UBC Faculty Research and Publications

Shrimp (Pandalopsis dispar) waste hydrolysate as a source of novel β–secretase inhibitors Li-Chan, Eunice C Y; Cheung, Imelda W Y; Byun, Hee-Guk Apr 25, 2016

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RESEARCH ARTICLEShrimp (Pandalopsis dispafk Bsaapo–iedhibis.iviall cases of AD. Consequently, blocking the production of Aβ (Kwak et al. 2005), hispidin frommycelial cultures of Phellinushydroxyl–con-g et al. 2004).he remainingf the shrimpsing (CheungLi-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 DOI 10.1186/s41240-016-0008-xapplicable to industries such as pharmaceutical, functionalUniversity, Gangneung 210-702, Republic of KoreaFull list of author information is available at the end of the articleet al. 2012). Many studies have examined the potentialuse of these underutilized materials, termed shrimpprocessing by–products, for functional properties that are* Correspondence: hgbyun@gwnu.ac.kr2Department of Marine Biotechnology, Gangneung–Wonju National2004). β–secretase (EC 3.4.23.46), an aspartic peptidase alsoknown as memapsin 2 and BACE1, is the first protease thatprocesses APP in the pathway leading to Aβ production. Ex-cessive levels ofAβ in the brain are closely related toADpatho-genesis, so much research has been focused on developingas β–secretase inhibitors. Moreover, severaltaining inhibitors have been reported (CumminShrimp have a high market value but theads and shells, which account for half oweight, are typically removed after procesby specific inhibition of the β–secretase required for Aβ gener-ation is a major focus of research into AD therapy (Citronlinteus (Park et al. 2004), and several compounds isolated fromSanguisorbae radix (Lee et al. 2011) have also all been studiedoid precursor protein (APP), plays an early and crucial role inBackgroundSuccessful public health policies and socioeconomic develop-ment have resulted in increasing number of elderly populationglobally, which are accompanied by challenges to address vari-ous health issues of an aging society. The World HealthOrganization reported in 2012 that 35.6 million people world-wide are living with dementia or Alzheimer's disease (AD), andthat this number will triple to 115.4 million by 2050 (WorldHealth Organization and Alzheimer’s Disease International,2012). Much of AD research has been focused on the amyloidcascade hypothesis, which states that amyloid beta (Aβ), a pro-teolytic derivative of the large trans–membrane protein amyl-drugs that can inhibit β–secretase and thereby reduceAβ levelsas a therapeutic treatment for AD. High–throughput screen-ing of compound collections and natural product extracts,together with drug design building on structure–activity rela-tionships, have led to the discovery and development of bothpeptide and non–peptide inhibitors of the enzyme. These in-clude compounds such as the peptidic β–secretase inhibitorOM99–1, other aspartic protease inhibitors, an eight–residuetransition state inhibitor OM99–2 (Chen et al. 1995), and amore potent eight–residue transition state inhibitor OM00–3(Turner et al. 2001). Chitosan derivatives from crab shell ex-hibited weak β–secretase inhibition (Byun et al. 2005), whilecatechins from green tea, ellagic acid from pomegranatehydrolysate as a source oβ–secretase inhibitorsEunice C. Y. Li-Chan1, Imelda W. Y. Cheung1 and Hee-GuAbstractIn this study, purified peptides from shrimp waste hydrolyagainst β–secretase. During consecutive purification usingperformance liquid chromatography on a C18 column, aHis (629 Da) was isolated and identified from SWH24 by Q92.70 μM. The β–secretase inhibition patterns of the purifsynthesized β–secretase inhibitory peptides, Leu–Phe–HisThe result of this study suggests that the β–secretase inhcandidates to develop nutraceuticals and pharmaceuticalKeywords: Alzheimer’s disease, β–secretase inhibitory act© 2016 Li-Chan et al. Open Access This articlInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/zeOpen Accessr) wastenovelyun2*tes (SWHs) were examined for their inhibitory effectsSephadex G–25 column chromatography and hightent β–secretase inhibitory peptide Asp–Val–Leu–Phe–TOF MS/MS and the IC50 value was determined to bepeptides were found to be competitive. Amongad higher β–secretase inhibitory activity than the others.tory peptide derived from SWH24 could be potentialty, Shrimp waste hydrolysates, Peptidee is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.food, and nutraceuticals (Dey and Dora 2014; Cheung andLi–Chan 2010).Functional peptides can be produced from enzymatic hy-drolysis of various bio–resource proteins. Bioactive peptidesare usually 3–10 amino acid residue chains whose activityis based on their amino acid composition and sequence(Stachel et al. 2004), and whose functions include regulatorybe detected. Assays were performed in 96–well blacktic acid (TFA) on an HPLC system (Agilent Technolo-Li-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 Page 2 of 7effects related to nutrient uptake, immune defense (Chenet al. 1995), and antioxidant activity (Mendis et al. 2005).Moreover, some peptides can influence higher brain func-tions, such as learning and memory, in humans and animals(Mclay et al. 2001). However, there is a paucity of informa-tion on bioactive peptides from food–derived products,which may have potential to serve as β–secretase inhibitors.The objective of this study was to isolate and characterizeβ–secretase inhibitory peptides purified from shrimp wastehydrolysates, and to elucidate the active component pep-tide(s) and the mode of inhibition of β–secretase.MethodsMaterialsShrimp processing by–products (including shells, headsand tails recovered from hand–peeling of cooked shrimpPandalopsis dispar) in frozen form were donated by AlbionFisheries Ltd. (Vancouver, BC, Canada). Shrimp wasteshydrolysis was performed under experimental conditionsaccording to Cheung and Li-Chan (2010). Protamex®(Bacillus amyloliquefaciens and Bacillus licheniformis,1.5 AU/g), a product from Novozymes North America Inc.(Salem, NC), was donated by Neova Technologies Inc.(Abbotsford, BC, Canada). β–secretase and MCA–EVKMDAEFK–(DNP)–NH2 (β–secretase substrate I)was purchased from Sigma Chemical Co. (St. Louis,MO). All other reagents used in this study were reagentgrade chemicals.Preparation of shrimp waste hydrolysates(SWHs)SWHs were prepared using Protamex enzyme for hydro-lysis of the shrimp waste under varying conditions ofwater:substrate ratio (1:1, 1:1.5, 1:2 or 1:2.5), percent en-zyme (2, 4, 6 or 8 % w/w protein contents of shrimpprocessing by–products) and time of hydrolysis (1, 4, 8or 24 h) (Table 1). The lyophilized hydrolysates wereTable 1 The conditions for hydrolysis of shrimp processing by–productsSample Hydrolysis conditionsProtease W:S Enzyme(%) Time(h) pHSWH1 Protamex 1.5:1 4 1 8.4SWH4 Protamex 2.5:1 6 4 8.3SWH8 Protamex 2:1 2 8 8.3SWH24 Protamex 1:1 8 24 8.0gies, USA). The peak showing potent inhibitory activitywas finally purified into a single peptide on a reversedphase HPLC analytical C18 column (4.6 × 250 mm,plates using a Spectrofluorometer (Molecular Devices).β–secretase and β–secretase substrate I were incubatedin a final volume of 200 μl in assay buffer (50 mMsodium acetate, pH 4.5). The hydrolysis of β–secretasesubstrate I was followed at 37 °C for 30 min, bymeasuring the accompanying increase in fluorescence.Readings (excitation 325 nm, emission 393 nm) weretaken every 60s. The inhibition ratio was obtained bythe following equation: Inhibition (%) = [1– {(S–S0)/(C–C0)} × 100], where C is the fluorescence of a con-trol (enzyme, assay buffer, and substrate) after 60 minof incubation, C0 is the fluorescence of control at zerotime, S is the fluorescence of tested samples (enzyme,sample solution, and substrate) after 60 min of incuba-tion, and S0 is the fluorescence of the tested sample atzero time. All data are the means of triplicateexperiments.Purification of β–secretase inhibitory peptideThe potent fraction as determined from β–secretaseinhibitory activity assay was further purified by sizeexclusion chromatography on a Sephadex G–25 gelfiltration column (25 × 750 mm) equilibrated with dis-tilled water. Separated fractions were monitored at215 nm, collected at a volume of 7.5 ml and mea-sured for β–secretase inhibitory activity. The most ac-tive fraction was then injected into a preparativereversed phase HPLC column (YMC, ODS C18, 10.0 ×250 mm, 5 μm) and separated using a linear gradient ofacetonitrile (0–40 % v/v) containing 0.1 % trifluoroace-stored at −80 °C until use. The SWHs was providedfrom Li–Chan’s laboratory in UBC, Canada.Measurement of β–secretase inhibitory activityβ–Secretase inhibitory activity was measured followingJohnston et al. (2008), using a commercially availablefluorogenic substrate, MCA–EVKMDAEFK–(DNP)–NH2. This substrate corresponded to the wild–typeAPP sequence, derivatised at its N–terminus with afluorescent 7–methoxycoumarin–4–yl acetyl (MCA)group, and on its C–terminal lysine residue with a2,4–dinitrophenyl (DNP) group. In the intact peptidethe fluorescence of the MCA group was abolished byinternal quenching from the DNP group. Upon cleav-age by β–secretase activity the MCA fluorescence could5 μm) using a linear gradient of acetonitrile (0–20 % v/v) in 0.1 % TFA.Amino acid sequence of purified peptideTo identify molecular weight and amino acid sequenceof the purified peptide, all MS/MS experiments wereperformed on a Q–TOF tandem mass spectrometer(Micromass Co., Manchester, UK) equipped with anano–ESI source. The peptide solution was desaltedusing Capcell Pak C18 UG120 V (4.6 × 250 mm,5 μm, Shiseido, Tokyo, Japan). The purified peptide dis-solved in methanol/water (1:1, v/v) was infused into theESI source and molecular weight was determined bydoubly charged (M + 2H)2+ state in the mass spectrum.Following molecular weight determination, peptide wasautomatically selected for fragmentation and sequenceinformation was obtained by tandem MS analysis.synthesis facility, PepTron Inc. (Daejeon, Korea). TheJapan).Statistical analysisEach experiment was performed at least three times andresults were presented as the mean ± SD. Statistical com-parisons of the mean values were performed by analysisof one–way ANOVA (SPSS 12, IBM, IL, Chicago, USA),followed by Duncan’s multiple–range test using SPSS(12) software. Differences were considered significant atp < 0.05.Results and discussionsβ–secretase inhibitory activity of SWHsβ–secretase inhibitory activity was measured using anassay that we developed and validated using a commer-0/ml)2.0 af βLi-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 Page 3 of 7Time (s)0 1000 2000 3000 400Relative fluorescence units0500100015002000250030003500ControlSWH 1SWH 4SWH 8SWH 24aFig. 1 Effect of shrimp waste hydrolysates (SWHs) on the inhibitory activity opeptides were synthesized using the Fmoc–solid phasemethod with a peptide synthesizer (PeptrEX–R48, PeptronInc., Daejeon, Korea). These synthetic peptides werepurified by RP–HPLC using a Capcell Pak C18 column(Shiseido, Japan). Elution was performed with a water–acetonitrile linear gradient (0–80 % of acetonitrile) contain-ing 0.1 % (v/v) TFA. Elution was monitored at 220 nm onHPLC instrument (Prominence HPLC, Shimadzu, Tokyo,Determination of β–secretase inhibition patternFor the Lineweaver-burk plot, the data were plotted asmean values of 1/v, the inverse of the increase in fluores-cence intensity per min (min/DRFU) of three independenttests with different concentrations of fluorescent substrate.The assay was performed in the presence of purifiedpeptide (final concentration of 0, 25, 50 and 100 μg/ml).Synthesis of β‑secretase inhibitory peptidesThe peptides were chemically synthesized in the peptidesubstrate I was incubated with hydrolysates (0.5 mg/ml) or buffer (control). b ICaverages (ANOVA, Duncan’s test)SampleSWH1 SWH4 SWH8 SWH24IC50 value (mg0.00.51.01.5cbd–secretase. a Relative fluorescence unit of SWHs under 10 mM β–secretasecially available fluorogenic substrate. Figure 1a showsthe kinetics of β–secretase inhibitory activity fromSWHs and a control. A fluorescent signal (in relativefluorescence units) was found over 0–60 min with hy-drolysates (or buffer) at 0.5 mg/ml and 10 mM β–secre-tase substrate I. The fluorescence of β–secretaseincubated in the absence of membrane protein was sub-tracted at each time point. There appeared to be a linearincrease in signal beginning after 5 min that continuedfor up to 1 h. Among hydrolysates, SWH24 was particu-larly potent. As seen in Fig. 1b, the lowest IC50 valuewas exhibited by SWH24 at 0.54 mg/ml. These discrep-ancies may be attributed to differences in substrate spe-cificity and conditions for optimal activity of the enzymepreparations, as well as to differing peptide sequencesand structural factors affecting reactivity of the proteinsubstrates. The findings underline the importance ofselecting the appropriate combination of experimentalconditions to release bioactive peptide sequences(Cheung and Li–chan 2010). Protamex hydrolysates haspreviously been reported to be effective in various bio-activity such as producing potent ACE inhibitory pep-tides from marine source (He et al. 2007).2.5b50 value (mg/ml) of SWHs. Letters indicate significantly (P< 0.05) differentPurification of β–secretase inhibitory peptideThe use of Sephadex G–25 chromatography led to afraction with greatly improved β–secretase inhibitory ac-tivity (Fig. 2–I). First, fraction D from SWH24 had thehighest β–secretase inhibitory activity, with an IC50value of 0.19 mg/ml. The lyophilized fraction D was fur-ther separated into eight sub–fractions by HPLC on anODS column with a linear gradient of acetonitrile (0–50 %) (Fig. 2–II). Finally, the purified fraction B2 wasfound to have the highest β–secretase inhibitory activity(Fig. 2–III). The active fraction B2 was subjected to re–chromatography on the HPLC column using a isocraticelution with 22.5 % acetonitrile for 30 min, at a flow rateof 1.0 ml/min (Fig. 2–IV). The IC50 value of this purifiedpeptide was 58.31 μg/ml. The β-secretase inhibitory ac-tivity of purified peptide was increased by 10.52-foldcompared to the SWH24 (0.54mg/ml), using the fourstep purification procedure.Identification of β–secretase inhibitory peptideThe amino acid sequence of the purified β–secretaseinhibitory peptide were identified using MS/MS. ForSWH24, the sequence was found to be Asp–Val–Leu–Phe–His (629 Da) for fraction B2, with an IC50 value of92.70 μM (Fig. 3). The amino acid sequence of thispeptide is critical in its β–secretase inhibitory activity.ywecoliton aLi-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 Page 4 of 7Fig. 2 Purification steps of β–secretase inhibitory peptide from SWH24 bGel filtration chromatogram of hydrolysates prepared with SWH24 (b, lofraction volume of 7.5 ml. The fractions isolated by Sephadex G–25 Gelupper panel (a). II,III,IV HPLC chromatogram of potent β–secretase inhibperformed with linear gradient of acetonitrile at a flow rate of 1.0 ml/mimonitored at 215 nm (b, lower layer). The fractions showing β–secretase inlayer (a)Sephadex G–25 column chromatography and HPLC. I Sephadex G–25r layer). Separation was performed with 1.5 ml/min and collected at aumn were separated (A ~ D) and β–secretase activity determined asry activity of separated fraction from previous step. Separation wasnd Grom–sil 120 ODS–5 ST column (5 μm, 10 × 250 mm). Elution washibitory activity were determinded IC50 (mg/ml) as shown in upperMax. 503.4 cps.50M514.25194.13629.3095.07415.19184.1477.064.0OHOFHNONHNHurLi-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 Page 5 of 7Kimura et al. (2010) investigated the synthetic β–secretaseinhibitor, KMI–370 (IC50 value = 3.4 nM), whose activity100 150 200 250 300 304080120160200240280320360400440480520Intensity, cps-18.01 5-87.99 302.11-63.00-187.05-144.05HHNOOHODNHOVHNOLNHFig. 3 Identification of molecular weight and amino acid sequence of the pon a Q–TOF tandem mass spectrometer equipped with a nano–ESI sourcewas greater than that of the purified peptide (IC50 value= 92.70 μM). Because its molecular weight was muchsmaller than those of the others, it was considered suitablefor absorption in the intestine. Lee et al. (2007) found thatthe amino acid sequence of a purified β–secretase inhibi-tor peptide from Saccharomyces cerevisiae was Gly–Pro–Leu–Gly–Pro–Ile–Gly–Ser with N–terminal sequenceanalysis. The molecular weight of the purified β–secretaseinhibitor was estimated to be 697 Da by LC–MS, and itsβ–secretase inhibitory activity IC50 value was 2.59 μM. Inspite of having the highest inhibition efficiency, they re-ported that this octapeptide needs a reduced molecularweight to overcome metabolic instability. The purified β–secretase inhibitory peptide acted competitively with asubstrate according to the Lineweaver–burk plots (Fig. 4).This strongly suggests that the purified peptide might havean affinity for the active site of an enzyme where the sub-strate also binds; the substrate and inhibitor compete foraccess to the enzyme's active site. Derivatives of these pep-tides are expected to be useful in the prevention of ADthrough the development of novel peptidic inhibitors.Availability of protein/ligand structures has opened up thepossibility of structure–based design of β–secretase inhib-itors. Prototypical aspartic acid protease inhibitors arepeptides of high molecular weight, and contain a sec-ondary alcohol that acts as a transition-state mimeticvia the formation of hydrogen bonds with the catalyticaspartic acid groups (Bursavich and Rich 2002). Potenttransition state–mimetic β–secretase inhibitors have400 450 500 550 600 650ass, Da176.1285.087 166.13ified peptide from SWH24 by HPLC. MS/MS experiments were performedbeen reported by several groups, and the area has beenreviewed recently (Hong et al. 2005). OM99–2, a syn-thesized peptidyl inhibitor of human brain β–secretase(Hong et al. 2005), was utilized to learn the interactionsof the β–secretase active site. The inhibitor was boundin the substrate–binding cleft located between the1/[S]-0.1 0.0 0.1 0.2 0.3 0.4 0.51/[V]0.00.20.40.60.81.0Control g/ml 50 g/ml 25 g/ml Fig. 4 Lineweaver–burk plots for determining inhibition pattern ofthe purified inhibitor against β–secretase. The intersection of thethree lines on the vertical axis signified that the purified β–secretaseinhibitor was a competitive inhibitorN– and C–terminal lobes. Six of the eight OM99–2residues (P4 ~ P'2) are bound in the active site of β–secretase in an extended structure and their respectivebinding sites (S4 ~ S'2) are well–designated by atomiccontacts with the inhibitor side.β–secretase inhibitory activity of synthetic peptidesThe peptide Asp–Val–Leu–Phe–His was purified fromLi-Chan et al. Fisheries and Aquatic Sciences  (2016) 19:11 Page 6 of 7SWH24. Based on it, five synthetic peptides wereprepared in order to study their β–secretase inhibitoryactivity relative to their amino acid sequences. Theywere further purified using a reversed–phase HPLC.The resulting IC50 values of the synthetic peptides areshown in Table 2. Among the synthetic peptides, theIC50 value of Leu–Phe–His was 34.11 μM. Moreover,the IC50 values of the synthetic peptides were improvedover the original peptide isolated from SWH24 (Asp–Val–Leu–Phe–His, IC50, 92.70 μM). Synthesized Leu–Phe–His acted competitively according to the Linewea-ver–Burk plot (data not shown). In both peptides (Asp–Val–Leu–Phe–His and Leu–Phe–His), leucine is likely tobe the important residue for β–secretase inhibition. In theβ–secretase inhibitory mechanism, leucine plays an im-portant role in the Swedish mutant APP, which has a mu-tation at the P2–P1 positions from Lys–Met to Asn–Leu.Generally, β–secretase has eight (P1 ~ P4 and P1' ~ P4')residues that are important in the catalytic domain, deter-mined by its crystal structure. Inhibitory activities againstβ–secretase when the P2 position was changed to severalother amino acids have been described (Hong et al. 2005).In the case of hydrophilic amino acids (Asp, Asn, Glu, andGln) in the P2 position, the inhibitory activities were weak(β–secretase inhibitory activity of 25–36 %). However,with hydrophobic amino acids like leucine in the P2 pos-ition, significant inhibitory activity was present (β–secre-tase inhibitory activity of >90 %). These results suggestedthat a hydrophobic interaction at the P2 site of β–secre-tase was more effective than a hydrophilic one, in spite ofthe hydrophilic property of the P2 site. Leucine wasemployed as the P2 moiety for the synthetic β–secretaseinhibitor. The isolated and synthesized peptides may notbe directly considered as potential drug candidates, sincethey have relatively groups. However, this is the firstTable 2 β–secretase inhibitory activity of synthesized peptidesSynthesized Peptide IC50 value (μM)Asp–Val–Leu–Phe–His 101.54 ± 11.54aAsp–Val–Leu 41.93 ± 4.14cAsp–Val 67.46 ± 7.83bLeu–Phe–His 34.11 ± 9.01cPhe–His 104.76 ± 8.67aa-c, Letters indicate significantly (P < 0.05) different averages (ANOVA,Duncan’s test)report on the β–secretase inhibiting activity of marine or-ganisms. These isolated and synthesized peptides fromSWH24 could be useful in the study of the mechanisms ofAlzheimer’s disease.ConclusionIn conclusion, the hydrolysate of shrimp waste proteingenerated by proteinases treatment followed by consecu-tive purification of gel filtration and reversed-phase HPLCresulted in a novel β-secretase inhibitory peptide ofDVLFH. The purified peptide acted as a competitive in-hibitor against β-secretase with an IC50 value of 92.70 μMand molecular weight of 629 Da. We were synthesizednovel β-secretase inhibitory peptide base on amino acidsequences of DVLFH. Among the synthesized peptides,LFH had higher β–secretase inhibitory activity the othersynthesized peptides. Our present results proposed thatthe β–secretase inhibitory peptides derived shrimp wasteprotein could be used as nutraceutical ingredients and alz-heimer’s disease medicine. The manufacturing of hydroly-sates and peptides loaded with bioactive peptide-richprotein from shrimp by–products could be a new possibil-ity for functional foods.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsHGB and ECYL conceived and designed the study. IWYC prepared thesamples and assisted with data collection. HGB performed theexperiments, analyzed the data, and drafted the manuscript. All authorsread and approved the final manuscript.AcknowledgementThis study was supported by Gangneung–Wonju National University.Author details1Food, Nutrition & Health Program, Faculty of Land & Food Systems, TheUniversity of British Columbia, 2205 East Mall, Vancouver, BC V6T 1Z4,Canada. 2Department of Marine Biotechnology, Gangneung–Wonju NationalUniversity, Gangneung 210-702, Republic of Korea.Received: 4 January 2016 Accepted: 28 January 2016ReferencesBursavich MG, Rich DH. 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Subsitespecificity of memapsin 2 (β–secretase): implications for inhibitor design.Biochem. 2001;40:10001–6.World Health Organization and Alzheimer’s Disease International. Dementia:a public health priority. World Health Organization, 2012.•  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submit

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