Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp. Tina Kyndt 1,2 , Els J. M. Van Damme 1 , Jozef Van Beeumen 3 and Godelieve Gheysen 1,2 1 Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Belgium 2 Institute for Plant Biotechnology for Developing Countries (IPBO), Ghent University, Belgium 3 Laboratory of Protein Biochemistry and Protein Engineering, Ghent, Belgium Articulated laticifers, containing a milky latex, are pre- sent in all organs of members of the small plant family Caricaceae [1]. The two economically most important genera of this family are the commonly grown tropical species Carica papaya and the group of highland papa- yas (Vasconcellea spp.), of which many are locally tol- erated and ⁄ or semicultivated for their fruit production. Although they used to be classified in one single genus (Carica), recent phenetic and phylogenetic results [2] have shown a clear separation between C. papaya and the 21 species of Vasconcellea, confirming their classifi- cation into two separate genera [3]. Experimental evidence has shown that latex gener- ally contributes to protecting the plant against pre- dators [4,5] in both a mechanical (by wound coagulation) and chemical (by the presence of toxic substances) manner. While proteinase inhibitors (PIs) are generally believed to actively contribute to plant defence mechanisms [6], Konno et al. [5] recently pro- vided evidence that the cysteine proteinases (and not the proteinase inhibitors) stored in the laticifers of papaya are the active compounds in its defence against herbivorous insects. Caricaceae latex contains huge amounts of cysteine proteinases: up to 30% of Keywords Caricaceae; cDNA cloning; cysteine proteinase; latex; purification Correspondence G. Gheysen, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium Fax: +32 92646219 Tel: +32 92645888 E-mail: godelieve.gheysen@ugent.be (Received 24 July 2006, revised 19 October 2006, accepted 13 November 2006) doi:10.1111/j.1742-4658.2006.05592.x Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activ- ity is correlated with a higher concentration of enzymes in the latex of Vas- concellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases pre- sent in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investiga- tion, several cDNA sequences coding for cysteine proteinases in Vasconcel- lea · heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. · heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I–VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkatÆmg protein )1 ) were isolated. After further purifi- cation, three of them were N-terminally sequenced. The observed microhet- erogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp. Abbreviations AA, amino acid; BAPNA, a-N-benzoyl- L-arginine 4-nitroanilide; MP, maximum parsimony; PI, proteinase inhibitor; VXH, Vasconcellea · heilbornii. FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 451 the total latex [3], at a molar concentration that probably exceeds 1 mm [7]. Cysteine proteinases are proteolytic enzymes that depend upon a cysteine resi- due for activity. Within this group of enzymes, at least seven different evolutionary origins have been determined, allocating them to seven clans, each con- sisting of several related families [8]. Because of their economic importance in the bever- age, food and pharmaceutical industries, constituents of the latex of C. papaya have been investigated thor- oughly. The four cysteine proteinases present in the latex of C. papaya, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (formerly known as proteinase W; EC 3.4.22.30) and glycyl endopepti- dase (or papaya proteinase IV; EC 3.4.22.25), all belong to the C1 family of clan CA, the largest clan of cysteine peptidases. Although papain is a minor con- stituent among the papaya proteinases, this latex enzyme has been most extensively studied in the past [9,10]. Amino acid sequences of papaya cysteine pro- teinases have been determined both at the protein level [9,11,12] and through sequencing of the corresponding cDNA clones [13–16]. Two similar but distinct cDNAs have been shown to code for caricain [14] and at least five similar but distinct cDNAs code for chymopapain [16]. In the genus Vasconcellea, only the latex of Vas- concellea cundinamarcensis has been studied in detail until now. These studies [17–20] suggested the existence of six to seven cysteine proteinases in latex from V. cundinamarcensis, some of which may be isoforms. Five of them were sequenced at the amino acid and ⁄ or nucleotide level. It has been reported [21] that the activity of freeze-dried latex from this species was between five- and eight-fold higher than that of C. papaya latex, while crude babaco (Vasconcellea · heilbornii ‘babaco’) latex revealed an equivalent or slightly higher proteolytic and lipolytic activity than that of papaya [22]. In a larger study, comparing the proteolytic activity of C. papaya latex with that from Vasconcellea stipulata, some V. · heilbornii genotypes, babaco, and V. cundinamarcensis [23], a four- to 13- fold higher proteolytic activity was reported for these Vasconcellea spp. Even though the different studies are not consistent about the level of proteolytic activity, probably due to varying experimental conditions, they confirm the potential of Vasconcellea spp. for commer- cial proteinase production. In this study, we report on the identification and characterization of cDNA sequences coding for cys- teine proteinases in the latex of V. stipulata and V. · heilbornii. Based on these sequences, the evolution of the cysteine proteinases within the Caricaceae was investigated. Furthermore, proteolytic enzymes were purified from the latex of V. · heilbornii and the N-ter- minal amino acid sequence characterized. Results and Discussion Amidase activity of total latex Proteolytic activity, measured as amidase activity per milligram dried latex using the BAPNA (a-N-benzoyl- l-arginine 4-nitroanilide) substrate, was evaluated by Scheldeman et al . [23] for several Vasconcellea spe- cies. They reported that Vasconcellea cundinamarcensis, V. stipulata and V. · heilbornii latex show a proteolytic activity that is approximately 4–13 times higher than the papaya reference. Two factors might play a role in the higher activity observed in Vasconcellea spp.: (1) a higher protein content in their latex; and (2) the pres- ence of other cysteine proteinases or isoforms in the latex. To investigate these two hypotheses, the protein concentration per milligram of dried latex, as well as the amidase activity per milligram of protein, was measured for three species (Table 1). The results show that the protein concentration in Vasconcellea latex is indeed slightly higher than in the papaya reference. Proteolytic activity, measured by BAPNA degradation and expressed as amounts of nkat per milligram pro- tein (where nkat is amount of enzyme that hydrolyses 1 nmol BAPNA per second), is found to be 1.25 to two times higher in latex of Vasconcellea fruits than in latex of C. papaya. Hence, when analyzing equal amounts of protein, the latex of Vasconcellea fruits still displays a stronger proteolytic effect, although the differences are not as pronounced as reported by Scheldeman et al. [23]. However, our results are obtained with latex from one single plant of each spe- cies, grown under suboptimal, but consistent, green- house conditions, while Scheldeman et al. [23] investigated several (2–8) wild plants of each species. It is possible that wild plants reveal a higher proteolytic activity or protein content than greenhouse plants. In addition, it has been demonstrated that repeated mechanical wounding of the fruit profoundly affects Table 1. Protein concentration and amidase activity of latex of Vasconcellea and C. papaya fruits. Species Protein concentration (mgÆg latex )1 ) Proteolytic activity (nkatÆmg protein )1 ) Proteolytic activity (nkatÆmg latex )1 ) C. papaya 26.69 6.50 0.17 V. monoica 36.31 8.09 0.29 V. stipulata 40.57 12.98 0.53 V. · heilbornii 31.75 8.65 0.27 Cysteine proteinases of Vasconcellea spp. T. Kyndt et al. 452 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS the protein content and activation of proteolytic enzymes in its latex [24]. While the fruits used in this experiment were all tapped for the first time, we are not aware of the frequency of tapping in other studies. These and probably several other, as yet unknown, factors affecting the activity and concentration of latex proteins complicate comparisons with previous studies. Our results (Table 1), obtained using equal condi- tions for all plants, clearly show that the higher pro- teolytic activity is only to a certain extent due to a higher protein concentration in latex of Vasconcellea fruit. The presence of other, possibly more proteolyti- cally active enzymes will be evaluated in the following experiments. cDNAs coding for cysteine proteinases in V. · heilbornii and V. stipulata Using a PCR approach with a cysteine proteinase primer (CyPr), specifically designed for this study, different cDNAs were isolated from the fruits of V. · heilbornii: VXH-A, VXH-B, VXH-C, VXH-D, and from that of V. stipulata: VS-A and VS-B. The amino acid sequence of all six cDNA-sequences was deduced in silico and analyzed. A detailed comparison was made with the available sequence data for cysteine proteinases from Caricaceae found in the GenBank database (Fig. 1). These sequences included complete cDNAs from papain, glycyl endopeptidase, two iso- forms of caricain and five isoforms of chymopapain from C. papaya. From the genus Vasconcellea, only the latex of V. cundinamarcensis has previously been studied in detail. Isolation and preliminary characteri- zation of the cysteine proteinases using ion-exchange chromatography showed four enzymatically active peaks in its latex, designated CC-I to CC-IV [17]. CC-III was almost completely sequenced [19] and has been suggested to correspond to chymopapain from papaya [17]. Further purification of the heterogeneous CC-I [18] into the two closely related components CC-Ia and CC-Ib was carried out using reverse-phase HPLC under denaturing conditions. Amino acid sequencing of both CC-Ia and CC-Ib confirmed their equivalence with papain. Based on the observation that CC-I and papain have catalytic constants of the same order of magnitude on BAPNA and chromozyme [17], the marked increase in proteolytic activity of V. cundi- namarcensis latex can be explained by the expression of several molecular forms of CC-I, in contrast to the single papain in papaya [18]. Another cysteine protein- ase, CC-23, was purified from V. cundinamarcensis latex and its corresponding DNA fragment cloned by Pereira et al. [20]. The N-terminal sequence appeared to be different from the N-terminal sequences reported for CC-I to CC-IV. The authors suggested the existence of six to seven cysteine proteinases in latex from V. cun- dinamarcensis, some of which may be isoforms, as in the case of chymopapain. For V. cundinamarcensis, one incomplete DNA-sequence (no stop codon) called CC-23, and five amino acid sequences were traced in the database: CC-Ia, CC-Ib, CC-II, CC-III, CC-IV. CC-II and CC-IV were only N-terminally sequenced [17]. Although CC-Ia (213 amino acids), CC-Ib (213 amino acids) and CC-III (214 amino acids) have a calculated mass corresponding to mass spectrometric results [18,19], they might be incomplete at the 3¢ end. Papaya proteinases are naturally synthesized with N-terminal signal and pro-peptides. The pro-regions aid the folding of the mature enzymes and act as selective high affinity inhibitors to prevent inappropri- ate proteolysis within the plant [25]. The enzymes are present in the latex as inactive precursors and are activated in response to wounding of the plant [26]. Similar to other known cysteine proteinases from Car- icaceae, the deduced protein sequences of VXH-A, -B, -C and -D, and VS-A and -B, were predicted to con- tain a signal peptide. For most of them, this signal peptide contains 26 amino acids. However, the signal peptide prediction software SignalP predicts VXH-B to be cleaved after 22 residues. The proregion of the pri- mary translation products contains 108 amino acids (or 112 amino acids in the case of VXH-B). Only for VS-A, a stop codon was found in the cDNA-sequence obtained. This cDNA encodes a mature protein of 188 amino acids with a calculated molecular weight of 20.3 kDa. As no stop codon was found in the other sequences, it is very likely that these sequences are not complete at the 3¢ end. The characteristic features of all known papain class cysteine proteinases are present in the amino acid sequences from V. stipulata and V. · heilbornii: the cat- alytic triad C 25 ,H 159 ,N 175 , stabilizer Q 19 ,D 158 (yellow stars in Fig. 1), six cysteines forming three disulfide bridges (blue stars in Fig. 1), as well as the well-con- served 173 IKNSWG 178 motif (numbering of amino acids according to their position in mature papain). One putative N-glycosylation site occurs in the prore- gion of VS-A and VXH-A: 115 NWS 117 . The glycan in the propeptide might aid in the protection against deg- radation, or in targeting or maturation of the enzyme, as was shown before in cathepsin C, which also belongs to the papain family of cysteine proteinases [27]. The overall similarity at the amino acid level between all known cysteine proteinases from Carica- ceae is 73%. Tables 2 and 3 show the percentage sequence similarity between the obtained cDNA T. Kyndt et al. Cysteine proteinases of Vasconcellea spp. FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 453 Fig. 1. Alignment of translated cDNAs of V. stipulata (VS), V. · heilbornii (VXH), V. cundinamarcensis (CC) and C. papaya. The transition between signal peptide and proregion is indicated by a red arrow. The black arrow shows the beginning of the mature enzyme. Cysteine res- idues involved in disulfide bridges are indicated with blue stars. Yellow stars show amino acids which are important for proteolytic activity. Cysteine proteinases of Vasconcellea spp. T. Kyndt et al. 454 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS sequences and all known cysteine proteinases from the Caricaceae. VXH-A and VS-A are almost identical (99%) at the amino acid and nucleotide level. A single amino acid substitution is present at position 122 of VXH-A, where a serine is replaced by a proline in VS-A. As is the case for CC-Ia, CC-Ib [18], CC23 [20], CCIII [19], and papain [11], VS-A, VS-B and VXH-A lack the insertion of four amino acids between position 168 and 169, which is present in all other cysteine proteinases of plant or animal origin. Unfortunately, Table 2. Percentage sequence similarity between the cDNA sequences of V. stipulata and V. · heilbornii, and known cysteine proteinases from Caricaceae. Values >85% are indicated in bold. Chymo, Chymopapain; Gly endo, glycyl endopeptidase. CC-Ia CC-Ib CC-II CC-III CC-IV CC23 Chymo ChymoII ChymoIII ChymoIV ChymoV Papain Caricain CaricainII Gly endo VS-A 90.4 95.2 76.7 63.4 76.7 65.3 61.5 61.5 61.2 61.5 60.1 69.2 70.6 70.9 68.1 VS-B 61.5 63.9 93.0 82.1 86.0 88.6 71.4 71.1 70.8 72.6 70.8 60.8 66.6 66.9 66.6 VXH-A 91.6 96.1 76.7 63.5 76.7 65.1 61.2 61.2 60.9 61.2 59.8 69.8 71.3 71.6 68.1 VXH-B 93.1 87.8 76.7 61.4 74.4 65.4 61.7 61.3 60.9 62.6 59.8 68.3 70.5 70.8 67.4 VXH-C 59.9 61.2 97.7 75.7 81.4 82.2 67.7 67.4 67.0 64.6 62.8 61.2 63.6 63.9 62.5 VXH-D 89.4 100.0 76.7 61.3 74.4 64.3 61.6 61.6 61.2 61.7 59.9 67.3 69.0 69.3 66.1 Fig. 1. (Continued). T. Kyndt et al. Cysteine proteinases of Vasconcellea spp. FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 455 this part of the sequence is not available for CC-II and CC-IV from V. cundinamarcensis and VXH-B, C and D. However, based on the close evolutionary relation- ships between Vasconcellea spp. [2]., we assume that other cysteine proteinases from this genus will also have this deletion. Molecular evolution of cysteine proteinases in Caricaceae The evolutionary relationships between the amino acid sequences are represented by the 50% Majority Rule Consensus tree of 3 Maximum Parsimonious (MP) trees shown in Fig. 2. The relatively high bootstrap values express a high degree of confidence in the gener- ated clustering. Glycyl endopeptidase was chosen as the outgroup because of its low degree of similarity with the other sequences. The MP tree reveals three major clusters. Cluster I combines the five chymo- papain isoforms of C. papaya with VXH-C, VS-B, CC-III and CC23. These results confirm the hypothesis of Walraevens et al. [17] that CC-III is a chymopapain homologue and predict VXH-C and VS-B to be the corresponding genes in V. · heilbornii and V. stipulata, respectively. In addition, this suggests CC23 to be a Table 3. Percentage pairwise sequence similarity between the cys- teine proteinase cDNA sequences of V. stipulata and V. · heilbornii. Values higher than 85% are indicated in bold. VS-A VS-B VXH-A VXH-B VXH-C VXH-D VS-A 100.0 VS-B 66.1 100.0 VXH-A 99.7 67.3 100.0 VXH-B 91.0 64.7 91.3 100.0 VXH-C 63.8 86.5 64.4 63.0 100.0 VXH-D 95.7 65.9 96.0 91.4 63.0 100.0 Fig. 2. The 50% Majority Rule Consensus tree of three Maximum parsimonious trees of cysteine proteinases of V. stipulata (VS), V. · heilbornii (VXH), V. cundinamarcensis (CC) and C. papaya. (Tree length ¼ 498, consistency index ¼ 0.8193, retention index ¼ 0.8811). Bootstrap values are indi- cated above the branches. Cysteine proteinases of Vasconcellea spp. T. Kyndt et al. 456 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS paralogue of CC-III, possibly also coding for a chymo- papain-like enzyme. Cluster II contains papain, next to VXH-A, VXH-B, VXH-D, VS-A, CC-Ia and CC-Ib, suggesting them to be papain homologues. Earlier observations [17,18] already suggested CC-I (containing CC-Ia and CC-Ib) to be the heterogenic papain homologue of V. cundina- marcensis. This heterogeneity was suggested to be responsible for the higher enzymatic activity found in latex of V. cundinamarcensis [18]. Our study reveals three predicted papain homologues in the highly enzy- matically active V. · heilbornii latex, but only one in V. stipulata. However, it is possible that further searches will reveal more than one papain homologue in the latex of V. stipulata. Although the different parologues and orthologues make the picture rather complex, the close phylo- genetic relationship between the cysteine proteinase sequences from V. cundinamarcensis and the newly determined sequence data from V. · heilbornii and V. stipulata, again confirm the evolutionary divergence between C. papaya and the Vasconcellea spp. [2]. From the proposed evolutionary relationships of cys- teine proteinases it can be deduced that the common ancestor of the genera Carica and Vasconcellea already contained at least two different cysteine pro- teinases in its latex (papain and chymopapain). After the divergence of these genera, their genes have evolved into different paralogues. Within the genus Vasconcellea, the evolutionary pathway of cysteine proteinases are probably obscured by different factors: (1) the close relationship between the three analyzed species, with V. stipulata and V. cundinamarcensis being involved in the hybrid origin of V. · heilbornii [28], and (2) their reported recent speciation [2]. Since no caricain or glycyl endopeptidase homologues have yet been found in V. cundinamarcensis, V. stipulata and V. · heilbornii, it is not possible to draw conclu- sions about their evolution. Fractionation of the proteinases from V. · heilbornii In an attempt to purify the proteinases from V. · heil- bornii, latex was collected and subjected to ion exchange chromatography. Figure 3 displays a typical elution profile from the Mono S 5 ⁄ 50 GL column of the total dialysed soluble fraction of the latex of V. · heilbornii. In general, five peaks with apparent microheterogeneity can be distinguished: VXH-I, VXH-II, VXH-III, VXH-IV and VXH-V, all showing amidase activity (Table 4). Amidase activity was inhib- ited by the addition of the cysteine proteinase inhibitor E-64. This observation clearly showed that the proteo- lytic activity was due to cysteine proteinases solely. Specific amidase activity of VXH-I toVXH-V ranged from 7.5 to 23.3 nkatÆmg protein )1 , which is 4.5- to 14-fold higher than the activity of chymopapain from papaya latex (1.68 nkatÆmg )1 ) [28]. The activities observed are comparable with the 14.2 nkat ⁄ mg found for CC28 (or CC-IV) from V. cundinamarcensis [29], but significantly lower than the activity of CC23, also found in the latex of this species, which was reported to be 84 nkatÆmg )1 [20]. SDS ⁄ PAGE analysis of the protein fractions obtained after ion exchange chromatography revealed the presence of smaller, contaminating polypeptides next to proteins of expected molecular size for cysteine proteinases (results not shown). Therefore, additional chromatographic steps had to be performed to remove these small polypeptides. Unfortunately, Vasconcellea plants growing in a greenhouse produce small fruits that contain only low amounts of latex. In addition, as they do not bear L Fig. 3. Ion exchange chromatography of V. · heilbornii latex on Mono S 5 ⁄ 50 GL column. Buffer: 50 m M NaAc, pH 5.0; flow rate: 2 mLÆmin )1 ; gradient: 0–1 M NaCl, pH 5.0. The triangles show absorbance (A 280 ) of each fraction. The black line repre- sents the conductivity. T. Kyndt et al. Cysteine proteinases of Vasconcellea spp. FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 457 fruits all year round, our purification was hampered by the limited amount of starting material available. Therefore, fractions VXH-I–VXH-V from the first ion exchange chromatography were pooled, and rechroma- tographed on a gel filtration column in an attempt to remove the smaller proteins. Size-exclusion chromato- graphy yielded essentially one large peak (data not shown) which was divided into pools A and B. Whereas the later fractions (pool B) revealed two pro- tein bands of 27 and 30 kDa after SDS ⁄ PAGE, the earlier fractions (pool A) showed an extra protein band of higher molecular weight (33 kDa) (Fig. 4). SDS ⁄ PAGE results confirm that the smaller contamin- ating proteins have been removed after gel filtration. The size of the polypeptides present in pools A and B is equivalent to or slightly larger than the molecular mass reported for papain (23 kDa), chymopapain (27 kDa) [30], caricain (24 kDa) [11], CC-IV (28 kDa) [29] and CC23 (23 kDa) [20]. Subsequently, the pro- teins in pools A and B were re-fractionated using ion- exchange chromatography (Fig. 5A,B). Pool A yielded three major peaks. Comparison of the elution profiles in Figs 3 and 5A,B suggests that these peaks corres- pond to VXH-I, VXH-III and the second part of VXH-IV (VXH-VIb). Pool B contained only two peaks (Fig. 5B), corresponding to VXH-III and the earlier part of VXH-IV (VXH-IVa). Apparently peaks VXH-II and VXH-V are not present in pools A and B after the gel filtration analysis. Four of these recovered peaks were selected for N-terminal protein sequence analysis: VXH-I and VXH-IVb from pool A, VXH-III and VXH-IVa from pool B. The microheterogeneity observed during all purifica- tion steps, indicating that there are multiple isoforms of the proteolytic enzymes in the latex of V. · heilbor- nii, was previously also reported for C. papaya [3] and V. cundinamarcensis [18]. Comparison of N-terminal sequences of V. · heilbornii cysteine proteinases The N-terminal amino acid sequences of the proteins VXH-I, VXH-III, VXH-IVa and VXH-IVb are shown in Fig. 6, and are compared with the known N-terminal sequences of the cysteine proteinases of C. papaya and V. cundinamarcensis. The cysteine pro- teinases of V. · heilbornii hold the generally conserved P 2 ,Q 19 and the 11 GAVTP 15 -motif located at the N-ter- minus of papain-like cysteine proteinases, leaving no doubt that these proteins belong to the papain super- family. Sequencing of VXH-I and VXH-III yielded two signals of equal intensity at positions 7 and 17, respectively, suggesting that these pools might contain different isoforms. The N-terminal sequences of VXH- IVa and VXH-IVb reveal different amino acids at positions 9, 18 and 20, confirming that peak VXH-IV holds at least two different cysteine proteinases. All N-terminal sequences show between 65 and 100% similarity, with an average of 80%. Such a high degree of homology makes it difficult to decide which form of VXH corresponds to which papaya or V. cun- dinamarcensis proteinase. The identical N-terminal sequence of VXH-IVb, CC-III and CC-IV suggests that these might be homologous proteins, but complete sequencing is necessary to confirm this result. Based on the 100% identity between the deduced amino acid sequence of VXH-C and the N-terminal sequence of VXH-I (Fig. 1) we can assume that cDNA clone VXH-C encodes VXH-I. Table 4. Amidase activity of pooled fractions VXH-I–VXH-V, meas- ured by BAPNA degradation. nkat: amount of enzyme that hydro- lyses 1 nmol BAPNA per second. Pool nkatÆmg protein )1 VXH-I 23.3 VXH-II 12.5 VXH-III 22.1 VXH-IV 15.0 VXH-V 7.5 Fig. 4. SDS ⁄ PAGE electrophoresis of pool A and pool B from the latex of V. · heilbornii. M: Protein molecular weight marker; mas- ses are indicated on the left. Cysteine proteinases of Vasconcellea spp. T. Kyndt et al. 458 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS Conclusion This study confirms a higher degree of proteolytic activity in the latex of three Vasconcellea spp. in com- parison with C. papaya. This is due to a higher protein content and the presence of other, more active, cys- teine proteinases in their latex. Fractionation of V. · heilbornii latex revealed that this species contains several highly proteolytic cysteine proteinases. Further- more, sequence analyzes at the amino acid and cDNA level showed a high degree of homology between cys- teine proteinases from different species of Caricaceae. The large number of different cDNA-sequences, and the observed microheterogeneity during the purifica- tion procedure, imply that V. stipulata and V. · heil- bornii express several isoforms of cysteine proteinases in their latex, which may be responsible for the higher proteolytic activity. The amount of latex that can be collected from the (generally smaller) fruits of the wild Vasconcellea plants is definitely lower than the latex yield of papaya (personal observations). Consequently, future Vasconcellea breeding programmes should select for varieties with a higher latex yield, to obtain a commer- cially interesting latex production. Experimental procedures Latex collection Unripe fruits of plants grown in the greenhouse were the source of latex used in this study. Latex was collected by making several incisions into the surface of the unripe fruit using a sharp blade. An equal volume of 100 mm thio- ureum was added to avoid oxidation, as recommended by Azarkan et al. [31], and the latex was stored at )20 °Cin the dark, until use. Electrophoresis Protein samples were electrophoresed in 15% polyacryl- amide denaturing gels (SDS ⁄ PAGE) [32] after boiling for 5 min at 95 °C. Electrophoresis was performed for 90 min at 150 V. Gels were stained with 0.1% Coomassie blue A B L L Fig. 5. Second ion exchange chromatography after size-selection of pool B from the latex of V. · heilbornii. Columns: mono S 5 ⁄ 50 GL; buffer: 50 m M sodium acetate, pH 5.0; flow rate: 2 mLÆmin )1 ; gradient: 0–1 M NaCl, pH 5.0. The triangles show absorbance (A 280 ) of each fraction. The black line indicates the conductivity. (A) Fractionation of pool A; (B) fractionation of pool B. T. Kyndt et al. Cysteine proteinases of Vasconcellea spp. FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 459 R-250 dissolved in 42% methanol)17% acetic acid fol- lowed by destaining in 15% ethanol)7.5% acetic acid. Amidase activity For analysis of amidase activity, 80 lL of the sample was preincubated for 10 min at 37 °C in 100 lL activation buf- fer, containing 2.5 mm dithiothreitol, 25 mml-cysteine, 5mm EDTA, 100 mm sodium phosphate, 100 mm sodium citrate and 100 mm borate in 25 mm Tris ⁄ HCl, pH 7. The enzymatic hydrolysis of l-BAPNA substrate (Sigma- Aldrich, Steinheim, Germany) was measured following incubation with 20 lL10mm BAPNA (in 1% dimethyl sulfoxide) for 20–60 min at 37 °C. The reaction was stopped by adding 20 lL 50% acetic acid, and the release of 4-nitro- aniline was determined spectrophotometrically at 410 nm (e 410 ¼ 8800 mol )1 Æcm )1 ). The hydrolysis time was adjusted in order to avoid A 410 exceeding 0.80. One unit of activity (nkat) is the amount of enzyme that hydrolyses 1 nmol of substrate per second under the above-cited conditions. Cloning and sequencing of cysteine proteinase cDNAs RNA was extracted using the Qiagen Rneasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from unripe fruits. The RNA was transformed into double-stranded cDNA by LD PCR with the Creator SMART TM cDNA Library Con- struction kit (BD Biosciences, Heidelberg, Germany). Dur- ing this procedure, adapters are ligated to the 5¢ and 3¢ end of the cDNAs. The 3¢ ends of cysteine proteinases were spe- cifically amplified using an internal cysteine proteinase specific primer (CyPr: 5¢-AAGGAGCYGTNACTCCT GTAA-3¢), derived from a central conserved region present in all known cysteine proteinases from Caricaceae, and a primer complementary to the previously built-in 3¢ adapt- ers. The total PCR volume of 20 lL consisted of 2 lL10· diluted cDNA (from LD PCR), 2 lL CyPr-primer (10 lm), 2 lL3¢ adapter primer (10 lm), 2 lL Pfx amplification buf- fer (Invitrogen, Paisley, UK), 2 lL dNTPs (5 mm), 0.4 lL Pfx polymerase, 0.4 lL MgSO 4 (50 mm) and 9.2 lL water. The PCR programme involved an initial denaturation for 4 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 90 s at 68 °C, and a terminal extension of 10 min at 68 °C. PCR products were separated on 1% agarose 0.5· TAE (20 mm Tris-Acetate, 0.5 mm EDTA) gels and purified using the QIAquick Gel Extraction Kit (Qiagen). As the high-fidelity Pfx polymerase was used in the PCR reaction, terminal 3¢ A-ends had to be added to the PCR products before they were cloned into pGEM-T plasmids (Promega Benelux, Leiden, The Netherlands) following the manufac- turer’s instructions. Insert sequencing was carried out in both directions using T7 and SP6 primers. Inserts were se- quenced using the ABI prism BigDye TM Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an automated sequencer (ABI prism 377, Applied Biosystems). Subsequently, 5¢ ends of each 3 ¢ sequence were amplified selectively by using a sequence- specific primer, in combination with a primer based on the previously built-in 5¢ adapters of the double stranded cDNA. PCR conditions and followed procedures were iden- tical to those used during sequencing of the 3¢ end. cDNA-sequences obtained were submitted to the Gen- Bank database (DQ836121-DQ836126). They were trans- lated in silico using bioedit 7.0.1 [33]. signalp and netnglyc (Expasy Proteomics Server) were used to predict the presence of signal peptides and possible N-glycosylation sites, respectively. All available cysteine proteinase amino acid sequences from Caricaceae were aligned using bioedit 7.0.1. CC-II and CC-IV sequences were deleted from the dataset because their short sequences might result in incorrect phylogenetic analyz- es. After deletion of constant characters from the alignment, parsimony analyzes were performed with paup* v4.0b10 [34] using the heuristic search option with random sequence addi- tion (100 random replications) and TBR branch-swapping. The sequence of Glycyl endopeptidase was used as an out- group. The consistency index and retention index were calcu- lated. Support for the different clades was tested by bootstrap analysis (100 replicates using heuristic search, random sequence addition and TBR branch-swapping). Fig. 6. N-Terminal amino acid sequences of proteinases from V. · heilbornii (VXH) and V. stipulata (VS), compared with the known sequences from V. cundinamarcensis (CC) and C. papaya (Cp). In the case of double signals, both amino acids are specified. Cysteine proteinases of Vasconcellea spp. T. Kyndt et al. 460 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... Walraevens V, Vandermeers-Piret M, Vandermeers A, Gourlet P & Robberecht P (1999) Isolation and characterization of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis Hook Biol Chem Hoppe-Seyler 380, 485–488 Jaziri M, Kleinschmidt T, Walraevens V, Schnek AG & Looze Y (1994) Primary structure of CC-III, the glycosylated cysteine proteinase from the latex of Carica candamarcensis... 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 461 Cysteine proteinases of Vasconcellea spp 17 18 19 20 21 22 23 24 25 T Kyndt et al Carica papaya prochymopapain isoforms in Escherichia coli Plant Sci 145, 41–47 Walraevens V, Jaziri M, Van Beeumen J, Schnek AG, Kleinschmidt T & Looze Y (1993) Isolation and characterization of the cysteine- proteinases from the latex of Carica candamarcensis... Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen)’ and by FWO-Vlaanderen (project number 3G005100) We thank W Peumans for his extensive assistance during the protein purification I Vandenberghe is acknowledged for the N-terminal protein sequencing The authors are also grateful to the Department of Plant Production (Ugent) for the greenhouse accommodation provided for the Vasconcellea. .. (3 · 70 cm) The column was run in 20 mm 1,3-diamino propane, pH 11 Ion-exchange chromatography of two pools (A and B) that contained protein bands of the expected mass range was then repeated on the Mono S 5 ⁄ 50 GL column (5 · 50 mm) under the same conditions as described above 3 4 5 6 7 N-Terminal protein sequencing Following SDS ⁄ PAGE and semi-dry blotting of the selected fractions, the samples... papain and papaya proteinase IV are selective high-affinity inhibitors of the mature papaya enzymes Prot Eng 8, 59–62 Moutim V, Silva LG, Lopes MTP, Fernandes GW & Salas CE (1999) Spontaneous processing of peptides during coagulatio of latex from Carica papaya Plant Sci 142, 115–121 Santilman V, Jadot M & Mainferme F (2002) Importance of the propeptide in the biosynthetic maturation of rat cathepsin... C, Wintjens R, Vincentelli J, Azarkan M & Looze Y (2001) Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism Cell Mol Life Sci 58, 556–570 Konno K, Hirayama C, Nakamura M, Tateishi K, Tamura Y, Hattori M & Kohno K (2004) Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex. .. sodium acetate, pH 5.0 The column was eluted using a linear gradient of 0–1 m NaCl in 50 mm sodium acetate (pH 5) Protein concentration and amidase activity of all fractions was measured SDS ⁄ PAGE of fractions with high activity revealed the polypeptide composition As many of smaller contaminating polypeptides were observed, apart from bands of the expected mass (22–30 kDa), further purification was performed... Moussaoui A, Van Wuytswinkel D, Dehon G & Looze Y (2003) Fractionation and purification of the enzymes stored in the latex of Carica papaya J Chrom B 790, 229–238 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment, ed and analysis program for Windows 95 ⁄ 98 ⁄ NT... cysteine peptidases Biol Chem Hoppe-Seyler 382, 727– 733 Mitchell REJ, Chaiken IM & Smith EL (1970) The complete amino acid sequence of papain: additions and corrections J Biol Chem 245, 3485–3492 Drenth J, Jansonius JN, Koekoek R, Swen HM & Wolthers BG (1968) Structure of papain Nature 218, 929–932 Dubois T, Kleinschmidt T, Schnek AG, Looze Y & Braunitzer G (1988) The thiol proteinases from the latex of. ..T Kyndt et al Cysteine proteinases of Vasconcellea spp Chromatographic procedures The latex of V · heilbornii was exhaustively dialyzed against cold H2O, centrifuged for 10 min at 3000 g, and the supernatant filtered through a Whatman paper The filtered solution was chromatographed on an AKTA fplc system (Amersham Biosciences, Uppsala, Sweden) using a Mono S 5 ⁄ 50 GL column (5 · . but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases pre- sent in papaya latex, . their latex; and (2) the pres- ence of other cysteine proteinases or isoforms in the latex. To investigate these two hypotheses, the protein concentration