- ther separated into eight sub–fractions by HPLC on an Identification of β–secretase inhibitory peptide ODS column with a linear gradient of acetonitrile (0– The amino acid sequence of the purified β–secretase 50 %) (Fig. 2–II). Finally, the purified fraction B2 was inhibitory peptide were identified using MS/MS. For found to have the highest β–secretase inhibitory activity SWH24, the sequence was found to be Asp–Val–Leu– (Fig. 2–III). The active fraction B2 was subjected to re– Phe–His (629 Da) for fraction B2, with an IC value of chromatography on the HPLC column using a isocratic 92.70 μM (Fig. 3). The amino acid sequence of this elution with 22.5 % acetonitrile for 30 min, at a flow rate peptide is critical in its β–secretase inhibitory activity. Fig. 2 Purification steps of β–secretase inhibitory peptide from SWH24 by Sephadex G–25 column chromatography and HPLC. I Sephadex G–25 Gel filtration chromatogram of hydrolysates prepared with SWH24 (b, lower layer). Separation was performed with 1.5 ml/min and collected at a fraction volume of 7.5 ml. The fractions isolated by Sephadex G–25 Gel column were separated (A ~ D) and β–secretase activity determined as upper panel (a). II,III,IV HPLC chromatogram of potent β–secretase inhibitory activity of separated fraction from previous step. Separation was performed with linear gradient of acetonitrile at a flow rate of 1.0 ml/min and Grom–sil 120 ODS–5 ST column (5 μm, 10 × 250 mm). Elution was monitored at 215 nm (b, lower layer). The fractions showing β–secretase inhibitory activity were determinded IC (mg/ml) as shown in upper layer (a) Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 5 of 7 520 Max. 503.4 cps. 514.25 O O O H H H N N N H N N OH H H HO O O D L H V F 194.13 629.30 95.07 415.19 184.14 77.06 176.12 -18.01 -87.99 54.0785.08 166.13 302.11 -187.05 -63.00 -144.05 100 150 200 250 300 350 400 450 500 550 600 650 Mass, Da Fig. 3 Identification of molecular weight and amino acid sequence of the purified peptide from SWH24 by HPLC. MS/MS experiments were performed on a Q–TOF tandem mass spectrometer equipped with a nano–ESI source Kimura et al. (2010) investigated the synthetic β–secretase aspartic acid groups (Bursavich and Rich 2002). Potent inhibitor, KMI–370 (IC value = 3.4 nM), whose activity transition state–mimetic β–secretase inhibitors have was greater than that of the purified peptide (IC value been reported by several groups, and the area has been =92.70 μM). Because its molecular weight was much reviewed recently (Hong et al. 2005). OM99–2, a syn- smaller than those of the others, it was considered suitable thesized peptidyl inhibitor of human brain β–secretase for absorption in the intestine. Lee et al. (2007) found that (Hong et al. 2005), was utilized to learn the interactions the amino acid sequence of a purified β–secretase inhibi- of the β–secretase active site. The inhibitor was bound tor peptide from Saccharomyces cerevisiae was Gly–Pro– in the substrate–binding cleft located between the Leu–Gly–Pro–Ile–Gly–Ser with N–terminal sequence analysis. The molecular weight of the purified β–secretase 1.0 inhibitor was estimated to be 697 Da by LC–MS, and its Control β–secretase inhibitory activity IC value was 2.59 μM. In g/ml spite of having the highest inhibition efficiency, they re- 0.8 50 g/ml ported that this octapeptide needs a reduced molecular 25 g/ml weight to overcome metabolic instability. The purified β– 0.6 secretase inhibitory peptide acted competitively with a substrate according to the Lineweaver–burk plots (Fig. 4). This strongly suggests that the purified peptide might have 0.4 an affinity for the active site of an enzyme where the sub- strate also binds; the substrate and inhibitor compete for 0.2 access to the enzyme's active site. Derivatives of these pep- tides are expected to be useful in the prevention of AD 0.0 through the development of novel peptidic inhibitors. -0.1 0.0 0.1 0.2 0.3 0.4 0.5 Availability of protein/ligand structures has opened up the 1/[S] possibility of structure–based design of β–secretase inhib- itors. Prototypical aspartic acid protease inhibitors are Fig. 4 Lineweaver–burk plots for determining inhibition pattern of the purified inhibitor against β–secretase. The intersection of the peptides of high molecular weight, and contain a sec- three lines on the vertical axis signified that the purified β–secretase ondary alcohol that acts as a transition-state mimetic inhibitor was a competitive inhibitor via the formation of hydrogen bonds with the catalytic Intensity, cps 1/[V] Li-Chan et al. Fisheries and Aquatic Sciences (2016) 19:11 Page 6 of 7 N– and C–terminal lobes. Six of the eight OM99–2 report on the β–secretase inhibiting activity of marine or- residues (P4 ~ P'2) are bound in the active site of β– ganisms. These isolated and synthesized peptides from secretase in an extended structure and their respective SWH24 could be useful in the study of the mechanisms of binding sites (S4 ~ S'2) are well–designated by atomic Alzheimer’s disease. contacts with the inhibitor side. Conclusion β–secretase inhibitory activity of synthetic peptides In conclusion, the hydrolysate of shrimp waste protein The peptide Asp–Val–Leu–Phe–His was purified from generated by proteinases treatment followed by consecu- SWH24. Based on it, five synthetic peptides were tive purification of gel filtration and reversed-phase HPLC prepared in order to study their β–secretase inhibitory resulted in a novel β-secretase inhibitory peptide of activity relative to their amino acid sequences. They DVLFH. The purified peptide acted as a competitive in- were further purified using a reversed–phase HPLC. hibitor against β-secretase with an IC value of 92.70 μM The resulting IC values of the synthetic peptides are 50 and molecular weight of 629 Da. We were synthesized shown in Table 2. Among the synthetic peptides, the novel β-secretase inhibitory peptide base on amino acid IC value of Leu–Phe–His was 34.11 μM. Moreover, 50 sequences of DVLFH. Among the synthesized peptides, the IC values of the synthetic peptides were improved 50 LFH had higher β–secretase inhibitory activity the other over the original peptide isolated from SWH24 (Asp– synthesized peptides. Our present results proposed that Val–Leu–Phe–His, IC , 92.70 μM). Synthesized Leu– 50 the β–secretase inhibitory peptides derived shrimp waste Phe–His acted competitively according to the Linewea- protein could be used as nutraceutical ingredients and alz- ver–Burk plot (data not shown). In both peptides (Asp– heimer’s disease medicine. The manufacturing of hydroly- Val–Leu–Phe–His and Leu–Phe–His), leucine is likely to sates and peptides loaded with bioactive peptide-rich be the important residue for β–secretase inhibition. In the protein from shrimp by–products could be a new possibil- β–secretase inhibitory mechanism, leucine plays an im- ity for functional foods. portant role in the Swedish mutant APP, which has a mu- Competing interests tation at the P2–P1 positions from Lys–Met to Asn–Leu. The authors declare that they have no competing interests. Generally, β–secretase has eight (P1 ~ P4 and P1' ~ P4') residues that are important in the catalytic domain, deter- Authors’ contributions mined by its crystal structure. Inhibitory activities against HGB and ECYL conceived and designed the study. IWYC prepared the samples and assisted with data collection. HGB performed the β–secretase when the P2 position was changed to several experiments, analyzed the data, and drafted the manuscript. All authors other amino acids have been described (Hong et al. 2005). read and approved the final manuscript. In the case of hydrophilic amino acids (Asp, Asn, Glu, and Gln) in the P2 position, the inhibitory activities were weak Acknowledgement This study was supported by Gangneung–Wonju National University. (β–secretase inhibitory activity of 25–36 %). However, with hydrophobic amino acids like leucine in the P2 pos- Author details ition, significant inhibitory activity was present (β–secre- Food, Nutrition & Health Program, Faculty of Land & Food Systems, The University of British Columbia, 2205 East Mall, Vancouver, BC V6T 1Z4, tase inhibitory activity of >90 %). These results suggested Canada. Department of Marine Biotechnology, Gangneung–Wonju National that a hydrophobic interaction at the P2 site of β–secre- University, Gangneung 210-702, Republic of Korea. tase was more effective than a hydrophilic one, in spite of Received: 4 January 2016 Accepted: 28 January 2016 the hydrophilic property of the P2 site. Leucine was employed as the P2 moiety for the synthetic β–secretase inhibitor. The isolated and synthesized peptides may not References be directly considered as potential drug candidates, since Bursavich MG, Rich DH. Designing non–peptide peptidomimetics in the 21st century: inhibitors targeting conformational ensembles. J Med Chem. 2002; they have relatively groups. However, this is the first 45:541–58. Byun HG, Kim YT, Park PJ, Lin X, Kim SK. Chitooligosaccharides as a novel β– secretase inhibitor. Carbohyd Polym. 2005;61:198–202. Table 2 β–secretase inhibitory activity of synthesized peptides Chen J, Suetsuna K, Yamauchi F. 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Fisheries and Aquatic Sciences – Springer Journals

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