Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds

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Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds Tejinder Pal Khaket a,b , Suman Dhanda a , Drukshakshi Jodha a , Jasbir Singh a,∗ a b

Department of Biochemistry, Kurukshetra University, Kurukshetra 136119, India Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Haryana, India

a r t i c l e

i n f o

Article history: Received 25 February 2016 Received in revised form 11 April 2016 Accepted 13 April 2016 Available online xxx Keywords: Exopeptidase Gly–Arg–4m␤NA Vigna radiata seeds Protease DPP-I PAGE

a b s t r a c t Dipeptidyl peptidases (DPPs) are widely distributed exopeptidases that hydrolyse the dipeptide moieties from the N-termini of oligopeptide chains. In the present study, DPP-I was purified from germinated moong bean seeds via acid and ammonium sulphate precipitation followed by successive chromatographies, that is, gel filtration (pH 7.4), cation exchange (pH 5.9) and anion exchange (pH 7.5). The purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and in situ gel assay. Purified plant DPP-I is a monomeric enzyme with a molecular weight of 38 kDa. It works optimally at pH 7.0 and 40 ◦ C, and it exhibits stability at pH ranging from acidic to slightly alkaline. Plant DPP-I preferentially hydrolyses glycine–arginine–4-methoxy-␤-naphthylamide and various other synthetic dipeptidyl substrates, but none of the studied endopeptidase and monopeptidase substrates. Inhibitory studies revealed the role of Cys and His amino acids in the catalytic mechanism. Functional studies of DPP-I revealed the significant role of this glycoproteinous enzyme in protein mobilization during germination. © 2016 Published by Elsevier Ltd.

1. Introduction Proteases are widely distributed hydrolases that cleave peptide bonds of proteins/oligopeptides. Thus, they are involved in various physiological functions such as providing raw material for new protein synthesis, removing misfolded/abnormal proteins, promoting the maturation of the zymogen form of enzymes/peptide hormones, inactivating regulatory peptides, and generating regulatory peptides for central processes [1,2]. Dipeptidyl peptidases (DPPs) are also widely distributed exopeptidases that hydrolyse dipeptide moieties from the N-termini of oligopeptide chains. Ten such activities have been identified, but only DPP-I to DPP-IV have been fully characterized based on their substrate specificity, subcellular localization and physiochemical parameters [3]. In plants, only DPP-II, DPP-III and DPP-IV have been purified and studied [3–5], and no specific function was assigned to plant DPP-III and DPP-IV. However, their mammalian counterparts are well studied. Animal DPP-III is reportedly involved in the hydrolysis of peptide hormones such as enkephalin and angiotensin. Thus, it has emerged as a major therapeutic target for analgesic and antihypertensive drugs [6]. DPP-IV is responsible for the degradation of incretins such as

glucagon-like peptide 1 (GLP-1). Therefore, it plays a central role in glucose metabolism [7]. DPP-IV also acts as a suppressor in the development of cancer and tumours [8,9]. DPP-I, also known as cathepsin C (EC: 3.4.14.1), is a wellestablished lysosomal cysteine protease present in animals, which exhibits broad exopeptidase activity and can progressively remove dipeptides from the amino termini of various proteins and polypeptides [10]. It is involved in the maturation of regulatory peptides such as the activation of peptide hormones and neuropeptides, which support its role in bioactive peptide generation, which in turn is of great therapeutic potential. It also plays a key role in the pathobiology of sepsis, abdominal aortic aneurysm, rheumatoid arthritis and other inflammatory disorders such as lung fibrosis, cystic fibrosis and chronic obstructive pulmonary disease [11–15]. Although proteases are involved in most cellular processes, proteolysis is yet to be elucidated in plant biology, and only little is known about the substrate specificity, physiological role or cellular location of many putative proteases or the corresponding processes. Research on aminopeptidases, which might be involved in biogenesis and disposition of bioactive peptides, has advanced with increasing interest in the role of bioactive peptides in plant development and stress response. Thus, the purification and characterization of such enzymes are necessary so as to understand their role in plants. Thus, the purification and biochemical char-

∗ Corresponding author. E-mail address: [email protected] (J. Singh). http://dx.doi.org/10.1016/j.procbio.2016.04.010 1359-5113/© 2016 Published by Elsevier Ltd.

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acterization of a DPP-I homologue from moong bean have been performed in this study to elucidate its physiological role.

2. Materials and methods 2.1. Materials Moong bean seeds (Pusa (P) Baisakhi) were collected from the Indian Agriculture Research Institute (IARI), Regional Station, Karnal (India). Glycine–arginine–4-methoxy-␤-naphthylamide (Gly–Arg–4m␤NA) and other synthetic substrates such as CMSephadex, Sephadex G-100, DEAE Sephadex, Fast Garnet GBC (o-amino-azo-toluene diazonium), and various inhibitors were obtained from Sigma-Aldrich, USA. Tris buffer, ␤-mercaptoethanol, ammonium sulphate, disodium hydrogen phosphate dehydrate and sodium phosphate monobasic were procured from Himedia, Mumbai, India. Sodium chloride, dimethyl sulphoxide (DMSO) and HCl were obtained from Rankem (Gurgaon, India) and polyvinyl pyrrolidone (PVP) from SRL India. The protein samples were concentrated using an Amicon ultrafiltration cell (Model 8200) with a YM 10 membrane under a nitrogen pressure of 5 psi. Marker proteins ranging from 14.3 to 97.4-kDa in molecular weight were obtained from Bangalore Genei, India. All centrifugation steps were performed in an Eppendorf centrifuge (Model 5415 R) procured from Eppendorf (India) and Dynamica Velocity RangeRefrigerated Bench Top Centrifuge (18R) from Dynamica (Asia) Limited, Hong Kong. To read the optical absorbance, an ultraviolet (UV)/visible double-beam spectrophotometer (Model-HaloDB-20) from Dynamica (Asia) Limited (Hong Kong) was used. The gel electrophoresis apparatus was obtained from Bangalore Genei, India.

2.2. Methods 2.2.1. Enzyme assay DPP-I activity was measured using Gly–Arg–4m␤NA as the substrate. Assay buffer (875 ␮l) (50 mM Tris-HCl, pH 7.0) and 0.1 ml of the enzyme sample were mixed and incubated at 37 ◦ C for 10 min. The reaction was initiated by adding 25 ␮l of the substrate (1 mg/ml DMSO), and the mixture was incubated for 20 min at 37 ◦ C. The reaction was stopped by adding 1.0 ml of stopping reagent (1 M sodium acetate buffer, pH 4.2) and 0.5 ml of coupling reagent (0.1% Fast Garnet GBC in water). The pink colour was extracted with 2.0 ml of n-butanol and estimated by recording the absorbance at 520 nm. In the blank, enzyme was added after the addition of coupling reagent. The enzyme activity was calculated in terms of nanomoles of −4m␤NA released per minute per millilitre of the enzyme as follows: Activity(nmol/min/ml) =

OD520 × 109 × 2.0 × 10−3 × 10 ε×t

where ε is the molar extinction coefficient of −4m␤NA under assay conditions of reaction time (t) of 36,600 min and volume of n-butanol 2.0 × 10−3 l. A multiplication factor of 10 was used to calculate the enzyme activity per millilitre, as 0.1 ml of the enzyme was used for the reaction and 109 was used for converting moles to nanomoles. One unit of enzyme activity was defined as the amount of enzyme that releases 1 nmol of 4-methoxy-␤-naphthylamine per minute from the substrate, under the assay conditions. Specific activity is expressed as enzyme units per milligram of the total protein.

2.2.2. Protein estimation Protein content was estimated by Lowry’s method [16] using bovine serum albumin as the standard. 2.2.3. Purification of enzyme The purification of DPP-I was carried out at 4 ◦ C as per the following procedure. 2.2.3.1. Crude enzyme preparation. First, 100 g of moong bean seeds germinated for 48 h was homogenized in chilled Tris-HCl buffer (50 mM, pH 7.4) and centrifuged at 4800g for 15 min. The supernatant (S1 ) was used to purify DPP-I. All purification steps were performed at 4 ◦ C. 2.2.3.2. Acid precipitation. Acid precipitation of unwanted proteins was performed by reducing the pH of S1 from 7.4 to 5.0 by dropwise addition of chilled 1 N HCl while stirring continuously. The mixture was kept overnight at 4 ◦ C; thereafter, the acid-precipitated proteins were removed by centrifugation at 4800g for 60 min. The supernatant (S2 ) was used for further purification. 2.2.3.3. Ammonium sulphate fractionation. The supernatant (S2 ) was subjected to ammonium sulphate fractionation (0–65%) and kept overnight under refrigerated conditions. The precipitated proteins were separated by centrifugation at 4800g for 60 min at 4 ◦ C. The pellet was redissolved in a minimum volume of 50 mM TrisHCl buffer at pH 7.4 and was dialysed against the same buffer. This sample was subjected to successive chromatographies. 2.2.3.4. Gel filtration chromatography on Sephadex G-100. The dialysed sample from 2.2.3.3 was loaded onto a Sephadex G100 column (75 × 1.25 cm) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.4. Fractions of 3 ml each were collected at a flow rate of 0.5 ml/min. The protein content of each fraction was measured spectrophotometrically by reading the absorbance at 280 nm, and the DPP-I activity was screened by its standard assay. Fractions with DPP-I activity were pooled, concentrated and dialysed against sodium acetate buffer (50 mM, pH 5.9). 2.2.3.5. Cation exchange chromatography on CM-Sephadex C-50. The dialysed sample of gel filtration was loaded onto a CM-Sephadex C-50 column (30 × 1.25 cm) pre-equilibrated with sodium acetate buffer (50 mm, pH 5.9). The column was run at a flow rate of 1 ml/min. Fractions of 3 ml each were collected and screened for the presence of a protein by reading the absorbance at 280 nm. The DPP-I enzyme activity was screened by its standard assay. DPP-I did not bind to CM-Sephadex; instead, it was eluted in unbound fractions. The fractions with DPP-I activity were pooled, concentrated and dialysed against Tris-HCl buffer (20 mM, pH 7.5). 2.2.3.6. Anion exchange chromatography on Q-Sepharose. The dialysed sample of the previous step was loaded onto a Q-Sepharose column (strong anion exchanger) (30 × 1.25 cm) pre-equilibrated with Tris-HCl buffer (20 mM, pH 7.5). All unbound proteins were eluted by washing the column with Tris-HCl buffer (20 mM, pH 7.5) at a flow rate of 1 ml/min. Bound proteins were eluted with a NaCl gradient from 0.0 to 1.0 M. The gradient was prepared by taking 100 ml of Tris-HCl buffer (20 mM, pH 7.5) in a stirred vessel and 100 ml of Tris-HCl buffer (20 mM, pH 7.5) containing 1 M NaCl in a non-stirred vessel. Fractions of 3 ml each were collected and monitored for proteins (OD280 nm), and also assayed for DPP-I activity. Fractions with DPP-I were pooled, concentrated and analysed for purity.

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Fig. 1. Elution profile of plant DPP-I on Q-Sepharose chromatography.

2.3. Polyacrylamide gel electrophoresis and in situ gel assay

2.6. Physicochemical characterization

The homogeneity, purity and in situ gel assay of enzyme was performed on 10% Davis gel electrophoresis [17]. For the in situ gel assay, polymerized gel was pre-run for 2 h before loading the sample. Purified DPP-I was loaded, and gel was cut into two halves after a complete run. One half was stained with Coomassie Brilliant Blue and the other half was stained for DPP-I enzyme activity with assay buffer and Gly–Arg–4m␤NA substrate at 37 ◦ C, with colour being developed with Fast Garnet GBC (1 mg/ml). Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) (12%) was performed according to Laemmli [18] to determine the molecular weight and subunit composition of the purified enzyme.

2.6.1. pH optima The pH dependence of the purified enzyme was determined via assay in the pH range of 4–11 using 50 mM sodium acetate (4.0–5.5), sodium phosphate (6.0–6.5), Tris-HCl (7.0–8.5), glycine–NaOH (9.5–10.0) and sodium phosphate–NaOH (11.0) as assay buffers

2.4. Molecular weight determination by matrix-assisted laser desorption/ionization-time of flight The purified protein sample was mixed with sinapinic acid (SA) matrix in a 1:1 ratio, and 2 ␮l was spotted onto the matrix-assisted laser desorption/ionization (MALDI) plate. After the sample was air-dried, it was analysed on an AB SCIEX 5800 MALDI TOF/TOF instrument, and further analysis was conducted with Data Explorer software for obtaining the intact mass. 2.5. Peptide mass fingerprinting For peptide mass fingerprinting, the purified protein was digested overnight with trypsin (MALDI grade) at 37 ◦ C. Then, the peptide mass spectra were obtained using a MALDI-TOF/TOF mass spectrometer (Bruker Ultraflex III TOF/TOF), and a MASCOT server (www.matrixscience.com) was used to identify the protein via peptide mass fingerprinting.

2.6.2. Temperature optima Optimum temperature of the purified enzyme was determined by assaying at different temperatures in the range of 0–75 ◦ C at pH 7.0. 2.6.3. pH and temperature stability The pH stability of purified DPP-I was determined in 50 mM buffers of varying pH, such as sodium acetate (pH 4.0–5.0), sodium phosphate (pH 6.0–6.5), Tris-HCl (pH 7.0–8.5), glycine–NaOH (9.5–10.0) and sodium phosphate–NaOH (11.0), with incubation at 37 ◦ C for 10 min. Then enzyme activity was measured at optimum pH (7.0). The temperature stability of both enzymes was determined by incubating the purified enzyme at different temperatures, that is, 0, 10, 20, 28, 32, 37, 40, 45, 50, 55, 60, 65 and 75 ◦ C for 10 min; then, the enzyme was assayed at 40 ◦ C 2.6.4. Kinetic parameters The kinetic constants Km and Vmax of purified DPP-I were measured by the Lineweaver–Burk [18] and Hanes plots [19] at 40 ◦ C using Gly–Arg–4m␤NA as the substrate (concentration ranging from 1 to 300 ␮M).

Table 1 Purification Table of DPP-I from germinated moong bean seeds. Values are expressed as mean ± SD of three replicates. Steps

Totalprotein(mg)

Total activity(nmol/min/ml)

Specific activity(nmol/min/mg protein)

Purificationfold

Homogenatesupernatant Acid-ppt supernatant 0–65%(NH4 )2 SO4 fraction Sephadex G-100 pool pool CM-Sephadex Q-Sepharose

2076 ± 86.0 1328 ± 13.5 103.4 ± 6.6 33.76 ± 0.64 13.72 ± 0.45 1.2 ± 0.4

3685 ± 195.0 2871.95 ± 72.9 1792.98 ± 21.45 1033.21 ± 73.6 691.14 ± 13.3 500.37 ± 9.0

1 1.22 9.79 17.28 28.45 360.97

1.77 2.16 17.34 30.6 50.37 203.93

% Yield 100 77.83 48.59 28 18.73 11.73

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bated with different concentrations of inhibitors for up to 10 min. The inhibitory effect was determined by assaying the enzyme in the absence of the inhibitor (control). 2.6.7. Investigation of active site by varying pH To determine the catalytic amino acid residues present at the active site, a graph was plotted between log Vmax versus pH. The curve was extrapolated to determine the pKa of amino acids involved in enzyme catalysis. 2.6.8. Effect of thiol compounds The effect of thiol compounds was studied by incubating purified DPP-I with various concentrations (0.1 mM to 1.5 mM) of thiol compounds such as DTE, DTT, cysteine, reduced glutathione (GSH) and ␤-ME for 10 min at 37 ◦ C, following which the assay was initiated by adding the substrate. The control assays were run without the thiol compound. 2.6.9. Effect of different metal ions on DPP-I activity To study the effect of metal ions, DPP-I was incubated with different concentrations of chloride salts of different metal ions such as Ca2+ , Zn2+ , Co2+ , Mg2+ , NH4 + , Ba2+ , K+ and Hg2+ . 2.6.10. Effect of chloride on DPP-I activity To study the effect of chloride ions, chloride ions were removed from purified DPP-I by dialysis against 50 mM sodium phosphate of pH 7.4. Dialysed DPP-I was mixed with different concentrations of NaCl (0, 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600 and 700 mM) and assayed. The results were expressed as percent activity compared with control. 2.6.11. Glycoprotein analysis Glycoprotein bearing hexosyl, hexuronosyl or pentosyl residues react with sulphuric acid to form furfural derivatives, which in turn react with thymol to form a coloured product that is stable for a few hours at ambient temperature [20,21]. The carbohydrate content of the enzyme was determined by a phenol sulphuric acid method using glucose as the standard [22].

Fig. 2. Gel analysis of purified DPP-I. (A) PAGE with Coomassie staining (Lane 1) and activity staining of in situ gel assay (lane 2).(B) SDS-PAGE (12%) with ␤-ME (Lane 2) and molecular weight markers in the range of 14.3–97.4 kDa (Lane 1).

2.6.5. Substrate specificity The substrate specificity of DPP-I was checked with various chromogenic substrates (4-methoxy-␤-naphthylamide (−4m␤NA) and ␤-naphthylamide (␤NA)). The enzyme was incubated at 40 ◦ C in the standard reaction mixture with different monopeptide substrates such as l-Leu–␤NA, l-Val–␤NA, lSer–␤NA, H-Gly–␤NA, l-Phe–␤NA and l-Tyr–␤NA; dipeptide substrates such as Gly–Arg–4m␤NA, Gly–Phe–␤NA, Gly–Arg–␤NA, Phe–Arg–␤NA and Gly–Ala–␤NA; and various endopeptidase substrates such as Z-Phe–Arg–␤NA, Z-Ala–Arg–Arg–4m␤NA and Z-Val–Lys–Lys–Arg–4m␤NA (each at a final concentration of 150 ␮M). The relative enzyme activity was calculated with respect to Gly–Arg–4m␤NA as the substrate. A blank was also prepared separately for each reaction.

2.6.12. Storage stability of DPP-I The purified DPP-I was stored in aliquots of 100 ␮l in Tris-HCl (50 mM, pH 7.4, with 10% glycerol) at −20 ◦ C. The enzyme was assayed up to 6 months at 1-month intervals, and the residual activity was calculated. 2.7. Role of DPP-I in proteolysis 2.7.1. Effect of germination time on DPP-I activity The effect of germination time on DPP-I activity and protein content was studied in dry and imbibed seeds (in deionized water for 6 h) and seeds germinated at 25 ◦ C for 6, 8, 16, 24, 48, 72, 96, 120, 144 and 168 h. Then, 10% homogenate was prepared for all dry and germinated seeds as specified in Section 2.2.3.1. The DPP-I activity and protein content were determined in each supernatant. 2.7.2. Total free amino acids The content of free amino acids was estimated by the ninhydrin method [23]. The concentration of free amino acids in the sample was determined from the standard plot of l-glycine. The values were expressed in micrograms per millilitre. 3. Results and discussion 3.1. Purification of enzyme

2.6.6. Effect of inhibitors To predict the involvement of amino acid residues in enzyme catalysis, different chemical modifiers (inhibitors) were used to study the effect of modification via enzyme assays. DPP-I was incu-

DPP-I was purified from Vigna radiata (P-Baisakhi) seeds via successive chromatographies up to apparent homogeneity. The elution profile of DPP-I on Q-Sepharose chromatography is shown in

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Fig. 3. Peptide mass fingerprinting of DPP-I.

Fig. 1, and the results of purification are summarized in Table 1. DPP-I from V. radiata (P-Baisakhi) was purified 204-fold with a protein yield of 11.73%. The specific activity increased from 1 to 361 nM/min/mg protein after the final chromatographic step (Table 1). The purification fold of plant DPP-I was higher than DPP-I from porcine skeletal muscle, rat lysosome, chicken kidney, rat liver and Schistosoma japonicum, but lower than that obtained from goat brain, rabbit lungs and human spleen [24–29]. The percent yield of purified plant DPP-I was greater than that of rat brain and S. japonicum [30,31]. The homogeneity of purified DPP-I was checked on 10% polyacrylamide gel stained with Coomassie Brilliant Blue R-250 and

activity staining gel, that is, via an in situ gel assay (Fig. 2A). A single band on native PAGE and a corresponding stained band on the in situ gel assay confirmed the purity of DPP-I. Another single band was also obtained for purified DPP-I with reducing SDS-PAGE (Fig. 2B). Then the molecular weight of purified DPP-I from V. radiata (P-Baisakhi) was calculated from the graph of log molecular weight versus relative mobility of marker proteins (bands). The molecular weight was found to be ∼38.01 kDa for purified plant DPP-I. The molecular weight of the purified enzyme was also determined by MALDI-TOF analysis. The MALDI-TOF results revealed a single peak corresponding to ∼38.501 kDa. These results complemented the observation of SDS-PAGE. Thus, both

Fig. 4. pH optima (. . .䊉. . .) and pH stability (–䊉–) of purified DPP-I.

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Fig. 5. Temperature optima (–䊉–) and temperature stability (. . .䊏. . .) of purified DPP-I enzyme.

SDS-PAGE and MALDI-TOF confirmed the monomeric nature of DPP-I with a molecular weight of ∼38 kDa. The molecular weight of plant DPP-I is close to the molecular weight of matured DPP-I from flatworm [22]. However, the molecular weight of mammalian (humans, rabbit and rat) DPP-I was reported to range from 180 to 210 kDa with subunits of 21–24 kDa [25–28,32–35]. However, gene expression studies predicted the monomeric nature of DPPI from humans, rat, mice, Plasmodium falciparum and fluke, which was processed into prodomain, heavy chain and light chain on maturation [29,36]. These studies also suggested acquired structural variation of DPP-I during evolution. However, like other C1-family peptidases, plant DPP-I is a monomeric protein with a molecular weight of ∼38 kDa. However, the possibility of DPP-I forming a multimeric structure at higher concentration or under different cellular conditions cannot be ruled out.

3.2. Peptide mass fingerprinting by MALDI-TOF Trypsin-digested peptide fragments of DPP-I were subjected to MALDI-TOF analysis. Peptide fragment (21) peaks obtained on MALDI were further analysed on the matrix server (Fig. 3). No significant similarity was observed with the available plant protein database. The lack of significant similarity in the protein sequence showed that the amino acid sequence was unique to DPP-I in plants. 3.3. Physicochemical characterization 3.3.1. pH optima and stability The DPP-I enzyme activity was determined in the pH range of 4–11. Purified DPP-I worked optimally at pH 7.0, as shown in Fig. 4. DPP-I exhibited >70% activity at pH 6.0 and 8.0. DPP-I

Table 3 Effect of different inhibitors on plant DPP-I activity. Values are expressed as mean ± SD of three replicates.

Table 2 Substrate specificity of purified plant DPP-I enzyme. Values are expressed as mean ± SD of three replicates. Substrates

% Activity

Gly–Arg–4m␤NA Gly–Phe–␤NA Gly–Arg–␤NA Phe–Arg–␤NA Gly–Ala–␤NA Lys–Ala–4m␤NA, Arg–Arg–4m␤NA, Gly–Pro–Leu–␤NA, Z-Phe–Arg–␤NA, Z-Ala–Arg–Arg–4m␤NA, Z-Val–Lys–Lys–Arg–4m␤NA, l-Trp–␤NA, l-Leu–␤NA, l-Val–␤NA l-Ser–␤NA H-Gly–␤NA l-Phe–␤NA l-Tyr–␤NA

100 96.53 ± 0.99 90.14 ± 2.16 87.24 ± 0.58 56.70 ± 0.89 NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL

Inhibitors

Concentration

% inhibition

NEM

0.5 mM 1.0 mM 0.5 mM 1.0 mM 0.05 mM 0.1 mM 1.5 mM 3.0 mM 0.1 mM 0.5 mM 0.025 mM 0.05 mM 0.1 mM 0.5 mM 0.25 mM 0.5 mM 1.0 mM 0.5 mM 1.0 mM 0.5 mM 1.0 mM 1% 5%

36.33 ± 1.33 53.59 ± 1.79 38.15 ± 2.1 40.84 ± 1.04 56.1 ± 1.69 60.94 ± 2.24 21.02 ± 0.78 25.37 ± 1.32 3.67 ± 0.11 7.18 ± 0.14 18.29 ± 0.91 54.85 ± 1.22 56.91 ± 1.58 0 54.85 ± 2.45 60.51 ± 2.06 63.73 ± 1.97 23.11 ± 2.1 1.19 ± 0.23 6.1 ± 0.7 71.87 ± 0.19 83.77 ± 0.98 85.76 ± 1.1

Iodoacetate Leupeptin DTNB Bestatin AEBSF

PMSF 4-Nitrophenyl iodoacetamide

4-Choloromercuribenzoic acid 1,10-phenanthroline Antipain DEPC

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Fig. 6. Determination of kinetic parameters for hydrolysis of Gly–Arg–4m␤NA by DPP-I. Michaelis–Menton plot (a), Hanes plot (b) and Lineweaver–Burk plot (c).

from rabbit, S. japonicum and rat also showed optimal activity at similar pH. pH optima of 6.0 or slightly higher were also noted in R. norvegicu, M. musculus, B. taurus and P. falciparum

[31,36–40]. DPP-I from Pseudomonas sp. worked optimally at pH 8.0 [36,37]. But DPP-I from humans, rabbit, goat, pig and Marsupe-

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Fig. 7. Investigation of catalytic residues by plotting logVmax versus pH.

naeus japonicus showed maximum activity at an acidic pH range (5.0–5.5)[25,27,28,32,35,38–42]. DPP-I was found to be stable in a narrow pH range of 6–7.5 with >90% activity, and >70% activity was retained at pH 5.0 and 8.0. On both sides of this pH range, the activity decreased steeply. DPP-I homologues from humans and chicken were also found to be stable at a pH range of 5–6.5 [24,26,35,43]; however, DPP-I from goat brain showed pH stability at a broad pH range of 4.0–8.0 [25]. 3.3.2. Temperature optima and stability To determine the effect of temperature on DPP-I activity, the enzymatic activity was measured at pH 7.0 at various temperatures from 0 to 75 ◦ C. The optimum temperature of plant DPP-I was found to be 40 ◦ C. It showed ∼80% activity up to 34 and 60 ◦ C, beyond which the DPP-I activity decreased abruptly (Fig. 5). Our results are in agreement with previous reports on the tempera-

ture optima of DPP-I from rabbit, humans and pig, which ranged between 45 and 55 ◦ C [25,26,28]. However, DPP-I homologues from several other species such as humans (liver and kidney cells), rat (liver, brain, bone), B. taurus (spleen), dog (mast cell), P. falciparum, S. japonicum and M. japonicus had an optimum temperature of 37 ◦ C [30–32,38–45]. The activation energy (Ea ) of plant DPP-I was calculated by the Arrhenius plot and was found to be 21.763 KJ/mol for Gly–Arg–4m␤NA. These results cannot be compared, as it is calculated for the first time for DPP-I. Plant DPP-I was stable up to 50 ◦ C with ∼80% activity, but the activity decreased steeply thereafter (Fig. 5), and only 42% activity was left at 70 ◦ C. 3.3.3. Kinetic characterization The kinetic parameters for plant DPP-I were determined under standard assay conditions using different concentrations of Gly–Arg–4m␤NA as the substrate. Km and Vmax were

Fig. 8. Effect of thiol compounds on purified DPP-I enzyme activity.

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Fig. 9. Effect of NaCl on DPP-I activity.

estimated to be 30 ␮M and 5.57 nM/min/ml, respectively, for Gly–Arg–4m␤NA by the Michaelis–Menton equation, Hanes plot, and Lineweaver–Burk plot (Fig. 6 a, b and c). However, higher Km (∼0.148 mM) values were reported for DPP-I homologues from pig and goat with the same substrate, although in this case Vmax was also high [26]. The turnover rate (Kcat ) and catalytic coefficient (Kcat /Km ) were calculated to be 0.077 s−1 and 2.56 × 103 s−1 M−1 .

to a greater extent than the Gly–Arg–␤NA chromogen derivative. Thus, the enzyme activity was considered to be 100% with this substrate (reference). DPP-I showed higher hydrolytic activity for substrates with Arg, Phe and Ala residues at the penultimate position, and Gly and Phe at the P2 position. DPP-I was also identified in pig and P. falciparum using Gly–Arg–4m␤NA as a substrate [28,36]. Our results are in agreement with earlier studies of DPP-I from

3.3.4. Substrate specificity Among the dipeptide substrates under study, purified DPP-I showed the highest hydrolytic activity towards Gly–Arg–4m␤NA followed by Gly–Phe–␤NA, Gly–Arg–␤NA, Phe–Arg–␤NA and Gly–Ala–␤NA (Table 2). Gly–Arg–4m␤NA was hydrolysed

Table 4 Effect of metal ions on plant DPP-I activity. Values are expressed as mean ± SD of three replicates. Metal ions

Concentration (mM)

% activity

Control FeCl3

0 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2

100 98.05 ± 59.97 ± 48.55 ± 92.34 ± 85.92 ± 74.49 ± 101 ± 102.34 ± 98.05 ± 115 ± 130.9 ± 126.73 ± 102 ± 104.83 ± 61.76 ± 120 ± 141.37 ± 116.97 ± 125 ± 141.37 ± 108.05 ± 112 ± 124.35 ± 132.44 ± 115 ± 125.19 ± 104.84 ± 115 ± 123.52 ± 126.73 ± 115 ± 125.19 ± 99.12 ±

CuCl2

HgCl2

FeSo4

ZnCl2

MgCl2

KCl

NH4 Cl

BaCl2

CaCl2

MnCl2 Fig. 10. Detection of glycoproteinous nature of DPP-I enzyme by phenol–sulphuric method.

0.82 0.45 1.07 1.13 0.98 1.02 0.23 0.45 0.21 0.98 1.41 0.87 0.37 0.92 2.3 2.98 1.47 2.15 0.5 1.08 0.79 1.62 1.77 0.88 1.76 1.22 1.1 2.1 1.98 1.24 1.77 0.88 1.25

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rabbit and goat, in which Gly–Arg–4m␤NA was suggested to be the best substrate for DPP-I [25,27]. However, DPP-I showed varying substrate specificity with species; for example, porcine DPP-I preferably hydrolysed Ala–Arg–AMC [28]. Plant DPP-I did not hydrolyse the Lys–Ala, Arg–Arg and Gly–Pro moieties, which are the usual substrates for DPP-II, DPP-III and DPPIV, respectively. This is because DPP-I preferentially hydrolyses Nterminal dipeptides with a penultimate basic residue such as Arg or proline, whereas dipeptides containing a basic amino acid at the P2 position are resistant to attack, as proven in other studies on DPP-I [8,10,26,38,39]. Plant DPP-I did not hydrolyse any of the studied monopeptide substrates: l-Leu–␤NA, l-Val–␤NA, l-Ser–␤NA, H-Gly–␤NA, lPhe–␤NA, l-Tyr–␤NA and N-terminal-blocked substrates (Table 2). Thus, DPP-I exhibited neither monopeptidase nor endopeptidase activity. DPP-I of humans, rat and bovine also did not show monoor endopeptidase activity [28,39].

3.3.5. Effect of inhibitors Amino acid residues involved in catalysis were partially characterized by amino acid-modifying agents/protease inhibitors, as summarized in Table 3. The decrease in enzyme activity was dependent on the inhibitor concentration. Among various class-specific inhibitors, diethylpyrocarbonate (DEPC) showed the highest inhibitory effect on DPP-I activity followed by antipain, 4-nitrophenyl iodoacetamide, leupeptin, 4-(2-aminoethyl) benzenesulphonyl fluoride (AEBSF) and N-ethylmaleimide (NEM) (Table 3). DEPC (5%), antipain (1 mM) and 4-nitrophenyl iodoacetamide (1 mM) inhibited DPP-I by ∼84%, ∼72% and ∼64%, respectively. DEPC (ethoxyformic anhydride) is useful for histidine-specific modification of the active site in the pH range of 5.5–7.5. It causes the substitution of one nitrogen position on the imidazole ring [48]. Antipain is modifier considered to be specific to cysteine and histidine residues, whereas 4-nitrophenyl iodoacetamide is a validated inhibitor of cysteine proteases. DPP-I from rat and goat brain was also inhibited by antipain [27]. Leupeptin is a mixed inhibitor of cysteine, serine and aspartic proteases. Leupeptin strongly inhibited plant DPP-I (∼61% at 1 mM). Inhibition of DPP-I by leupeptin has been reported previously from goat, rat and humans [27,34,43]. Plant DPP-I was inhibited by NEM (1 mM), iodoacetate (0.5 mM) and p-chloromercuribenzoic acid (PCMB) (1 mM) by ∼53.6%, 38.15% and 23.11%, respectively. NEM also inhibited DPP-I from goat and humans [27,43]. Among these inhibitors, iodoacetate is a useful reagent for rapid modification of cysteinyl residues specific to the SN2 reaction mechanism into carboxyamidomethyl derivatives. Our results are in line with the inhibitory effect of iodoacetate on DPP-I from porcine skeletal muscle and goat brain [27,28]. Comparable inhibition of DPP-I by PCMB was also observed for human placenta and chicken liver [24]. By contrast, DPP-I from pig [28], beef spleen [49] and rabbit lungs [25] was found to be insensitive to PCMB, indicating variations in different species and/or tissue sources of DPP-I. Our results are also in accordance with the early classification of this enzyme as a cysteine peptidase with involvement of histidine residue in catalysis [24–27,49]. 5,5 -Dithiobis (2-nitrobenzoic acid) (DTNB) inhibited DPP-I slightly at a concentration of 3 mM. Thiols react with DTNB, cleaving the disulphide bond to give 2-nitro-5-thiobenzoate (TNB− ), which ionizes to TNB2− dianion in water at neutral and alkaline pH. Bestatin, an aminopeptidase inhibitor, had little effect on DPP-I (∼7.18% inhibition) activity. 1, 10-Phenanthroline showed a negligible inhibitory effect even at a concentration of 1 mM. It is a metallopeptidase inhibitor that acts via the removal and chelation of metal ions required for catalytic activity, leaving an inactive

apoenzyme. These results suggest that metal ions are not necessary for catalysis by plant DPP-I. AEBSF, a serine class protease inhibitor, was found to inhibit >50% DPP-I activity even at 0.05 mM. Some cysteine proteases were also found to be sensitive to AEBSF [3]. Sensitivity to AEBSF may be due to cross-reactivity with other amino acid residues. However, it is difficult to explain AEBSF inhibition mechanistically at this stage, and further structural studies on the complex of plant DPP-I and AEBSF are needed. Phenylmethanesulphonyl fluoride (PMSF) (sulphonyl fluorides) (serine class protease inhibitor) had no effect on DPP-I activity. DPPI from humans and rat also did not show any sensitivity toward PMSF [26,34]. DPP-I from rabbit lungs was, however, highly sensitive to PMSF [24]. The lack of inhibition by PMSF suggested that the serine residue was not involved in the protease catalysis. Cysteine, histidine and aspartic acid were proposed to have a role in the catalytic mechanism of human DPP-I [33,46,47]. Inhibition of plant DPP-I by antipain, DEPC, iodoacetamide, PCMB, NEM and leupeptin supported the role of cysteine and histidine in catalysis [24–26,34]. 3.3.6. Amino acid residues on active site The pKa values of amino acids involved in catalysis were ∼6.0 and ∼8.3, which correspond to the pKa values of His and Cys, respectively (Fig. 7). The imidazole ring of histidine can bind and release protons in the course of enzymatic reactions. These results are in agreement with inhibition studies, but these results should be confirmed because the pKa values (R group) of amino acids depend on temperature, ionic strength and the micro-environment of catalytic groups. The nucleophilicity of catalytic cysteine is typically dependent on a catalytic triad of Asp, His and Cys, commonly referred to as the charge relay system. Many cysteine peptidases use a simpler dyad mechanism, where His is paired with the catalytic Cys. Other cysteine peptidases mediate catalysis via novel triads of residues, such as a pair of His residues combined with nucleophilic serine. Some amino acid residues such as Tyr, Phe, Gly, Gln, Asn and Ser residues are conserved in the active site of DPP-I in different species [46,47,50]. Our studies also suggest the possible involvement of Cys, Asp and His triad in the catalysis of plant DPP-I. 3.3.7. Effect of thiol compounds ␤-ME, cysteine and dithioerythritol increased the activity of plant DPP-I by up to 20%, 18% and 10%, respectively (Fig. 8). Activation of DPP-I by ␤-ME and cysteine may be due to their similar oxidation–reduction potential. Increased activity of DPP-I from pig, rat and goat was also observed in the presence of ␤-ME and cysteine [27,28,39]. However, DTT strongly activated porcine DPP-I compared with cysteine or ␤-ME [27]. DTT, GSH and thioglycolic acid decreased plant DPP-I activity at all studied concentrations. Both DTT and GSH caused a 10% decrease in DPP-I activity at a concentration of 1 mM, but a logarithmic decrease was reported thereafter. Thioglycolic acid severely affected the DPP-I activity, causing ∼67% reduction at a concentration of 1.5 mM. Together with thiol requirement, the need for halide ion was also reported for DPP-I from pig and rabbit [25,38]. Surprisingly, there was no halide requirement for plant DPP-I. 3.3.8. Effect of metal ions NH4 + , Ca2+ , Fe2+ , Mg2+ , K+ and Ba2+ increased the activity of plant DPP-I by approximately 32%, 26%, 26%, 16%, 8% and 4%, respectively, at 0.2 mM concentration (Table 4). However, the NH4 + and SO4 2− groups inhibited the activity of the porcine enzyme. Like porcine skeleton muscle [28], halide is not necessary for the activity of plant DPP-I. In the presence of Fe3+ , Zn2+ , Cu2+ , Hg2+ and Mn2+ , the DPP-I activity was found to decrease due to the gradual oxidation of the −SH groups (Table 4). Slight inhibition was observed for Hg2+ and

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Fig. 11. Effect of germination time on protein content.

Fe2+ but none was observed with Cu2+ , Cd2+ and Ca2+ for porcine DPP-I [28]. Ethylenediaminetetraacetic acid (EDTA) (potent metal chelator) did not affect DPP-I activity even up to a concentration of 10 mM (Table 4). Similar results were obtained for DPP-I in other studies [25,43]. These results suggest that metal ions caused enzyme activation. 3.3.9. Effect of chloride ions DPP-I was activated by chloride ions, with its activity increasing up to 30% at 25 mM NaCl (Fig. 9) and decreasing thereafter. Cathepsin C (CTSC) appears to be unique among the C1 family of cysteine

peptidases in activating enzymes using univalent anions. Chloride was found to be the most potent activator in this family [10,39].

3.3.10. Glycoprotein analysis The carbohydrate content of purified enzyme was studied by the phenol–sulphuric method and measured from a standard graph of known glucose concentration. The estimated carbohydrate content of DPP-I was 4%. Protein glycosylation was also studied by the thymol–sulphuric acid method. Dark-brown spots of DPP-I on native gel confirmed the glycosylation of DPP-I (Fig. 10). Both spectrophotometric and gel analyses confirmed purified DPP-I to be a glycoprotein. In general, N-terminal glycation of the

Fig. 12. Effect of germination time on free amino acid content.

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Fig. 13. Effect of germination time on DPP-I activity.

heavy chain was reported for mammalian DPP-I with different carbohydrate moieties in different species [26,45]. Two glycosylation sites were reported in kuruma prawn DPP-I [42], three to four in mammalian DPP-I [40,50] and five in flatworm DPP-I [51]. However, no specific glycosylation site was reported in the amino acid sequence of Chinese mitten crab and black tiger shrimp DPP-I [41,52]. In general, glycosylation may deliver the enzyme to the cellular site of activation and it may also be essential for the catalytic activity of the enzyme. N-glycosylation influences many properties of glycoprotein, including its stability, solubility, antigenicity, clearance rate, half-life, folding and intracellular trafficking [53]. 3.3.11. Storage stability Purified DPP-I was found to be stable for many months under experimental conditions. Approximately 86% and 60% activity was retained after 1 and 6 months of storage, respectively. DPP-I from humans and rat was stable after storage for many months at 4 and −15 ◦ C, respectively [26,31].

positively correlated with amino acid content (r > 0.5, p). This significant correlation among the activity of DPP-I, protein content and free amino acid content indicate the substantial role of DPP-I in protein mobilization during germination. DPP-I may be involved either directly in the final stage of hydrolysis or in the activation of other proteases by inducing conformational changes that increases the susceptibility of storage proteins to cleavage by other endonucleases. Further studies are under way in our laboratory for conclusive insights on physiological functions. 4. Conclusion DPP-I was purified to apparent homogeneity from germinated moong bean seeds. Plant DPP-I is a monomer with molecular mass of ∼38 kDa, working optimally at pH 7.0. It is a glycoproteinous cysteine protease with broad substrate specificity. However, it was found to preferentially cleave Gly–Arg–4m␤NA. Plant DPP-I might play a role in the activation of other proteases and generation of bioactive peptides.

3.4. Studies on physiological role of plant DPP-I Acknowledgements 3.4.1. Germination and proteolysis The role of plant DPP-I in protein turnover during germination was studied. Therefore, the protein content, amino acid content and activity of DPP-I were studied with respect to germination and evaluated using statistical methods. On germination, a significant change in protein and free amino acid contents was observed (Figs. 11 and 12). Germination time was negatively correlated with protein content and positively correlated with free amino acid content. This change in protein and free amino acid contents clearly supported proteolysis on germination also reported previously [54,55]. This proteolysis is considered to provide raw material (free amino acids) for embryonic plant growth. On germination, DPP-I activity was increased up to 48 h, although it decreased slightly thereafter (Fig. 13). The correlation analysis results revealed that DPP-I activity was significantly correlated (r > 0.6, p) with germination time. Change in DPP-I activity was also negatively correlated (r > 0.7, p) with protein content and

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Please cite this article in press as: T.P. Khaket, et al., Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.04.010