Analytical Biochemistry 449 (2014) 179–187

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Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures Tatiana A. Semashko a, Elena A. Vorotnikova b, Valeriya F. Sharikova a, Konstantin S. Vinokurov c, Yulia A. Smirnova d, Yakov E. Dunaevsky d, Mikhail A. Belozersky d, Brenda Oppert e,⇑, Elena N. Elpidina d, Irina Y. Filippova a a

Department of Chemistry, Moscow State University, Moscow 119992, Russia Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia ˆ R, C ˆ eské Bude˘jovice CZ-37005, Czech Republic Entomological Institute, Biology Centre AV C d A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia e USDA Agricultural Research Service, Center for Grain and Animal Health Research, Manhattan, KS 66502, USA b c

a r t i c l e

i n f o

Article history: Received 20 September 2013 Received in revised form 20 December 2013 Accepted 23 December 2013 Available online 3 January 2014 Keywords: Cysteine peptidases Substrates of peptidases Selective peptide substrates Multicomponent enzyme mixtures Enzymatic peptide synthesis

a b s t r a c t This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp = pyroglutamyl; Xaa = Phe or Val; and Y = pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. Published by Elsevier Inc.

Peptide hydrolases of the C1 (papain) family belong to clan CA of cysteine peptidases containing a catalytic diad, Cys and His. Two other active site residues are found: a Gln residue preceding the catalytic Cys and an Asn residue following the catalytic His. The Gln residue is believed to help in the formation of the ‘‘oxyanion hole,’’ and the Asn is believed to orientate the imidazolium ring of the catalytic His [1–3]. Cysteine peptidases have been identified and studied in detail in different organisms—viruses [4,5], bacteria [6,7], protozoa [8–16], plants [1,17–21], and mammals [3,17,22,23]. The ancestor of this family, a plant peptidase papain from papaya, as well as closely related peptidases from the tropical fruits bromelain (pineapple) and ficin (fig latex) have important applications in the food

⇑ Corresponding author. Fax: +1 785 537 5584. E-mail address: [email protected] (B. Oppert). 0003-2697/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ab.2013.12.032

industry, pharmacology, and scientific research [1,24,25]. A large group of C1 family cysteine peptidases are lysosomal cysteine cathepsins, described mainly in mammals and humans [3,26,27]. Cysteine cathepsins are responsible for nonselective lysosomal protein degradation, but they also participate in more specific processes such as activation of zymogens, hormone maturation, and antigen presentation, among others. However, cysteine peptidases also are indicated in the development of different pathologies such as osteoporosis, rheumatoid arthritis, atherosclerosis, cancer metastasis, and tumor invasion [26,28– 35]. Digestive cysteine peptidases also have been described in some insect pests, including some families of beetles, aphids, and thrips [36–45]. Given the fact that cysteine peptidases are widespread and important, highly specific and effective peptide substrates are needed. Although cysteine peptidases of the papain family have broad substrate specificity, activity assays for these enzymes have been limited to a set of Arg-containing substrates, starting from the

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most simple methyl p-nitrophenyl esters and amides of benzoylArg (Bz-Arg)1 [46–48]. However, these substrates are rarely used now because of inherent problems; they lack sensitivity, have low solubility, and sometimes spontaneously hydrolyze (in the case of p-nitrophenyl esters). The most popular cysteine peptidase substrates are the short chromogenic peptides Z-Phe-Arg-pNA (where pNA = p-nitroanilide), Z-Arg-Arg-pNA [17,49–52], and their fluorogenic analogs Z-Phe-Arg-AMC (where AMC = 4-amino-7-methylcoumaride), Z-Phe-Arg-AFC (where AFC = 4-amino-7-trifluoromethyl-coumaride), Z-Arg-Arg-AMC, and Z-Arg-Arg-AFC [50,53–56]. These compounds are moderately soluble and highly stable in water-based solvents, and they have kinetic characteristics desirable in peptidase assays. With these substrates, the values of Km for the model enzyme papain vary from 0.08 to 32 mM and the values of kcat vary from 0.2 to 34 s1 [50]. However, Arg- and Lys-containing substrates are also hydrolyzed by trypsin-like serine peptidases; therefore, these substrates are not selective toward C1 cysteine peptidases. In addition, the production of arginine-containing substrates by a complex multistage chemical synthesis is complicated [56]. Previously, we enzymatically synthesized the selective chromogenic peptide substrate Glp-Phe-Leu-pNA (where Glp = pyroglutamyl) [57]. This substrate is specific for papain-like cysteine peptidases and was successfully used in the purification and evaluation of cysteine peptidases of various origins and levels of purity [38,57]. However, the use of this substrate is limited by its low solubility in water and the need to use high concentrations of organic solvents (20% dimethylformamide or dimethyl sulfoxide). To avoid these problems, we found that chromogenic and fluorogenic analogues of this substrate, containing an Ala residue in the P1 position, were better substrates for cysteine peptidases [58,59], and one of them, Glp-Phe-Ala-pNA, was successfully used to characterize complexes of digestive peptidases from insects [43–45,60]. In the current article, we have broadened the set of substrates containing the Ala residue in the P1 position, modified the techniques of synthesis, and demonstrated the selectivity of the substrates for the characterization of C1 cysteine peptidases either in a homogeneous state or as part of complex multicomponent mixtures of digestive enzymes in the stored products pests Tenebrio molitor and Tribolium spp. (Coleoptera: Tenebrionidae). Materials and methods Enzymes Papain (EC 3.4.22.2), stem bromelain (EC 3.4.22.32), ficin (EC 3.4.22.3), cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.15), bovine trypsin (EC 3.4.21.4), and subtilisin Carlsberg (EC 3.4.21.62) were obtained from Sigma–Aldrich (USA). Thermolysin (EC 3.4.24.27) and a-chymotrypsin (EC 3.4.21.1) were obtained from Fluka (Switzerland). Porcine pepsin (EC 3.4.23.1) was purified as described previously [61]. Chemicals Acetonitrile (MeCN) for high-performance liquid chromatography (HPLC), special purity grade containing not more than 0.01% 1

Abbreviations used: Bz, benzoyl; pNA, p-nitroanilide; AMC, 4-amino-7methylcoumaride; AFC, 4-amino-7-trifluoromethyl-coumaride; Glp, pyroglutamyl; MeCN, acetonitrile; HPLC, high-performance liquid chromatography; DMF, dimethylformamide; TEA, triethylamine; TFA, trifluoroacetic acid; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; UV, ultraviolet; TLC, thin-layer chromatography; NMR, nuclear magnetic resonance; 3D, three-dimensional; UB, universal buffer; E-64, trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane; Abz, o-aminobenzoyl; CHTR, a-chymotrypsin; SL, subtilisin Carlsberg.

of water, was obtained from Lekbiofarm (Russia). Dimethylformamide (DMF) and triethylamine (TEA) of analytical grade (Reakhim, Russia) were further purified by the method in Ref. [62]. Trifluoroacetic acid (TFA) of analytical grade was obtained from Fluka. Acetic acid of analytical grade was obtained from Reakhim. Ethylenediaminetetraacetic acid (EDTA), calcium chloride, and dithiothreitol (DTT) were obtained from Sigma–Aldrich. Sephadex G-100 was obtained from Pharmacia (Sweden). L-Alanine 7-amido-4-(trifluoromethyl)coumarin trifluoroacetate (L-Ala-AFC  TFA), L-alanine 7-amido-4-methylcoumarin trifluoroacetate (L-Ala-AMC  TFA), L-alanine p-nitroanilide (L-Ala-pNA), and chromogenic substrates Bz-Arg-pNA, Z-Arg-Arg-pNA, and Z-Phe-Arg-pNA were obtained from Bachem (Switzerland). Derivatives of other amino acids and peptides were synthesized in our laboratory by standard techniques [63]. Synthesis of substrates by enzymes in solution To begin the synthesis of Glp-Phe-Ala-pNA, 1.5 mg of a-chymotrypsin was added to a solution containing 112 mg (0.53 mmol) of L-Ala-pNA and 154 mg (0.53 mmol) of pyroglutamyl-phenylalanine methyl ester (Glp-Phe-OCH3) [59] in 0.53 ml of DMF and 3 ml of 0.2 M Na2CO3–NaHCO3 buffer (pH 9.9). After 5 min, a voluminous precipitate was formed. The mixture was stirred for 1 h at 20 °C and left for 10 h at 0 °C. The precipitate was centrifuged, washed with 5% citric acid and water, and dried over NaOH in a vacuum. Synthesis with subtilisin Carlsberg was performed as described above except that 0.2 M Tris–HCl buffer (pH 8.2) was used instead of the sodium carbonate buffer. Glp-Phe-Ala-AFC was synthesized by the procedure similar to that described above for Glp-Phe-Ala-pNA, using 29 mg (0.1 mmol) Glp-PheOMe and 41 mg (0.1 mmol) CF3COOHAla-AFC. All other enzymes and buffers were the same. Synthesis of substrates by immobilized enzymes Alternatively, substrates Glp-Phe-Ala-pNA (I), Glp-Val-Ala-pNA (II), Glp-Phe-Ala-AMC (III), and Glp-Phe-Ala-AFC (IV) were synthesized using immobilized chymotrypsin and subtilisin. Preparations of immobilized enzymes on poly(vinyl alcohol)–cryogel carrier were obtained according to the procedure in Ref. [64]. All substrates (I–IV) were obtained in analytical quantities in an anhydrous medium of polar organic solvents (DMF–MeCN, 20:80, vol%) in one cycle for 24 h according to the previously described method [59] using Glp-X-OMe (where X = Phe or Val), and Ala-Y (where Y = pNA, AMC, or AFC) as starting compounds. GlpPhe-Ala-pNA (I) and Glp-Phe-Ala-AMC (III) were also synthesized during six and three consequent cycles of peptide production, respectively, according to the procedure described in Ref. [64]. After each cycle, the beads of biocatalyst were rinsed twice with the solvent mixture. Glp-Phe-Val-pNA and Glp-Phe-Ala-AFC were also obtained in preparative quantities as described previously [59]. The physic-chemical characteristics of substrates are presented in Table S1 of the online Supplementary material. Analyses of synthesized compounds Thin-layer chromatography (TLC) was performed on an Eastman Chromogram Sheet (Kodak, USA) in the following systems: (A) n-butanol–pyridine–water–acetic acid (15:12:10:3); (B) acetone–benzene–acetic acid (100:25:4). Compounds with a free a-amino group were detected with ninhydrin reagent, and peptides were visualized by spraying chlorinated plates with 0.05 M KI and exposure to ultraviolet (UV) light (254 and 290 nm) using UV analysis lamps (HP-UVIS, Sweden).

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HPLC (reverse phase) was carried out with an Altex model 110A chromatograph (USA) using a Nucleosil C18 column (4.6  250 mm; Biokhimmak, Russia) eluted with a linear gradient of 10 to 85% CH3CN in water for 35 min at a flow rate 1 ml/min. The eluent contained 0.1% TFA. The elution profile was monitored at 220, 280, or 315 nm. Amino acid analyses were performed on a Hitachi 835 automatic amino acid analyzer (Japan) after acidic hydrolysis of samples with 5.7 M HCl at 105 °C in evacuated ampoules for 24 and 48 h. Mass spectra were obtained by a Finnigan LCQ-IonTrap instrument (Thermo Electron, USA) using an electrospray ionization method. Nuclear magnetic resonance (NMR) spectra were obtained in dimethyl sulfoxide (DMSO)-d6 by a Brucker AC-300 spectrometer at a frequency of 300 Mhz. Chemical shifts were in parts per million (d, ppm) toward the inner standard of tetramethylsilane.

A¼

181

dI  kfl ; dt  cenz

where A is the activity of the preparation, lMsubstrate  M1enzyme  min1; dI/dt, units/min, is an initial rate of fluorophore release, determined as a coefficient of a linear plot of the kinetic curve (a function of fluorescence intensity I from time); kfl, lM  U1, is the concentration of a fluorophore at which the fluorescence intensity of the solution is equal to 1 unit (defined in a special experiment by plotting a calibration curve); kfl = 0.885 mM  U1 for 7-amino-4-methylcoumarin and 0.118 lM  U1 for 7-amino4-(trifluoromethyl)coumarin; and cenz is enzyme concentration in the sample, M (for cysteine peptidases, cenz was calculated by the concentration of active sites of the peptidase). Determination of kinetic parameters of substrate hydrolysis

Homology modeling of a three-dimensional (3D) bromelain structure was performed using chymopapain chain A (1YAL) as the closest homolog by SwissModel server [65]. Molecular docking was performed using SwissDock [66]. Data were visualized with VMD [67] and PyMOL [68].

The initial rates of hydrolysis of the chromogenic and fluorogenic substrates were determined by similar procedures as described above (see enzymatic assay), with substrate concentrations of 0.05–0.5 mM for Glp-Phe-Ala-pNA, 0.1–1.0 mM for Glp-Val-Ala-pNA, and 1.25–25 lM for Glp-Phe-Ala-AMC and GlpPhe-Ala-AFC. Enzyme concentrations in the reaction were similar to those in procedures described above. Calculations of the kinetic parameters were performed by nonlinear regression using the program OriginPro 7.5.

Enzyme assays using chromogenic pNA substrates

Active site titration of cysteine peptidases

For assays of commercially available enzymes, 5 ll of enzyme solution with concentrations of 0.2–0.56 lM (except for bromelain with a concentration of 1.8 lM) were placed into wells of a 96-well plate and diluted to a final volume of 195 ll with 0.1 M universal acetate–phosphate–borate buffer (pH 5.6) (UB) [69] containing 1 mM DTT. The mixture was preincubated at room temperature (23 °C) for 20 min before the addition of substrate. To initiate the reaction, 5 ll of 20 mM substrate solution was added (except for 10 mM Z-Phe-Arg-pNA in DMF), and initial absorption at 405 nm at time zero was measured and thereafter every 5 min for 60 min at 37 °C. The absorbance was measured by StatFax 2100 (Awareness Technology, USA) and ELx808 (BioTek, USA) microplate readers. Enzymatic activity was calculated by the formula

To identify the proportion of active cysteine peptidases in the commercial preparations, 5 ll of enzyme solution (cathepsin L, D280 = 0.03–0.08 OU; other commercial peptidases, D280 = 0.1 OU) was added to 40 ll of 0.1 M UB (pH 5.6) [69] containing 1 mM DTT, and 5 ll of the inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane (E-64) in 50% ethanol was combined in a well of a 96-well plate and incubated at room temperature (23 °C) for 20 min. The initial concentrations of E-64 were 0.17 to 1.7 lM for papain and ficin, 0.25 to 3.00 lM for cathepsin B, and 0.05–0.50 lM for cathepsin L. To initiate the reaction, the mixture was diluted with 145 ll of 0.1 M UB containing 1 mM DTT and 5 ll of 20 mM Glp-Phe-Ala-pNA solution in DMF at 37 °C, and initial absorption at 405 nm at time zero was measured, and thereafter every 5 min for 60 min. A linear plot of the dependence of enzyme residual activity on the inhibitor concentration was approximated by a straight line. The concentration of active sites was determined by the intersection of this line with the abscissa axis. Calculations were carried out by least squares regression using Microsoft Excel 2007.

3D modeling and molecular docking

A¼

dD405  kchr ; dt  cenz

where A is the activity of the preparation, lMsubstrate  M1enzyme  min1; dD405/dt, OU/min, is an initial rate of p-nitroaniline release, determined as a coefficient of initial linear plot of kinetic curve (a function of the change in absorbance at 405 nm over time); kchr = 154 lM  OU1 – concentration of p-nitroaniline at which the absorbance of the solution is equal to 1 optical unit (defined in a separate experiment by plotting a calibration curve); and cenz is enzyme concentration in the sample, M (for cysteine peptidases, cenz was calculated by the concentration of active sites of the peptidase). Enzyme assays using fluorogenic substrates The enzymatic activity with fluorogenic substrates was assayed similar to that described for chromogenic substrates, using 1 mM substrate stock solutions in DMF and measuring fluorescence intensity at kex = 355 nm and kem = 460 nm for Glp-Phe-Ala-AMC and at kex = 400 nm and kem = 505 nm for Glp-Phe-Ala-AFC. Fluorescence was measured on a Cary Eclipse fluorimeter (Varian, USA) as well as on a Fluoroskan Ascent microplate spectrofluorimeter (Thermo Scientific, USA). Enzymatic activity was calculated by the formula

Fractionation of T. molitor larvae midgut peptidases T. molitor larvae rearing, isolation of midgut proteins extract, and fractionation of larval digestive peptidases on a Sephadex G-100 column were performed as described previously [43,44]. Enzymatic activity in the fractions was assayed with chromogenic peptide substrates Glp-Phe-Ala-pNA and Z-Phe-Arg-pNA (pH 5.6 with 1 mM DTT or pH 7.9 and nonreducing) in 50-ll aliquots, as described in the previous sections. Inhibitory analysis of midgut extracts from Tribolium larvae Rearing of Tribolium castaneum, Tribolium confusum, and Tribolium brevicornis larvae was as described in Ref. [45]. For inhibition studies, aliquots of the total enzyme preparation (from the total midgut fraction) were preincubated with 0.1, 1, and 10 lM E-64 for 15 min at room temperature, and the substrates

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Bz-Arg-pNA, Z-Phe-Arg-pNA, and Glp-Phe-Ala-pNA in 0.1 M UB (pH 6.8) [69] containing 1 mM DTT were added to initiate the reaction (final substrate concentration of 0.25 mM), and residual activity was assayed as described previously. Results Design of substrates For this study, we designed four chromogenic and fluorogenic substrates—Glp-Phe-Ala-pNA (I), Glp-Val-Ala-pNA (II), GlpPhe-Ala-AMC (III), and Glp-Phe-Ala-AFC (IV)—specific for cysteine peptidases from the papain family with an alanine residue in the P1 position. We evaluated compounds I–IV as substrates for C1 cysteine peptidases by flexible molecular docking in comparison with the previously described substrate Abz-Phe-Ala-pNA (where Abz = o-aminobenzoyl) [59]. Fig. 1A–D predict the binding of substrates with the active centers of papain, cathepsin B, and cathepsin L 3D structures and a bromelain model. Fig. 1E summarizes the binding energy modules of the enzyme–substrate complexes. The molecular docking studies predicted that these proposed substrates would be hydrolyzed effectively by C1 cysteine peptidases. Synthesis of substrates Synthesis of the substrates was carried out by the following scheme, using the enzymes a-chymotrypsin (CHTR) or subtilisin Carlsberg (SL) for peptide bond formation: enzyme

Glp-XaaOMe þ H-Ala-Y ! Glp-Xaa-Ala-Y þ MeOH where Xaa = Phe or Val and Y = pNA, AMC, or AFC. The enzymatic synthesis of substrates was performed by two methods: (i) under the action of native CHTR and SL in DMF/aqueous buffer and (ii) with the use of modified enzymes in an anhydrous medium of polar organic solvents (DMF–MeCN, 20:80, vol%) (Table 1). In the first method, synthesis was possible only for Glp-Phe-Ala-pNA (I), with 82 and 66% yields, and Glp-PheAla-AFC (IV), with 50 and 18% yields, using CHTR and SL, respectively. The second method, using CHTR and SL immobilized on polyvinyl alcohol cryogel (PVAG), was more universal and resulted in synthesis of all substrates, albeit with differing efficiencies. The yield of Glp-Phe-Ala-pNA was high (70–80%) even after reusing the same sample of biocatalyst six times. The efficiency of synthesis of Glp-Phe-Ala-AMC was similar for two cycles; in 4 h, the yield was 50%, and it essentially was complete (100%) in 48 h. All synthesized peptides were stable crystalline compounds with high melting points (> 200 °C). All compounds were characterized by mass spectrometry, NMR, and amino acid analysis as well as chromatographic mobility values (Rf values) in several systems (TLC) and retention times (HPLC) (see Table S1 in Supplementary material). The resulting physico-chemical properties of the synthesized compounds I–IV demonstrate their chemical homogeneity and spectral purity. Hydrolysis of the substrates by model cysteine peptidases (from C1 family) The hydrolysis of newly synthesized substrates I–IV, determined as kcat/Km, was evaluated by cysteine peptidases from the C1 family, including plant enzymes papain, bromelain, ficin, and bovine and human lysosomal cathepsins B and L, respectively (Table 2). For all enzymes except bromelain, kinetic constants of hydrolysis were calculated by the concentration of the active enzymes, which was determined by titration with the cysteine

peptidase inhibitor E-64 [70] (data not shown). This titrant was ineffective for bromelain, making it difficult for a valid comparison of kinetic constants for this enzyme with those of the other peptidases. Of the tested enzymes, papain demonstrated the highest efficiency of hydrolysis. In general, plant enzymes papain and ficin were substantially more active in the hydrolysis of all substrates than were mammalian lysosomal cathepsins, and fluorogenic substrates were more efficiently hydrolyzed by all enzymes (Table 2).

Substrate selectivity: Comparison with commercial Arg-containing substrates Action of model peptidases from other classes Substrates I–IV were evaluated for their selectivity, that is, for resistance to hydrolysis by peptidases from other classes, including serine peptidases (trypsin, CHTR, and SL), aspartic peptidase (pepsin), and metallo peptidase (thermolysin). Enzymatic activity for all enzymes was measured under conditions optimal for cysteine peptidases: under mild acidic conditions (pH 5.6) in the presence of a reducing agent (1 mM DTT). All substrates (I–IV) were selective for cysteine peptidases of the C1 family and were not hydrolyzed by peptidases of other classes (Table 3). For comparison, we also tested the selectivity of commercial chromogenic substrates that are widely used for testing the enzymatic activity of C1 family cysteine peptidases: Z-Phe-Arg-pNA (V), Z-ArgArg-pNA (VI), and Bz-Arg-pNA (VII). Z-Phe-Arg-pNA was easily hydrolyzed by all cysteine peptidases, with papain being the most active. Z-Arg-Arg-pNA was cleaved by bromelain and cathepsin B, and Bz-Arg-pNA was hydrolyzed by all cysteine peptidases but at a lower rate. However, all commercial substrates were not selective because they were easily cleaved with trypsin.

Evaluation of cysteine peptidases during the chromatographic separation of a native multicomponent enzyme mixture During the separation of midgut contents from T. molitor larvae, the elution profiles of peptidase activities were monitored by the hydrolysis of Glp-Phe-Ala-pNA and the commercial substrate ZPhe-Arg-pNA under conditions optimal for cysteine peptidases (pH 5.6, 1 mM DTT) or trypsin-like peptidases (pH 7.9, no DTT) (Fig. 2). Enzyme activity with the substrate Glp-Phe-Ala-pNA was detected only under conditions optimal for cysteine peptidases and only in two peaks: TmCPI and TmCPII. Activity with the substrate Z-Phe-Arg-pNA was detected under both conditions and in more fractions, which corresponded to the elution not only of cysteine but also of trypsin-like peptidases.

Inhibition analysis of cysteine peptidase activity in multicomponent crude extracts We used the cysteine peptidase inhibitor E-64 [70] to demonstrate the selectivity of substrate (I) for cysteine peptidases in crude extracts from the gut of T. molitor larvae as well as other tenebrionid larvae of the genus Tribolium: T. castaneum, T. confusum, and T. brevicornis. Cysteine peptidase activity was measured by the hydrolysis of Glp-Phe-Ala-pNA compared with the commercially available substrates Z-Phe-Arg-pNA and Bz-Arg-pNA at pH 6.8 in the presence of DTT with or without E-64 (Fig. 3). At 1 lM E-64 concentration, which is characteristic for the inhibition of pure cysteine peptidases, only the enzyme activity with Glp-Phe-Ala-pNA was almost totally inhibited. Even with a 10 mM concentration of E-64, the residual enzyme activity with Z-Phe-Arg-pNA and Bz-Arg-pNA was still 40 to 90%.

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Fig.1. Modeling of substrates I–IV binding to papain (PDB ID 1CVZ) (A), bromelain model (UniProt ID O23799) (B), cathepsin B (PDB ID 1CSB) (C), and cathepsin L (PDB ID 1ICF) (D). Glp-Phe-Ala-pNA (I) is marked with yellow, Glp-Val-Ala-pNA (II) is marked with green, Glp-Phe-Ala-AMC (III) is marked with blue, Glp-Phe-Ala-AFC (IV) is marked with purple, and Abz-Phe-Ala-pNA is marked with red. The binding energy modules of the enzyme–substrate complexes is shown in panel E.

Table 1 Yields of the enzymatic synthesis of substrates by native enzymes in an aqueous–organic (DMF/aqueous buffer) mixture or by immobilized enzymes in an organic (DMF/MeCN) mixture using chymotrypsin or subtilisin. Substrate

Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC Note. CHTR, chymotrypsin; SL, subtilisin. a 24-h incubation, one cycle. b Yield is given for preparative variant.

Yield (%): DMF/MeCNa

Yield (%): DMF/aqueous buffer CHTR

SL

CHTR

SL

82 0 0 50

66 0 0 18

83 0 100 (46)b 40

44 32 11 25

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Table 2 Efficiency of the hydrolysis of substrates I–IV by cysteine peptidases. Peptidase

Substrate

Papain

Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC Glp-Phe-Ala-pNA Glp-Val-Ala-pNA Glp-Phe-Ala-AMC Glp-Phe-Ala-AFC

Ficin

Bromelain

Cathepsin B

Cathepsin L

kcat/Km (mM1 min1) (I) (II) (III) (IV) (I) (II) (III) (IV) (I) (II) (III) (IV) (I) (II) (III) (IV) (I) (II) (III) (IV)

481 ± 76 148 ± 24 4376 ± 162 5493 ± 312 129 ± 12 71 ± 6 1345 ± 83 1259 ± 83 7±1 8±1 116 ± 8 90 ± 4 39 ± 4 19 ± 1 582 ± 78 670 ± 33 33 ± 4 25 ± 2 328 ± 14 137 ± 1

Discussion Most peptidases of the papain C1 family are monomers with a molecular mass of 20–35 kDa, structurally composed of two domains, L and R, separated by a cleft where the active site is located [1,2,49,71]. The substrate binding site in this peptidase family consists of seven subsites (S1–S4 and S01 –S03 ), which are located at the junction of the L and R domains. The S2 subsite has the highest selectivity, with slightly less for S1 and S01 ; other binding subsites have significantly lower selectivity and specificity [71]. The S2 subsite is the only subsite with a complete substrate binding pocket [3]. Using substrate libraries for a series of human C1 cathepsins (L, V, B, S, K, and F), it has been shown that the majority of cysteine peptidases are characterized by strong affinity for substrates with a Lys and Arg at position P1 and, to a lesser extent, for the substrates with small and uncharged amino acid residues at P1 (glutamine, threonine, and alanine). In position P2, preferred amino acids are hydrophobic (Phe, Trp, Tyr, Leu, Ile, Val, and Met) [72–76]. In general, cysteine peptidases have rather broad specificity and are able to hydrolyze a wide range of substrates [1,3,71]. The structure of our proposed substrates Glp-Phe-Ala-pNA (I), Glp-Val-Ala-pNA (II), Glp-Phe-Ala-AMC (III), and Glp-Phe-Ala-AFC (IV) can be expressed by the general formula Glp-Xaa-Ala;Y (where Xaa = Phe or Val; Y = pNA, AMC, or AFC; and the arrow shows the expected position of substrate hydrolysis). These substrates have the hydrophobic residue Phe or Val in position P2,

Fig.2. Use of the synthesized substrate Glp-Phe-Ala-pNA and a commercial substrate, Z-Phe-Arg-pNA, for the detection of cysteine peptidases (pH 5.6, 1 mM DTT) and trypsin-like peptidases (pH 7.9, no DTT) during the fractionation of T. molitor larval digestive enzymes in a midgut extract on a Sephadex G-100 column.

which is crucial for the specificity of C1 peptidases [71]. The introduction of a Val residue in the P2 position was proposed according to the screening of the enzymatic activity of cysteine peptidases from the papain family, which indicated that, contrary to popular opinion about the preference for aromatic residues, position P2 may be occupied by small aliphatic amino acid residues [72,76]. We hypothesized that short substrates containing two amino acid residues would provide better selectivity and resistance to hydrolysis by peptidases from other classes despite some loss in efficiency of hydrolysis by cysteine peptidases. The docking studies indicated that all compounds bound to the active center of four different C1 peptidases in a conformation conducive to the further cleavage of substrates at the amide bond proposed for the hydrolysis. The N-terminal group of the substrates is a pyroglutamic acid residue (Glp). This residue is more hydrophilic than the majority of commonly used protecting groups and was introduced to increase the solubility of substrates in an aqueous mixture. A chromogenic pNA or fluorogenic AMC and AFC groups were added as C-terminal residues. These residues provided convenience and accuracy to control enzymatic activity by detecting changes of the spectral and fluorescent characteristics of the substrates during hydrolysis. The substrates were obtained by an enzymatic approach using CHTR or SL. These enzymes have substrate specificities that satisfy the structure of synthesized compounds. The enzymatic peptide bond formation guaranteed selectivity of the reaction, provided optical purity of the target compounds, and simplified the scheme of synthesis and isolation of products. The comparison of the two synthesis methods, by native and immobilized enzymes,

Table 3 Hydrolysis of synthesized (I–IV) and commercial (V–VII) chromogenic and fluorogenic substrates with cysteine peptidases from the C1 family and peptidases from other classes. Peptidase

A (Msubstrate/Menzyme ⁄ min) Glp-Phe-Ala-pNA (I) Glp-Val-Ala-pNA (II) Glp-Phe-Ala-AMC (III) Glp-Phe-Ala-AFC (IV) Z-Phe-Arg-pNA (V) Z-Arg-Arg-NA (VI) Bz-Arg-pNA (VII)

Papain 132.1 ± 0.0 Ficin 41.6 ± 0.0 Bromelain 2.2 ± 0.0 Cathepsin B 11.8 ± 0.0 Cathepsin L 11.1 ± 1.4 Trypsin 0.1 ± 0.0 a-Chymotrypsin 0 Subtilisin 0 Pepsin 0.7 ± 0.0 Thermolysin 0

39.4 ± 0.0 18.8 ± 0.0 2.6 ± 0.0 7.5 ± 0.0 4.9 ± 1.5 0.3 ± 0.1 0.5 ± 0.0 0.1 ± 0.0 0 0.5 ± 0.1

56.7 ± 0.0 16.3 ± 0.0 2.2 ± 0.0 3.0 ± 0.0 14.7 ± 0.1 0 0 0 0 0

58.8 ± 0.0 14.8 ± 0.0 1.9 ± 0.0 4.8 ± 0.0 4.7 ± 0.5 0 0 0 0 0

760.8 ± 0.0 133.9 ± 0.0 1.8 ± 0.0 89.5 ± 0.0 159.1 ± 21.1 27.3 ± 0.6 0 0 0 0.4 ± 0.1

2.9 ± 0.0 2.2 ± 0.0 8.9 ± 0.0 17.8 ± 0.0 13.0 ± 4.1 50.3 ± 1.2 0.7 ± 0.0 0.1 ± 0.0 0.4 ± 0.1 0

12.1 ± 0.0 3.5 ± 0.0 0.3 ± 0.0 3.0 ± 0.0 – 1.7 ± 0.1 0.1 ± 0.0 0 0 0

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Fig.3. Effect of E-64 on the activity of enzymes in midgut extracts from Tribolium castaneum, Tribolium confusum, Tribolium brevicornis, and Tenebrio molitor larvae with chromogenic commercial substrates Bz-Arg-pNA and Z-Phe-Arg-pNA and synthesized substrate Glp-Phe-Ala-pNA, assayed at pH 6.8.

demonstrated the advantage of synthesis with immobilized biocatalysts. The second approach was more efficient and allowed the repeated use of the catalyst. In general, the synthesis of Phecontaining derivatives was more effective with the use of CHTR, whereas Val-containing derivatives were more efficiently synthesized by SL. A demonstration of the hydrolysis of the substrates by model cysteine peptidases from the C1 family indicated that plant enzymes papain and ficin were substantially more active in the hydrolysis of all substrates than were mammalian lysosomal cathepsins. Fluorogenic substrates were more efficiently hydrolyzed by the model enzymes by approximately an order of magnitude more than chromogenic substrates. The selectivity of our substrates was compared with commercial Arg-containing substrates by the action of model peptidases from other classes, by an evaluation of cysteine peptidases during the chromatographic separation of a native multicomponent enzyme mixture, and by an inhibition analysis of cysteine peptidase activity in crude multicomponent extracts. The synthesized substrates containing an Ala residue in the P1 position were more selective for cysteine peptidase activity than the widely used commercial substrates that contain an Arg residue at position P1. The synthesized substrates have been indispensable in the study of digestive peptidases from stored-product pests, including larvae of the yellow mealworm T. molitor. T. molitor larvae have a multicomponent set of digestive peptidases located in the midgut, mostly cysteine-, trypsin-, and chymotrypsin-like peptidases [42– 45]. Cysteine digestive peptidases are engaged during the initial stages of proteolysis of food proteins and are mainly located in the anterior midgut with pH 5.6, and serine peptidases are primarily located in the posterior midgut with pH 7.9. During the chromatographic fractionation of proteins from T. molitor larvae midgut extract, only the use of our selective substrate enabled unambiguous identification of cysteine peptidase activity in the digestive complex of the insect. Recently, the 3D structure of two major cathepsins L from T. molitor was published [77]. Our data indicated that one of these cathepsins, pCAL3 AY332272, is the major digestive enzyme in this insect [78,79]. According to Beton and coworkers [77], its 3D structure is very close to that of human cathepsin L. The newly synthesized substrates and a cysteine peptidase inhibitor were used to evaluate enzymes in the most complicated mixtures–crude midgut extracts from T. molitor larvae as well as other tenebrionid larvae of the genus Tribolium: T. castaneum, T. confusum, and T. brevicornis, all of which have a similar set of

digestive peptidases with mainly quantitative differences. There are at least 25 genes encoding cysteine cathepsins in T. castaneum, including those tentatively identified as cathepsins L, B, O, and K, according to genome annotation studies [80]. Our studies indicate that the primary structure of the major digestive cathepsin L from T. castaneum is very close to pCAL3 AY332272 from T. molitor [80,81]. Our data demonstrate that the substrate Glp-Phe-Ala-pNA was superior to other commercially available cysteine peptidase substrates in the evaluation of biologically active enzymes in complex multicomponent mixtures. Overall, these studies demonstrate that the synthesized substrates are selective for C1 cysteine peptidases and can be useful in the analysis of complex systems containing peptidases from different classes. Acknowledgments This work was supported by funds from the Russian Foundation for Basic Research (grants 12-04-01562-a and 12-03-01057-a) and the International Science and Technology Center (ISTC, grant 3455). The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2013.12.032. References [1] N.D. Rawlings, A.J. Barrett, Introduction: the clans and families of cysteine peptidases, in: N.D. Rawlings, G.S. Salvesen (Eds.), Handbook of Proteolytic Enzymes, vol. 2, 3rd ed., Academic Press, London, 2013, pp. 1743–1776. [2] N.D. Rawlings, A.J. Barrett, A. Bateman, MEROPS: the peptidase database, Nucleic Acids Res. 38 (2010) D227–D233. [3] V. Turk, V. Stoka, O. Vasiljeva, M. Renko, T. Sun, B. Turk, D. Turk, Cysteine cathepsins: from structure, function, and regulation to new frontiers, Biochim. Biophys. Acta 2012 (1824) 68–88. [4] M. Allaire, M.M. Chernaia, B.A. Malcolm, M.N. James, Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases, Nature 369 (1994) 72–76. [5] G.N. Rudenskaya, D.V. Pupov, Cysteine proteinases of microorganisms and viruses, Biochemistry (Moscow) 73 (2008) 1–13. [6] G. Dubin, B. Wladyka, J. Stec-Niemczyk, D. Chmiel, M. Zdzalik, A. Dubin, J. Potempa, The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity, Biol. Chem. 388 (2007) 227–235.

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