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Purificationandcharacterizationofthe cysteine
proteinases inthelatexofVasconcellea 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 ofthe small plant family
Caricaceae [1]. The two economically most important
genera of this family are the commonly grown tropical
species Carica papaya andthe 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 thecysteineproteinases (and not
the proteinase inhibitors) stored inthe laticifers of
papaya are the active compounds in its defence
against herbivorous insects. Caricaceae latex contains
huge amounts ofcysteine 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. Inthe present study, we show that this higher activ-
ity is correlated with a higher concentration of enzymes inthelatexof Vas-
concellea fruits, but in addition also results from the presence of other
cysteine proteinases or isoforms. In contrast to thecysteineproteinases pre-
sent in papaya latex, which have been extensively studied, very little is
known about thecysteineproteinasesofVasconcelleaspp.In this investiga-
tion, several cDNA sequences coding for cysteineproteinasesin Vasconcel-
lea · heilbornii andVasconcellea 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 latexof V. · heilbornii in
order to characterize its cysteineproteinases 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 inthe N-terminal and cDNA sequences reveals the presence of
several distinct cysteine proteinase isoforms inthelatexofVasconcellea 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]. Cysteineproteinases 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 inthe bever-
age, food and pharmaceutical industries, constituents
of thelatexof C. papaya have been investigated thor-
oughly. The four cysteineproteinases 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 inthe 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 ofthe 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]. Inthe genus Vasconcellea, only thelatexof Vas-
concellea cundinamarcensis has been studied in detail
until now. These studies [17–20] suggested the existence
of six to seven cysteineproteinasesinlatex 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 ofVasconcelleaspp. for commer-
cial proteinase production.
In this study, we report on the identification and
characterization of cDNA sequences coding for cys-
teine proteinasesinthelatexof V. stipulata and
V. · heilbornii. Based on these sequences, the evolution
of thecysteineproteinases within the Caricaceae was
investigated. Furthermore, proteolytic enzymes were
purified from thelatexof V. · heilbornii andthe 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 inVasconcellea spp.: (1) a
higher protein content in their latex; and (2) the pres-
ence of other cysteineproteinases 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 inVasconcellealatex is
indeed slightly higher than inthe 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 inlatexofVasconcellea fruits than in
latex of C. papaya. Hence, when analyzing equal
amounts of protein, thelatexofVasconcellea 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 ofthe fruit profoundly affects
Table 1. Protein concentration and amidase activity oflatex 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 proteinasesofVasconcelleaspp. 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 ofthe 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 inlatexof Vasconcellea
fruit. The presence of other, possibly more proteolyti-
cally active enzymes will be evaluated inthe following
experiments.
cDNAs coding for cysteineproteinases 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 inthe 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 latexof V. cundinamarcensis has previously been
studied in detail. Isolation and preliminary characteri-
zation ofthecysteineproteinases 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 purificationofthe 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 ofthe 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 cysteineproteinasesinlatex 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 ofthe mature enzymes and act as
selective high affinity inhibitors to prevent inappropri-
ate proteolysis within the plant [25]. The enzymes are
present inthelatex as inactive precursors and are
activated in response to wounding ofthe plant [26].
Similar to other known cysteineproteinases 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 ofthe pri-
mary translation products contains 108 amino acids
(or 112 amino acids inthe case of VXH-B). Only for
VS-A, a stop codon was found inthe 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 inthe 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 inthe 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 inthe prore-
gion of VS-A and VXH-A:
115
NWS
117
. The glycan in
the propeptide might aid inthe protection against deg-
radation, or in targeting or maturation ofthe enzyme,
as was shown before in cathepsin C, which also belongs
to the papain family ofcysteineproteinases [27].
The overall similarity at the amino acid level
between all known cysteineproteinases from Carica-
ceae is 73%. Tables 2 and 3 show the percentage
sequence similarity between the obtained cDNA
T. Kyndt et al. CysteineproteinasesofVasconcellea 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 ofthe 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 proteinasesofVasconcelleaspp. T. Kyndt et al.
454 FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS
sequences and all known cysteineproteinases 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. CysteineproteinasesofVasconcellea spp.
FEBS Journal 274 (2007) 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 455
this part ofthe 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 Vasconcelleaspp. [2]., we assume that
other cysteineproteinases from this genus will also
have this deletion.
Molecular evolution ofcysteine 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 inthe 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 cysteineproteinasesof 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 proteinasesofVasconcelleaspp. 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 inthe 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 thelatexof V. stipulata.
Although the different parologues and orthologues
make the picture rather complex, the close phylo-
genetic relationship between thecysteine proteinase
sequences from V. cundinamarcensis andthe newly
determined sequence data from V. · heilbornii and
V. stipulata, again confirm the evolutionary divergence
between C. papaya andtheVasconcelleaspp. [2].
From the proposed evolutionary relationships of cys-
teine proteinases it can be deduced that the common
ancestor ofthe 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 inthe 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 ofthe proteinases
from V.
·
heilbornii
In an attempt to purify theproteinases 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 ofthelatex 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 ofthecysteine proteinase inhibitor
E-64. This observation clearly showed that the proteo-
lytic activity was due to cysteineproteinases 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 inthelatexof this species, which was reported
to be 84 nkatÆmg
)1
[20].
SDS ⁄ PAGE analysis ofthe 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. CysteineproteinasesofVasconcellea 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 ofthe 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 ofthe elution profiles
in Figs 3 and 5A,B suggests that these peaks corres-
pond to VXH-I, VXH-III andthe 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 inthelatexof 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 ofthe proteins
VXH-I, VXH-III, VXH-IVa and VXH-IVb are shown
in Fig. 6, and are compared with the known
N-terminal sequences ofthecysteineproteinases of
C. papaya and V. cundinamarcensis. Thecysteine 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 andthe 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 proteinasesofVasconcelleaspp. 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 inthelatexof three Vasconcelleaspp.in com-
parison with C. papaya. This is due to a higher protein
content andthe presence of other, more active, cys-
teine proteinasesin 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 ofcysteine proteinases
in their latex, which may be responsible for the higher
proteolytic activity.
The amount oflatex that can be collected from the
(generally smaller) fruits ofthe wild Vasconcellea
plants is definitely lower than thelatex 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 inthe greenhouse were the
source oflatex used in this study. Latex was collected by
making several incisions into the surface ofthe 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], andthelatex 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 thelatex 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. CysteineproteinasesofVasconcellea 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 ofthe 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, andthe 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 ofcysteine 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 ofcysteineproteinases 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 cysteineproteinases 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 inthe 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 ofthe double stranded
cDNA. PCR conditions and followed procedures were iden-
tical to those used during sequencing ofthe 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 ofproteinases from
V. · heilbornii (VXH) and V. stipulata (VS), compared with the
known sequences from V. cundinamarcensis (CC) and C. papaya
(Cp). Inthe case of double signals, both amino acids are specified.
Cysteine proteinasesofVasconcelleaspp. 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 andcharacterizationofthe CCI papain-like cysteineproteinases from thelatexof 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 thelatexof Carica candamarcensis... 451–462 ª 2006 The Authors Journal compilation ª 2006 FEBS 461 CysteineproteinasesofVasconcellea 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 andcharacterizationofthe cysteine- proteinases from thelatexof 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 ofthe 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 ofthe selected fractions, the samples... papain and papaya proteinase IV are selective high-affinity inhibitors ofthe 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 oflatex from Carica papaya Plant Sci 142, 115–121 Santilman V, Jadot M & Mainferme F (2002) Importance ofthe propeptide inthe biosynthetic maturation of rat cathepsin... C, Wintjens R, Vincentelli J, Azarkan M & Looze Y (2001) Revisiting the enzymes stored inthe laticifers of Carica papaya inthe context of their possible participation inthe 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 ofcysteine 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 ofthe expected mass (22–30 kDa), further purification was performed... Moussaoui A, Van Wuytswinkel D, Dehon G & Looze Y (2003) Fractionation andpurificationofthe enzymes stored inthelatexof Carica papaya J Chrom B 790, 229–238 Laemmli UK (1970) Cleavage of structural proteins during the assembly ofthe 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 thelatex of. ..T Kyndt et al CysteineproteinasesofVasconcellea spp Chromatographic procedures Thelatexof V · heilbornii was exhaustively dialyzed against cold H2O, centrifuged for 10 min at 3000 g, andthe 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