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
Trang 1proteinases in the latex of Vasconcellea spp.
Tina Kyndt1,2, Els J M Van Damme1, Jozef Van Beeumen3and Godelieve Gheysen1,2
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.
Trang 2the 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
Vasconcelleaspp 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 stipulataand 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)
Trang 3the 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 isoiso-forms 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 C25, H159, N175, stabilizer Q19, D158(yellow stars in Fig 1), six cysteines forming three disulfide bridges (blue stars in Fig 1), as well as the well-con-served 173IKNSWG178 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: 115NWS117 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
Trang 4Fig 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.
Trang 5sequences 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
Fig 1 (Continued).
Trang 6this 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.
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.
Trang 7paralogue 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.
Trang 8fruits 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
P2, Q19and the11GAVTP15-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.
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.
Trang 9This 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 C in the dark, until use
Electrophoresis Protein samples were electrophoresed in 15% polyacryl-amide denaturing gels (SDS⁄ PAGE) [32] after boiling for
5 min at 95C 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.
Trang 10R-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 37C in 100 lL activation
buf-fer, containing 2.5 mm dithiothreitol, 25 mm l-cysteine,
5 mm 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 lL 10 mm BAPNA (in 1% dimethyl
sulfoxide) for 20–60 min at 37C The reaction was stopped
by adding 20 lL 50% acetic acid, and the release of
4-nitro-aniline was determined spectrophotometrically at 410 nm
(e410¼ 8800 mol)1Æcm)1) The hydrolysis time was adjusted
in order to avoid A410exceeding 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 SMARTTMcDNA 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 lL 10· diluted cDNA (from LD PCR), 2 lL CyPr-primer (10 lm),
2 lL 3¢ 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 MgSO4(50 mm) and 9.2 lL water The PCR programme involved an initial denaturation for
4 min at 95C, followed by 30 cycles of 30 s at 95 C, 30 s
at 55C and 90 s at 68 C, and a terminal extension of
10 min at 68C
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 BigDyeTM 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 replicaaddi-tions) 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.