Báo cáo khoa học: Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus docx

Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus Akihiko Tsuji, Yayoi Kikuchi, Kentaro Ogawa, Hiroko Saika, Keizo Yuasa and Masami Nagahama Department of Biological Science and Technology, University of Tokushima Graduate School, Japan Genome analysis has indicated that plants, like animals, possess a variety of protease genes. The Arabidopsis thaliana genome has 680 protease (known and putative peptidase) sequences representing all six catalytic types: aspartic, cysteine, glutamic, metallo, serine and threonine peptidases (MEROPS peptidase database; http://merops.sanger.ac.uk/), reflecting the diverse functions of plant proteases. In A. thaliana, cDNAs encoding 10 members [vignain, brassicain, RD19A, pseudotzain, aleurain, cathepsin B-like cyste- ine protease (CBCP), RD21A, XCP1, XCP2 and SAG12] of the papain-like cysteine protease (C1A) family were identified [1]. Indeed, recent genetic studies have indicated the involvement of papain-like cysteine Keywords cathepsin B; cotyledon; cysteine protease; germination; occluding loop Correspondence A. Tsuji, Department of Biological Science and Technology, University of Tokushima Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan Fax: +81 88 655 3161 Tel: +81 88 656 7526 E-mail: tsuji@bio.tokushima-u.ac.jp (Received 29 July 2008, revised 1 September 2008, accepted 5 September 2008) doi:10.1111/j.1742-4658.2008.06674.x Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. How- ever, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analy- sis, and the loop is shorter. In an effort to investigate the enzymatic prop- erties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus)by chromatography through Sephacryl S-200, DEAE–cellulose, hydroxyapatite and organomercurial–Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS ⁄ PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4- methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocortico- tropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopepti- dase activities. In addition, CBCP was found to display carboxymonopepti- dase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO 2 ). Daikon CBCP is less sensitive (1 ⁄ 7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination. Abbreviations Abz, o-aminobenzoyl; ACTH, adrenocorticotropic hormone; Boc, t-butyloxycarbonyl; CA074, N-( L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)- L-isoleucyl-L-proline; CBCP, cathepsin B-like cysteine protease; E-64, trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane; MCA, 4-methylcoumaryl 7-amide; PVDF, poly(vinylidene difluoride); pyr, L-pyroglutamyl; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; Z, carbobenzoxy. FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS 5429 proteases in various physiological processes, such as programmed cell death and disease resistance [2]. However, with the exception of vignain, aleurain and XCP, the enzymatic properties of these enzymes have not been determined. Vignain was purified from germi- nating cotyledons of soybean [3], aleurain was purified from barley aleurone layers [4] and caster bean endosperm [5], and XCP from white clover was expressed in Escherichia coli and characterized [6]. CBCP was first identified as a gibberellin-regulated gene during wheat germination and showed highest homology with the mammalian cysteine protease, cathepsin B (EC 3.4.22.1) [7]. During germination, CBCP is expressed in aleurone cells surrounding the endosperm and plays a role in the mobilization of seed storage proteins and the support of seedling growth. CBCP has been cloned from various plants, including A. thaliana [1], barley [8] and Nicotiana rus- tica [9], and CBCP transcript expression profiles have been investigated. Recently, Gilroy et al. reported the involvement of CBCP in the plant disease resis- tance hypersensitive response [10]. The amino acid sequences predicted from cDNA sequences of the CBCPs display a high degree of identity in plants. Unlike most other cysteine proteases of the papain superfamily, cathepsin B displays both endopeptidase and exopeptidase (dipeptidyl carboxypeptidase) activi- ties [11–13]. A unique sequence of cathepsin B referred to as the occluding loop between Ile105 and Pro126 (IPPCEHHVNGSRPPCTGEGDTP, human mature cathepsin B), which is not present in papain, was shown to play an important role in the exopeptidase activity of cathepsin B [12–14]. This loop is highly con- served in cathepsin B from vertebrates and inverte- brates. Two histidine residues (His110 and His111) within this occluding loop provide positively charged anchors for the C-terminal carboxylate group of the substrate [15,16]. In contrast, plant CBCP possesses an occluding loop that differs from that of animal cathep- sin B, although amino acid sequence alignment showed that the cysteine residues involved in disulfide bridge formation and substrate binding, in addition to active site residues, are well conserved [7–9]. Plant CBCP has a shorter loop than animal cathepsin B and contains only one histidine residue. Deletion of either His110 or His111 in human cathepsin B markedly affects both cleavage specificity and sensitivity to endogenous inhibitors such as cystatin c and the propeptide of cathepsin B [14]. Differences between the cleavage specificity of plant CBCP and animal cathepsin B have yet to be determined. The enzymatic characterization of plant CBCP is a necessary first step towards identi- fying the physiological function of this protease. In this study, we purified CBCP from daikon radish (Raphanus sativus), in an effort to compare its enzy- matic properties with those of mammalian cathepsin B. Daikon radish and A. thaliana are in the same Brassic- aceae family. Therefore, it was expected that the amino acid sequence and developmental regulation of CBCP would be similar, although expression analyses of proteases in daikon radish have hitherto not been performed. Furthermore, it is easier to prepare suffi- cient amounts of cotyledons from daikon radish for the purification of CBCP than from A. thaliana.We investigated CBCP cleavage specificity and sensitivity to protease inhibitors. This article represents the first report detailing the enzymatic characterization of a plant CBCP. Results Purification of cysteine protease from the cotyledons of daikon radish The trans-epoxysuccinyl-l-leucylamide-(4-guanidino) butane (E-64)-sensitive enzyme expressed in cotyledons of daikon radish was purified 3200-fold by employing a series of column chromatographic procedures utiliz- ing Sephacryl S-200, DEAE–cellulose, hydroxyapatite and organomercurial–Sepharose, as described in Experimental procedures. The specific activity against Boc-Leu-Arg-Arg-4-methylcoumaryl 7-amide (MCA) of the final preparation was 22.9 lmolÆmin )1 Æmg )1 . About 100 lg of purified enzyme was obtained from 1 kg of cotyledons. The purified enzyme did not bind concanavalin A–Sepharose gel, suggesting that the enzyme does not contain high-mannose oligosaccha- rides. Purified enzyme eluted as a single symmetrical peak on HPLC with a Superose 12PC3.2 ⁄ 30 gel filtration column, and its molecular mass was esti- mated to be 21 kDa (data not shown). As shown in Figs 1A,B, the final preparation yielded a single pro- tein band on native PAGE in the absence of SDS, whereas four bands (28, 15, 13 and 11 kDa) were detected on SDS ⁄ PAGE. N-terminal sequences of these bands were examined and compared with sequences of other cysteine proteases. As shown in Fig. 1C, the N-terminal sequence of the 28 kDa protein (LPKSFDARTHWPQXT) showed the highest degree of identity with CBCP. The 15 and 11 kDa proteins had the same N-terminal sequence, suggesting that both the 15 and 11 kDa proteins represent pro- cessed or degraded forms of the 28 kDa enzyme. In contrast, the sequence of the 13 kDa protein (QLW- SESKHYS) differed from these sequences, although a similar sequence is present in the middle region of the Characterization of daikon CBCP A. Tsuji et al. 5430 FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS catalytic domain of CBCP from other plants, such as Arabidopsis (KLWSESKHYS, DDBJ accession num- ber AF370193) [1] and wheat (QVWEEKKHFS, DDBJ accession number X66012) [7]. Both sequences were located at position 123–132 starting from the N-terminus of the mature enzyme. As mentioned below, the sequence of the 13 kDa protein was present in the deduced amino acid sequence derived from dai- kon radish CBCP cDNA. Recently, Van der Hoon et al. labeled cysteine proteases in Arabidopsis leaves using biotinylated E-64, and a 30-kDa labeled protein was identified by MS as a CBCP [17]. It is highly likely that the 28 kDa protein represents a single-chain form of CBCP. The 15, 13 and 11 kDa fragments may be degradation products of CBCP produced in the course of the purification. Isolation of cDNA encoding the catalytic domain of daikon radish CBCP In order to classify the purified cysteine protease, cDNAs encoding the propeptide and catalytic domains of the enzyme were isolated by PCR as described in Experimental procedures. Figure 2 shows the compos- ite cDNA and predicted amino acid sequences of two PCR products. The N-terminal sequences of band a (LPKSFDARTHWPQCT) and band c (QLWSESK- HYS) of the purified enzyme match perfectly with the deduced sequence, residue numbers 104–118 and 226– 232, respectively. A signal peptide cleavage site was predicted to be located between Ala31 and Glu32, using the signalP3.0 server (http://www.cbs.dtu.dk/ services/SignalP/). Two possible glycosylation sites (Asn152 and Asn311) were also identified. The deduced sequences of the propeptide and catalytic domains showed high amino acid identity with CBCP from A. thaliana (90%, DDBJ accession number AF370193) [1], potato (72%, DDBJ accession number AY450641) [8] and tobacco (72%, DDBJ accession number DQ492287) [9]. The sequence of the daikon enzyme showed highest amino acid identity with human cathepsin B (47%, DDBJ accession number M14221) [17] among the animal cysteine pro- teases. Alignment of the cysteine protease sequence identified the putative active site residues (Cys132, His287 and Asn308), as shown in Fig. 3. The cysteine residues involved in disulfide bridge formation are completely conserved (Cys14–Cys43, Cys26– Cys71, Cys62–Cys128, Cys63–Cys67, Cys100–Cys132 and Cys108–Cys119 in human mature cathepsin B). Residues in the S1, S2 and S1¢ sites (S1, Gln23 and Gly74; S2, Glu245 and Gly198; S1¢, Val176) are also conserved. Ala77 and Ala173 in the S2 site of human mature cathepsin B are replaced by isoleucine and ser- ine, respectively. Thus, amino acid sequence alignment indicates that the purified enzyme from daikon radish is a CBCP. One striking difference between plant CBCP and animal cathepsin B occurs in the occluding loop region, which plays a critical role in the exopepti- dase activity of cathepsin B (Fig. 4). All animal cathepsin B enzymes, including those from human [18], cattle [19], mouse [18], chicken [20], Atlantic hali- but (DDBJ acession number DQ993253), yellow meal- worm [21] and Clonorchis sinensis (DDBJ accession number EF102086), possess similar occluding loop structures. The occluding loop structure is also con- served in invertebrates; however, the loop is either partially or entirely absent in CBCPs from some parasites and nematodes [22–25]. Two types of CBCP that have a loop containing a dihistidine motif A C B Fig. 1. Gel electrophoresis and N-terminal sequence of the purified enzyme. (A) Native PAGE of the purified enzyme (0.5 lg) in the absence of SDS. (B) SDS ⁄ PAGE of the purified enzyme (2 lg) and N-terminal sequence of bands a–d identified by Edman degradation. The marker proteins were as follows: b-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), glyc- eraldehyde-3-phosphate dehydrogenase (36 kDa), trypsin inhibitor (20 kDa) and egg cystatin (12.8 kDa). (C) Similarities of N-terminal sequences between the purified enzyme and other plant cysteine proteases. The cysteine proteases are: CBCP (DDBJ accession numbers: Arabidopsis, AF370193; wheat, X66012; potato, AY450641), RD21A (DDBJ accession number AK221689), XCP2 (DDBJ accession number AF191028), F9P14.12 (DDBJ accession number AC025290) and aleurain (DDBJ accession number AF233883). Identical amino acid residues are shown in bold. A. Tsuji et al. Characterization of daikon CBCP FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS 5431 Fig. 2. Nucleotide sequence of the cDNA encoding the purified enzyme and its deduced amino acid sequence. The deduced amino acid sequence is displayed below the nucleotide sequence in a one-letter code. The nucleotide sequences of primers corresponding to the N-ter- minal (Met1–Cys9) and C-terminal (Ala337–Lys343) sequences were derived from Arabidopsis CBCP (DDBJ accession number AF370193). The positions of primers used for PCR amplification are marked with bold lines. The amino acid sequences identified are underlined. Amino acids 1–31 and 32–103 comprise a putative signal peptide and propeptide, respectively. The essential active site residues Cys132, His287 and Asn308 are shown in bold. Possible glycosylation sites (Asn152 and Asn311) are double-underlined. The accession number of the sequence in the DDBJ ⁄ EMBL ⁄ GenBank nucleotide sequence databases is AB377273. Characterization of daikon CBCP A. Tsuji et al. 5432 FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS (Trypanosoma congolense-1) or a single histidine (T. congolense-6) were present in Trypanosoma [25], as shown in Fig. 4. Rehman and Jasmer classified nema- tode CBCPs into four groups on the basis of charac- teristics of the occluding loop region [22]. Group I members possess a similar occluding loop structure containing a dihistidine motif. Group II members lack a dihistidine motif but have a single histidine in the loop. Group III and IV members lack histidine in the loop. In Group IV members, loop-forming cysteines appear to be present, although seven or eight amino acids that would ordinarily form the terminal loop upon disulfide bridge formation are absent. In con- trast, all plant CBCPs possess a shorter occluding loop containing a single histidine residue. In an effort to identify the role of this short occluding loop in plant CBCP function, the cleavage specificity of dai- kon CBCP was analyzed. Fig. 3. Sequence similarities between plant CBCP and animal cathepsin B. The five sequences were aligned for maximum homology. The proteases are: daikon CBCP, Arabidopsis CBCP (DDBJ accession number AF370193, amino acids 1–344), tobacco (Nicotiana benthamiana) CBCP (DDBJ accession number DQ492887, amino acids 1–341), human cathepsin B (DDBJ accession number M14331, amino acids 1–335) and mouse cathepsin B (DDBJ accession number M14222, amino acids 1–335). Identical amino acid residues of the aligned sequences are marked with asterisks. Active site residues are indicated by arrows. The occluding loops of human and mouse cathepsin B are underlined. A. Tsuji et al. Characterization of daikon CBCP FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS 5433 Cleavage specificity of CBCP towards synthetic substrates The enzymatic properties of daikon CBCP were inves- tigated using synthetic substrates. When the effect of pH on the activity towards t-butyloxycarbonyl (Boc)- Leu-Arg-Arg-MCA was examined, the enzyme dis- played the highest activity at pH 5.5 and the enzyme was stable at acidic pH (pH 5–6.5). When the enzyme was incubated at 25 °C for 10 min at pH 8.0, most of the activity was lost. CBCP had optimal activity at 40 °C. The activity of the purified enzyme was also examined against various synthetic substrates and compared with that of human cathepsin B (Table 1). Of the substrates examined, Boc-Leu-Arg-Arg-MCA and l-pyroglutamyl (pyr)-Arg-Thr-Lys-Arg-MCA were shown to be good substrates for daikon CBCP. The enzyme hydrolyzed substrates with a basic amino acid at the P1 position, and showed preference for a positively charged amino acid at the P2 position. The k cat ⁄ K m values of Boc-Leu-Arg-Arg-MCA and pyr-Arg-Thr-Lys-Arg-MCA were 2.8-fold and 1.6-fold higher, respectively, than that of carbobenzoxy (Z)-Phe-Arg-MCA, the latter substrate being com- monly used for cathepsin B assays. For tripeptide sub- strates with dibasic amino acids (Arg-Arg) at the P1 Fig. 4. Alignment of occluding loop sequences of plant CBCP from daikon (DDBJ accession number AB377273), A. thaliana (DDBJ acces- sion number AF370193), rice (DDBJ accession number AY916493), barley (DDBJ accession number AJ310426), wheat (DDBJ accession number X66012), cowpea (DDBJ accession number AM748426), barrel medic (DDBJ accession number ABD149038), garden pea (DDBJ accession number AJ251536), potato (DDBJ accession number AY450641), sweet potato (DDBJ accession number AAK69541), tobacco (DDBJ accession number X81995), grape (DDBJ accession number CAO040249), coast spruce (DDBJ accession number EF083997), moss (DDBJ accession number EDQ82213), human (DDBJ accession number M14221), cow (DDBJ accession number L06075), mouse (DDBJ accession number M14222), chicken (DDBJ accession number U18083), Atlantic halibut (DDBJ accession number DQ993253), yellow meal- worm (DDBJ accession number DQ356051), Clonorchis sinensis (DDBJ accession number EF102086), Caenorhabditis elegans (cpr-6; DDBJ accession number L39894), T. congolense-1 (TcoCB1; DDBJ accession number EU233643) and T. congolense-6 (TcoCB6; DDBJ accession number EU233648). His110 and His111 (human cathepsin B numbering), which are essential for the exopeptidase activity of cathepsin B, are arrowed. Characterization of daikon CBCP A. Tsuji et al. 5434 FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS and P2 positions, the enzyme activity increased with the order Leu > Gln at the P3 position. Substrates with glycine at the P2 position, such as Boc-Ile-Glu- Gly-Arg-MCA and Boc-Gln-Gly-Arg-MCA, and sub- strates with a single amino acid, such as Z-Arg-MCA, were only marginally hydrolyzed by daikon CBCP (data not shown). The enzyme did not hydrolyze substrates for chymotrypsin-like enzyme (glutaryl-Ala- Ala-Phe-MCA) and aminopeptidase (Leu-MCA, Arg- MCA). In contrast, human cathepsin B is most active against Boc-Leu-Arg-Arg-MCA and Z-Phe-Arg-MCA. Both enzymes exhibit preference for leucine at the P3 position for tripeptide substrates with Arg-Arg at the P1 and P2 positions. The Boc-Leu-Arg-Arg-MCA to Boc-Gln-Arg-Arg-MCA k cat ⁄ K m ratios for daikon CBCP (18%) and human cathepsin B (20%) are simi- lar. The most remarkable difference in cleavage speci- ficity between daikon CBCP and human cathepsin B is reflected in the absolute value of k cat ⁄ K m . The k cat ⁄ K m values of Boc-Leu-Arg-Arg-MCA (10.8-fold), pyr- Arg-Thr-Lys-Arg-MCA (31.1-fold), Z-Phe-Arg-MCA (4.3-fold), Z-Arg-Arg-MCA (16.4-fold) and Boc-Gln- Arg-Arg-MCA (9.9-fold) for daikon CBCP were mark- edly higher than those for human cathepsin B. The difference appears to be largely due to an increase in k cat . These results suggest that daikon CBCP possesses higher endopeptidase activity than human cathepsin B. Hydrolysis of glucagon and adrenocorticotropic hormone (ACTH) The ability of the enzyme to hydrolyze model peptides was examined. Human glucagon and ACTH 1–24 were digested using the purified enzyme at 37 °C for 20 h. Cleavage products were separated by RP-HPLC, and peptide amino acid sequences were determined. As shown in Fig. 5A, three dipeptides (Leu26-Met27, Phe22-Val23 and Tyr13-Leu14), one tripeptide (Phe22- Val-Gln24) and larger fragments (His1-Lys12 and His1-Arg18) represented the hydrolysis products fol- lowing glucagon digestion. These results indicated that the Lys12-Tyr13, Leu14-Asp15, Arg18-Ala19, Asp21- Phe22, Val23-Gln24, Gln24-Trp25 and Met27-Asn28 bonds were cleaved by the purified enzyme. These results strongly suggested that Asn28-Thr29, Leu26- Met27, Gln24-Trp25 and Phe22-Val23 dipeptides were released sequentially by the dipeptidyl carboxypepti- dase activity of CBCP. Peaks 13, 23 and 29 were applied to the sequencer; however, no N-terminal amino acids were identified. It is highly likely that hydrophilic dipeptides dissociate from the glass fiber disk coated with polybrene used in the sequence analy- sis. ACTH was also digested, and sequences of the generated peptides were analyzed (Fig. 5B). Peak 31 eluted faster than control ACTH; however, its total sequence could not be determined. Digestion of ACTH resulted in cleavage of the His6-Phe7, Phe7-Arg8, Arg8-Trp9, Gly14-Lys15, Val20-Lys21, Lys21-Val22 and Val22-Tyr23 bonds. Although the C-terminal tri- peptide Val-Tyr-Pro24 was identified as a major cleav- age product in peak 11, the C-terminal dipeptide Tyr23-Pro24 was identified as a minor cleavage product in peak 7. The internal peptide fragments Arg8-Gly14 and Lys15-Val20 were generated by the digestion. It is highly likely that the Phe7-Arg8, Gly14-Lys15 and Lys21-Val22 bonds were cleaved by the endopeptidase activity of the enzyme. These results indicated that daikon CBCP possesses both endopepti- dase and dipeptidyl carboxypeptidase activities. In order to draw a comparison with the cleavage specific- ity of human cathepsin B, ACTH was digested with human cathepsin B under the same conditions for 20 h, and its cleavage products were analyzed. Two major products, the C-terminal peptide Tyr23-Pro24 and N-terminal peptide Ser1-Lys21 were identified, suggesting that dipeptide Tyr23-Pro24 was generated by the dipeptidyl carboxypeptidase action of cathep- sin B. The N-terminal peptide Ser1–Val22 was detected as a minor product. Therefore, daikon CBCP seemed to exhibit a stronger preference for hydrolyzing sub- strates via its endopeptidase activity as compared with human cathepsin B. Hydrolysis of b-casein Bovine b-casein was digested with the purified enzyme at pH 5.5, and SDS ⁄ PAGE was used to analyze the Table 1. Kinetic constants for the hydrolysis of peptide MCA sub- strates and Abz-Phe-Arg-Phe(4-NO 2 )-OH by CBCP and human liver cathepsin B. Enzyme activity was determined in 0.1 M acetate buf- fer (pH 5.5) at 37 °C. The concentration of daikon CBCP and human cathepsin B were determined by active site titration with E-64. Standard errors for the determination of K m and k cat were lower than 7%. Substrate Daikon CBCP Human cathepsin B k cat (s )1 ) K m (lM) k cat ⁄ K m (M )1 Æs )1 ) k cat ⁄ K m (M )1 Æs )1 ) Boc-Leu-Arg-Arg-MCA 14.2 19.2 740 000 68 400 Pyr-Arg-Thr-Lys-Arg-MCA 7.59 18.2 417 000 13 400 Z-Phe-Arg-MCA 5.23 20.0 262 000 60 800 Z-Arg-Arg-MCA 5.70 25.0 228 000 13 900 Boc-Gln-Arg-Arg-MCA 1.32 9.80 135 000 13 600 Abz-Phe-Arg-Phe (4-NO 2 )-OH 1.02 65.8 15 500 Not detected A. Tsuji et al. Characterization of daikon CBCP FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS 5435 A B C Fig. 5. Cleavage of glucagon, ACTH and b-casein by daikon CBCP. HPLC profiles of glucagon (A) and ACTH (B) digests. Peaks were collected and sequenced. (C) Time- course of b-casein digestion by daikon CBCP. The digestion products were sepa- rated by SDS ⁄ PAGE (15% gel), transferred to a PVDF membrane, and then sequenced. The amino acid sequences (one-letter code) of the peptides and cleavage sites are indi- cated by arrows. The arrow in parenthesis indicates a minor cleavage site. Characterization of daikon CBCP A. Tsuji et al. 5436 FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS time-course of the digestion. The generated fragments (bands a–e) separated by SDS ⁄ PAGE were transferred onto a poly(vinylidene difluoride) (PVDF) membrane and sequenced (Fig. 5C). Cleavage of the Gln54- Thr55, Ser57-Leu58, Gly94-Val95 and Lys99-Glu100 bonds had occurred. Although b-casein possesses two arginines at positions 25 and 207, the Ile26-Arg207 fragment was not found. Monopeptidyl carboxypeptidase activity of CBCP Cathepsin X is a lysosomal cysteine protease that belongs to the papain family and acts primarily as a monopeptidyl carboxypeptidase [26–28]. Superposition of the cathepsin X and cathepsin B structures indicates that His23 of the miniloop of cathepsin X occupies a region in space that partially overlaps with His110 of the occluding loop of cathepsin B [29,30]. The occlud- ing loop of daikon CBCP is shorter than that present in human cathepsin B and contains only a single histidine, like cathepsin X. These data suggested that daikon CBCP, like cathepsin X, possesses carb- oxymonopeptidase activity. This idea was also sup- ported by the data from the model peptide digestion suggesting that Trp25 of glucagon and Lys21 of ACTH were cleaved by CBCP via carboxymonopepti- dase activity. In an effort to determine whether daikon CBCP possesses carboxymonopeptidase activity, the quenched fluorogenic substrate o-aminobenzoyl (Abz)- Phe-Arg-Phe(4NO 2 )-OH, which contains a fluorescent N-terminal Abz group internally quenched by interac- tion with a nitrophenylalanyl residue at the P1¢ posi- tion [26], was used. In a control experiment, human cathepsin B activity towards this substrate was also assayed. Na ¨ gler et al. reported that the k cat ⁄ K m (m )1 Æs )1 ) towards this substrate for human cathepsin X was 1.23 · 10 5 at pH 5.0. The k cat ⁄ K m (m )1 Æs )1 ) for daikon CBCP was estimated to be 15 500 (k cat , 1.02 s )1 ; K m , 65.8 lm) (Table 1). On the other hand, the monopeptidyl carboxypeptidase activity of human cathepsin B detected was negligible. These results sug- gested that daikon CBCP, like cathepsin X, possesses monopeptidyl carboxypeptidase activity. Susceptibility to inhibitors The effects of various protease inhibitors on daikon CBCP were investigated. Although the enzyme activity was strongly inhibited by cysteine protease inhibitors such as leupeptin, antipain, E-64, egg white cystatin, chymostatin, Na-p-tosyl-l-lysine chloromethyl ketone (TLCK) and iodoacetamide (Table 2). Phenyl- methanesulfonyl fluoride, pepstatin, soybean trypsin inhibitor, o-phenanthroline and EDTA had no inhibi- tory effects. It should be noted that daikon CBCP, unlike cathepsin B, was not significantly inhibited by 1 lm N-(l-3-trans-propylcarbamoyl-oxirane-2-car- bonyl)-l-isoleucyl-l-proline (CA074). CA074 exhibited 10 4 -fold greater inhibition of cathepsin B than cathep- sin L and cathepsin H [31]. This specificity of CA074 has been attributed to the presence of two histidines in the occluding loop of cathepsin B [16,32]. CA074 is an irreversible inhibitor of cathepsin B, so the second- order rate constants of inactivation of daikon CBCP and human cathepsin B by CA074 were compared. When Boc-Leu-Arg-Arg-MCA was used as a substrate, product formation by daikon CBCP in the presence of CA074 approached an asymptote, indicating that dai- kon CBCP is also inhibited irreversibly by CA074. The second-order rate constants of daikon CBCP and human cathepsin B with CA074 were 57.0 m )1 Æs )1 and 4.02 · 10 5 m )1 Æs )1 , respectively, indicating that daikon CBCP was 7000-fold less sensitive than human cathep- sin B to CA074. Egg cystatin is a competitive reversible inhibitor of papain-like cysteine proteases. The K i value of 9.5 nm for the human cathepsin B–cystatin interaction was found to be consistent with values obtained by others [33]. On the other hand, the K i value for daikon CBCP was estimated to be 38 nm, indicating that daikon CBCP was slightly less sensitive to cystatin than human cathepsin B. Expression of CBCP during germination The expression of CBCP in the germinating embryo was analyzed at both the RNA and protein levels. Cot- yledons of embryos (3, 5 and 8 days after imbibition) were extracted and subjected to gel filtration on Seph- acryl S-200 (Fig. 6A). The Boc-Leu-Arg-Arg-MCA- cleaving activity in the second peak increased following germination, and the activity reached a maximum at Table 2. Effects of various cysteine protease inhibitors on the activity of CBCP. Activities toward Boc-Leu-Arg-Arg-MCA were assayed at pH 5.5 in the presence of various inhibitors. Protease inhibitor Concentration % Inhibition Leupeptin 1 l M 98.4 Antipain 1 l M 98.3 E-64 1 l M 95.4 CA074 1 l M 21.0 Egg cystatin 1 l M 92.7 Chymostatin 1 l M 57.2 TLCK 1 l M 80.4 Iodoacetamide 2 m M 90.6 A. Tsuji et al. Characterization of daikon CBCP FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS 5437 5 days after imbibition. The expression of CBCP was analyzed at the protein level using cystatin–Sepharose [34]. As shown in Fig. 6B, a single 28-kDa protein was clearly detected from cotyledons obtained 8 days after imbibition. From cotyledons obtained 3 days after imbibition, a faint 28 kDa band and additional bands were also detected. A clear 28 kDa band and addi- tional bands were observed from cotyledons obtained 5 days after imbibition. The identity of the 28 kDa protein was confirmed by amino acid sequencing, and the sequence LPXSFDAE, identical to the N-terminal sequence of CBCP, was obtained. Other sequences were not present. The time-course of cysteine protease activity assayed using Boc-Leu-Arg-Arg-MCA and expression of the 28 kDa protein are shown in Fig. 6C. Expression of mature CABP at 5 and 8 days after imbibition increased approximately threefold relative to that observed at 3 days after imbibition. Although the enzyme activity reached a maximum at 5 days and subsequently decreased, the expression levels of the 28 kDa protein at 5 and 8 days after imbi- bition were almost the same. The cystatin–Sepharose protein-binding profiles indicated the presence of other cysteine proteases that have smaller molecular masses than CBCP. It is likely that, as the expression of other cysteine proteases decreased markedly at 8 days, the Boc-Leu-Arg-Arg-MCA-cleaving activity also decreased. In an effort to determine whether the increase in CBCP activity results from transcriptional or post-transcriptional events, the expression of CBCP transcript was investigated by RT-PCR. As shown in Fig. 6D, the level of CBCP transcript in cotyledons was highest at 2 days after imbibition and decreased gradually during days 3–8. It is therefore highly likely that the expression of CBCP activity in cotyledons during germination is controlled by post-transcrip- tional events. Discussion The current investigation highlighted the cleavage spec- ificity and inhibitor sensitivity of daikon CBCP, which possesses a short occluding loop lacking a dihistidine motif. The unique occluding loop structure of cathep- sin B, which was previously used to identify the enzyme, also represents the point at which the cathep- sin B-specific inhibitor CA074 acts [16,32]. The occlud- ing loop plays an important role in the dipeptidyl carboxypeptidase activity of cathepsin B and interac- tion of the propeptide with the active site [13–15]. Deletion of the entire loop abolished exopeptidase activity while conserving endopeptidase activity [13]. AB D C Fig. 6. Expression analysis of daikon CBCP during germination. (A) Gel filtration of extract derived from daikon cotyledons (25 g) following imbibition (3, 5 and 8 days). The Boc-Leu-Arg-Arg-MCA-hydrolyzing activity at pH 5.5 was assayed. Fractions indicated by the horizontal bar were pooled, concentrated and used for cystatin–Sepharose analysis. (B) Identification of cysteine proteases by cystatin–Sepharose analysis. Proteins bound to cystatin–Sepharose were separated by SDS ⁄ PAGE (12% gel) and sequenced as described in Experimental procedures. (C) The Boc-Leu-Arg-Arg-MCA-hydrolyzing activity in the second peak in (A) and the intensities of the 28 kDa band in (B) are plotted against days after imbibition. (D) RT-PCR analysis of CBCP transcript in cotyledons of germinating embryos (0, 1, 2, 3, 4, 5, 6 and 8 days after imbi- bition). CBCP cDNA encoding the catalytic domain was amplified by PCR as described in Experimental procedures. Actin 2 transcript was amplified as an internal control. Characterization of daikon CBCP A. Tsuji et al. 5438 FEBS Journal 275 (2008) 5429–5443 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... method of Anastasi et al [38], and immobilized on Sepharose 4B [34] Purified human cathepsin B from liver was from Sigma (St Louis, MO, USA) All other chemicals used were of analytical grade Plants Fresh sprouts of daikon radish (R sativus) grown by hydroponics were purchased from a grocery store and used for purification of the enzyme For analysis of expression of the enzyme during germination, daikon. .. with enzyme, and substrate hydrolysis was monitored by excitation at 320 nm and emission at 420 nm at 25 °C using a Hitachi F-2000 spectrofluorometer Protein was determined by the method of Bradford, using BSA as a standard [40] Purification of cathepsin B-like cysteine protease from daikon radish All purification procedures were performed at 4 °C unless otherwise indicated Cotyledons of daikon radish... characteristics of cathepsin B-like proteins from nematodes: inferred functional diversity and phylogenetic relationship Mol Biochem Parasitol 102, 297–310 Sajid M & McKerrow JH (2002) Cysteine proteases of parasitic organisms Mol Biol Parasitol 120, 1–21 Dacks JB, Kuru T, Liapounova NA & Gedamu L (2008) Phylogenetic and primary sequence characterization of cathepsin B cysteine proteases from the oxymonad... acidification of the vacuole, followed by activation of cysteine proteases by removal of the propeptide [35,36] The autoprocessing activity of human cathepsin B is impaired following deletion of the occluding loop [13] The affinity of the propeptide of cathepsin B for the loop-deletion mutant of cathepsin B was higher than for the wild-type enzyme As shown in Fig 6, the increase in CBCP activity in cotyledons. .. region of the catalytic domain of plant CBCP The sequences of the sense and antisense primers were 5¢-CTACCTAAATCTTTTGATGCTAGAAC-3¢ and 5¢-TTCTTGCTTGAAGGCAAACCAGC-3¢, respectively Characterization of daikon CBCP Total RNA was isolated from the cotyledons of daikon radish 5 days after imbibition using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol One microgram of total... between Trypanosoma and daikon CBCP First, the segment of 11 amino acids between the two cysteines in the occluding loop of TcoCBc1 and TcoCBc6 is longer than that of daikon CBCP (four amino acids), although the amino acid sequences of CBCPs from T congolense are homologous with daikon CBCP (39% amino acid identity) Second, the inhibitor sensitivities of Trypanosoma CBCP and daikon CBCP differ markedly... annotation of a full-length Arabidopsis cDNA collection Science 296, 141–145 2 Schaller A (2004) A cut above the rest: regulatory function of plant proteases Planta 220, 183–197 3 Asano M, Suzuki S, Kawai M, Miwa T & Shibai H (1999) Characterization of novel cysteine proteases from germinating cotyledons of soybean [Glycine max (L.) Merrill] J Biochem 126, 296–301 4 Koehler S & Ho T-H D (1988) Purification and. .. or cystatin, but was inhibited by CA074 and E-64 These results indicate that CBCP from T congolense is an atypical CBCP in terms of its enzymatic properties Therefore, TcoCB6 and daikon CBCP are distinct from one another in terms of the role of the one histidine in the occluding loop As shown in Figs 3 and 4, the amino acids around the subsites of cathepsin B and the unusual occluding loop structure... Juliano L & Turk D (2000) Crystal structure of cathepsin X: a flip-flop of the ring of H23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease Structure 8, 305–313 ´ Sivaraman J, Nagler DK, Zhang R, Menard R & ¨ Cygler M (2000) Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine J Mol Biol 295, 939–951 Towatari... acids at the P1, P2, P3, P1¢ and P2¢ substrate positions was not observed for CBCP The sensitivity of cathepsin B and CBCP to CA074 clearly differed CA074 is a strong, irreversible inhibitor of cathepsin B [16,31,32] The second-order rate constant of inactivation for CBCP (57.0 m)1Æs)1) was Characterization of daikon CBCP markedly lower (7000-fold) than the constant for cathepsin B (4.02 · 105 m)1Æs)1), . Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus Akihiko Tsuji,. determined by the method of Bradford, using BSA as a standard [40]. Purification of cathepsin B-like cysteine protease from daikon radish All purification procedures