A cysteine protease is expressed as an inactive precursor in a pre-proenzyme form which contains a signal peptide pre-, an inhibitory Abbreviations CT, C-terminal; E-64, 1-[L-N-trans-epo
Trang 1modulates proteolytic activity through a partial inhibitory mechanism
Sruti Dutta, Debi Choudhury, Jiban K Dattagupta and Sampa Biswas
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India
Keywords
C-terminal extension; cysteine proteases;
modulation of proteolytic activity;
papain-like; thermostable
Correspondence
S Biswas, Crystallography and Molecular
Biology Division, Saha Institute of Nuclear
Physics, 1 ⁄ AF Bidhannagar, Kolkata
700 064, India
Fax: +91 332 337 4637
Tel: +91 332 337 5345
E-mail: sampa.biswas@saha.ac.in
(Received 14 March 2011, revised 16 May
2011, accepted 22 June 2011)
doi:10.1111/j.1742-4658.2011.08221.x
The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT) The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the speci-ficity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases Kinetic stability analyses (T50 and t1⁄ 2) and refolding experiments show similar thermal stability and refolding efficiency for both forms These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme
or in its folding mechanism
Structured digital abstract
l ErvC cleaves ErvC by protease assay (View interaction)
l trypsin cleaves ErvC by protease assay (View interaction)
Introduction
Papain-like cysteine proteases (EC 3.4.22) from plant
sources are of industrial and biotechnological
impor-tance because these enzymes are better suited to various
industrial processes [1] A cysteine protease is expressed
as an inactive precursor in a pre-proenzyme form which contains a signal peptide (pre-), an inhibitory Abbreviations
CT, C-terminal; E-64, 1-[L-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; Erv-C, ervatamin-C; pNA, p-nitroanilide; rmErv-C+CT, recombinant mature ervatamin-C with C-terminal extension; rmErv-CDCT, recombinant mature ervatamin-C without C-terminal extension; rproErv-C+CT, recombinant proervatamin-C with C-terminal extension; rproErv-CDCT, recombinant proervatamin-C without C-terminal
extension.
Trang 2pro-region and a mature catalytic domain [2–4]
Fol-lowing synthesis, the pre-peptide is removed during
passage to the lumen of the endoplasmic reticulum [2]
The inactive proenzyme subsequently undergoes
prote-olytic processing to produce an active mature enzyme
by autocatalytic cleavage of the propeptide part at the
N-terminus [2] It is known that the propeptide at the
N-terminus of the protease acts as an intramolecular
chaperone to mediate correct folding of the protease
[2] The mature catalytic domain of the enzyme of this
family has a molecular mass of 21–30 kDa and
shares a common fold with papain, the archetype
enzyme of the family These proteases are folded into
two compact interacting domains of comparable size,
delimiting a cleft which contains the active site residues
cysteine and histidine, forming a zwitterionic catalytic
dyad (Cys) His+) [5]
Sometimes a larger precursor is also synthesized
which contains a C-terminal (CT) extension⁄
propep-tide in addition to the abovementioned N-terminal
propeptide flanking the mature protease domain [6,7]
Unlike N-terminal propeptides, the role of
CT-exten-sions (or propeptides) is not yet well established
Sometimes an endoplasmic reticulum retention signal
Lys-Asp-Glu-Leu (KDEL) is found in the
CT-propep-tide which regulates the delivery of protease precursor
to other cellular compartments [8] Some other
mem-bers of the papain family from plant sources also
con-tain a larger CT-propeptide domain which shares a
similarity with animal epithelin⁄ granulin and the
func-tion of this domain is reported to be involved in leaf
senescence [6] In addition, a CT-propeptide without
any specific domain or motif is observed in some
papain-like cysteine proteases from plants like
Nicoti-ana tabacam (Q84YH7), Actinidia chinensis (P00785)
and Vicia sativa (Q41696) In most of the cases, such a
CT-extension contains the vacuolar sorting signal and
is cleaved inter- or intramolecularly after sorting [9]
No conserved sequence motif has been found in the
vacuolar sorting signal at CT-propeptide, rather an
amphipathic-like (hydrophobic and acidic) motif is
generally observed [9,10] at the core of such peptides
Other than plant systems, a CT-extension found in
a lysosomal cysteine protease (Lpcys2) of
Leish-mania pifanoi, plays a role in the regulation of enzyme
activity [11] CT-extension in mammalian and yeast
bleomycin hydrolase [12,13] is a key factor which
regu-lates their endo-peptidase or exo-aminopepetidase
activity by blocking the unprimed subsites in the
enzymes
Ervatamin-C (Erv-C) is a papain-like cysteine
prote-ase (EC 3.4.22) with high stability purified from the
latex of a tropical plant Ervatamia coronaria [14] The
3D structure of Erv-C reveals an extra disulfide bond, shorter loop regions and additional electrostatic inter-actions in the interdomain space, which are thought to
be responsible for its high stability [15] Sequencing
of the cDNA (from mRNA) of Erv-C from the leaf
of the plant in our laboratory [16], and comparison of the cDNA-derived amino acid sequence with other members of the family reveal that Erv-C is synthesized
as a precursor protein and in addition to the pre- (19 amino acids), pro- (114 amino acids) and mature (208 amino acids) parts, it contains an extension of 24 amino acids at the CT of the mature enzyme [16] (Fig 1A) This CT-extension was not observed, how-ever, when the mature Erv-C was purified directly from the latex of the plant [15]
In this article, we attempt to understand the role of this CT-extension in zymogen activation, enzyme activ-ity, folding and stability in vitro at the molecular level from structural and functional points of view
Results
Cloning, expression, purification and refolding of rproErv-CDCTand rproErv-C+CT
Both the proteins, recombinant proervatamin-C with-out the CT-extension (rproErv-CDCT) and recombinant proervatamin-C with the CT-extension
(rproErv-C+CT), were expressed in E coli as inclusion bodies with an apparent molecular mass of 41 and
43 kDa (Fig 1B), respectively, which is consistent with the estimated molecular masses of their deduced amino acid sequences Correct refolding was checked
by gelatin gel assay The condition and efficiency of refolding were almost similar for both forms with
> 90% recovery of the folded form from Ni-NTA purified protein for each (Table S1)
Activation to mature protease The purified refolded rproErv-C+CT could be con-verted into its mature active form (rmErv-C+CT) by using cysteine (20 mM) as the activator in 50 mM Tris buffer, pH 8.00, at 60C for 25–30 min, whereas the purified refolded rproErv-CDCT could be converted into its mature form (rmErv-CDCT) by the same activa-tor in 50 mM Na-acetate buffer, pH 4.5, at 60C for
45 min (Fig 2A) Thus the zymogen activation process occurs at different pH and time of maturation for the two enzymes The molecular mass of the mature enzyme rmErv-C+CT is higher ( 27 kDa) than that
of rmErv-CDCT ( 25 kDa) as observed in the SDS⁄ PAGE analyses (Fig 2A) This difference in
Trang 3molecular mass almost fits the theoretically calculated
value for the 24-amino-acid CT-extension We
expected, therefore, that the CT-extension continued to
remain attached with the mature enzyme even after
autocatalytic processing of rproErv-C+CT Gelatin gel
assay (Fig 2B) and western blot analyses (Fig 2C) also confirmed the retention of the CT-extension in the mature rmErv-C+CT
Because Erv-C isolated from the latex of the same plant does not show the CT-extension, the possibility
365 aa
I
34 aa
Protease domain
CT - ex
380 aa
II
N P
34 aa
N-Pro
356 aa
III
MSTLFIISILLFLASFSYA MDISTIEYKYDKSSAWRTDEEVKEIYELWLAKHDKVYSGLVEYEKRFEIFKDNLKFIDEH
SCWAFSTVSTVESINQIRTGNLISLSEQQLVDCNKKNHGCKGGAFVYAYQYIIDNGGIDTEANYPYKAVQGPCRAAK KVVRIDGYKGVPHCNENALKKAVASQPSVVAIDASSKQFQHYKSGIFSGPCGTKLNHGVVIVGYWKDYWIVRNSW GRYWGEQGYIRMKRVGGCGLCGIARLPYYPTKA AGDENSKLETPELLQWSEEAFPLA
IV A
B
66 kDa
45 kDa
36 kDa
29 kDa
24 kDa
20 kDa
3 2
1
Fig 1 (A) (I) Open reading frame of Erv-C precursor, pre-pro-ErvC (II) Recombinant ervatamin-C with C-terminal extension, rproErv-C+CT (III) rproErv-CDCT, recombinant ervatamin-C without C-terminal extension Red indicates vector portion and ‘aa’ stands for amino acids (IV) The amino acid sequence of the open reading frame The sequence of CT-extension is in red (B) Lanes 1 and 2, Purified proteins rproErv-C+CT and rproErv-CDCT, respectively; lane 3, Molecular mass markers.
Trang 4of removal of the CT-extension by other plant
prote-ases in vivo could not be ruled out To explore this
possibility, we performed a trans mode activation of
rproErv-C+CT in vitro using seven different proteases
(four cysteine proteases Erv-A, -B, -C and papain from
the plant latex; two serine proteases trypsin and
chy-motrypsin from bovine pancreas; one aspartic protease
pepsin from porcine stomach mucosa), each having
sequences specific for their cleavage in the amino acid
sequence of the CT-extension The activated mature
protein thus generated in each case shows a band at
the same position ( 27 kDa) like that in the
autoacti-vated enzyme, as observed in SDS⁄ PAGE analyses
(Fig S1), indicating that these external enzymes can
not cleave the CT-extension Even with a prolonged
digestion time (24 h), the same result was obtained for
all proteases except trypsin (Fig S1) Trypsin digestion
for 24 h resulted in a truncated protein at 26 kDa,
slightly above the activated mature rmErv-CDCT
( 25 kDa) This result probably indicates that trypsin
has some accessibility to its sites of specificity (Lys,
which is in position 7 of the CT-extension) (Fig S2)
and only after a prolonged incubation time can it
result in a band at a slightly lower molecular mass
position, as observed in the SDS⁄ PAGE analysis
(Fig S1)
Specific activity and optimum temperature of activity
The optimum temperature of activity, Topt (Fig 3), for both forms is 65C Interestingly, it was observed that rproErv-C+CTshows no activity below 45C and then activity rises sharply from 60C onwards, reaching a maximum at 65C In the case of rproErv-CDCT, a gradual increase in activity with temperature was observed until it reached its maximum at 65C
At Topt (65C), the specific activity of rmErv-CDCT
was found to be almost double that of rmErv-C+CT (Table 1) At 37C, however, rproErv-CDCT shows measurable proteolytic activity, whereas no activity was seen for rmErv-C+CT
Kinetic measurements of the recombinant proteins
Kinetic constants of rproErv-C+CT and rproErv-CDCT were measured at room temperature against N-ben-zoyl-Phe-Val-Arg-p-nitroanilide (pNA), a tripeptide substrate with a valine at the P2 position which is known to act as a substrate for Erv-C [17] The kinetic constants of the two recombinant enzymes (Table 1) clearly show that rproErv-CDCT has almost 10 times
97 kDa
66 kDa
66 kDa
45 kDa
29 kDa
20 kDa
+CT
rproErv-C +CT rproErv-C ΔCT
rmErv-C +CT rmErv-C ΔCT
rproErv-C ΔCT rmErv-C ΔCT rmErv-C +CT
29 kDa
20 kDa M
45 30 25 20 15 10 5 0 C M 45 30 25 20 15 10 5 0 C A
Fig 2 (A) Time course of activation to the mature form (I) rproErv-C+CT (II) rproErv-CDCTas discussed in Materials and methods Time intervals of 0–45 min are indicated for the respective lanes Untreated proteins (control) are labelled as ‘C’, ‘M’ denotes the molecular mass marker (B) Gelatin gel assay of activated rproErv-CDCT (lane 1) and rproErv-C+CT (lane 2) (C) Western blot analysis Lane 1, Erv-C purified from the plant latex; lanes 2 and 3, purified and refolded rproErv-C+CTand rproErv-CDCT [Correction added on 26 July 2011 after original online publication: in the figure, labelling for part C was changed from ‘rproErv-C+CT, rproErv-CDCT, rproErv-C+CTand rproErv-CDCTto rproErv-C+CT, rproErv-CDCT, rmErv-C+CT and rmErv-CDCT’].
Trang 5higher activity than rproErv-C+CT One can probably
conclude that the CT-extension has some inhibitory
effect on the activity of the enzyme against a small
peptide
Thermal stability
Temperatures of maximum proteolytic activity (Tmax)
for rproErv-C+CT and rproErv-CDCTare 50 and 45C
(Table 2 and Fig 4) and they retain > 90% activity
up to 65 and 60C, respectively These data indicate a
good thermotolerance for these enzymes The pattern
of retention and fall in activity beyond Tmaxwas more
or less the same for both enzymes The T50values for
rproErv-C+CT and rproErv-CDCT were 76 and 72C
(Fig 4), respectively The half lives (t1⁄ 2) at 65C were
400 min (Fig 5) for both enzymes
Molecular modeling studies
To gain insight into the stability and dynamic proper-ties of the structure, solvent MD simulation was
110
100 rproErv-C +CT
rproErv-C ΔCT
90
80
70
60
Temperature (°C)
50
40
30
20
10
0
30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Fig 3 Determination of optimum temperature of activity (Topt) of
rproErv-C+CT and rproErv-CDCT Purified proenzymes (10–20 lg)
were converted to their respective mature forms and the
percent-age residual enzyme activities were determined with respect to the
maximum activity using an azocasein assay at different
tempera-tures, as described in Materials and methods Each data point is an
average of three independent experiments having similar values
(Table S3).
Table 1 Kinetic constants using the substrate N-benzoyl-Phe-Val-Arg-pNA Specific activity using azocaesin and IC50value for the inhibitor E-64 ND, not determined.
kcat(s)1) a Km(lM) a
kcat ⁄ Km (s)1ÆmM)1)
Specific activity
at 37 C b (UÆmg)1)
Specific activity
at 65 C b (UÆmg)1)
IC50 against E-64 (nM) c
a Given standard errors were calculated based on nonlinear fitting of the Michaelis–Menten saturation curve using the software Graphpad PRISM (http://www.graphpad.com/prism) b Each value of specific activity of rproErv-C+CT and rproErv-CDCT is a mean of three independent experiments ± SD.cGiven standard deviations were calculated from linear regression plot of residual activity and inhibitor concentration.
Table 2 Kinetic stabilities ND, not determined.
Tmax (C) T50 (C)
t1 ⁄ 2 at
65 C (min) Topt (C) rproErv-C+CT
(activity at 65 C)
rproErv-CDC (activity at 65 C)
Native mature Erv-C (latex) [14,32]
100
rproErv-C +CT rproErv-C ΔCT
80 60
Temperature (°C)
40 20
0
40 45 50 55 60 65 70 75 80 85 90
Fig 4 Effect of temperature on activity of rproErv-C+CT and rpr-oErv-CDCT Each purified proenzyme (10–20 lg) was treated for
10 min at different temperatures followed by activation of the pro-proteins to their respective mature forms The percentage residual enzyme activities (at each temperature) were determined with respect to the maximum activity using an azocasein assay at 65 C
as described in Materials and methods Each data point is an aver-age of three independent experiments having similar values (Table S3).
Trang 6performed The total energy of the whole system and
root mean square deviation (RMSD) from the starting
structure are essential to determine the sustainability
and convergence of MD simulation Figure 6A shows
the RMSD of backbone atoms of the CT-extension in
association with a mature domain as well as in an
iso-lated form The graph shows that the RMSD reached
below 0.4 A˚ when the extension is attached to the
mature domain and is > 1 A˚ when the extension is on
its own The fluctuation in the radius of gyration was
also analyzed (Fig 6B) as a measurement of the
over-all stability of the CT-extension for both forms These
analyses show that the extension achieves a relatively
more stable conformation when it is attached to the
mature domain
The modeled structure of the CT-extension with the
mature catalytic domain (rmErv-C+CT) shows that the
extension peptide blocks some of the unprimed
sub-sites of the enzyme (Fig 7) The interface area of the
CT-extension and the mature catalytic domain is
1037 A˚2, which is 56% of the total surface area of
the CT-extension The Leu side chain at the position
23 of the CT-extension occupies specificity pocket S2
of the catalytic domain and is stabilized mainly by
hydrophobic interactions with S2 subsite residues A67,
F68, A131, L155 and L201 (Fig 8) [17] Residue
Phe21 of the CT-extension is buried inside a
hydro-phobic pocket of mature domain formed by V69, L201
and Y203 (Fig 8B) Other residues of the CT-exten-sion are stabilized by electrostatic and hydrophobic interactions with the mature catalytic domain of the enzyme Superposition of the crystal structure of the complex of mature Erv-C (without CT-extension) with the inhibitor 1-[L-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane (E-64; Protein Data Bank ID
2PRE) and the modeled structure of Erv-C with CT-extension (rmErv-C+CT) reveals that the Leu of
100
rproErv-C +CT rproErv-C ΔCT
90
80
70
60
Time in min
50
40
30
20
10
0
0 10 20 30 40 50 60
120 180 240 300 360 420
Fig 5 Time course of thermal inactivation of rproErv-C+CT and
rproErv-CDCT at 65 C An aliquot of the purified proenzymes
(10–20 lg) was treated at 65 C for 0 min to 8 h followed by
activa-tion of the pro-proteins to their respective mature forms At each
indicated time, the residual enzyme activity was determined using
an azocasein assay at 65 C, as described in Materials and
meth-ods, and values are expressed as percentage of the initial activity
of the respective enzymes The experiment was carried out in
duplicate for each data point (Table S3).
Fig 6 2 ns molecular dynamics trajectory of CT-extension part in association with the mature Erv-C domain (red) and in an isolated form (black) (A) Backbone RMSD and (B) radius of gyration.
Fig 7 Surface presentation of mature Erv-C (A) Mature Erv-C without the CT-extension (Protein Data Bank ID 2PNS ), the catalytic cleft is marked in red (B) Mature Erv-C with modeled CT-exten-sion, the CT-extension is displayed in magenta.
Trang 7E-64 at P2 position of the inhibitor lies close to Leu23
of the CT-extension of the rmErv-C+CT molecule with
a minimum distance of 1.9 A˚ between two atoms of the two leucines (Fig 8A) Because the Leu23 residue
of the CT-extension of the rmErv-C+CT molecule is buried inside the S2 pocket, it restricts access of Leu at the P2 position of E-64 and results in a higher IC50 value (482.5 nM) of E-64 inhibition compared with rmErv-CDCT (349.1 nM) (Table 1) in which the exten-sion is truncated Modeling studies also reveal that although this CT-extension blocks some of the unprimed subsites beyond S2, access to the catalytic centre (Cys-His dyad) is not totally blocked
Discussion
It is known that the autocatalytic processing of papain-like cysteine proteases from pro- to mature form generally occurs at acidic pH [18] The 3D struc-tures of papain-like cysteine proteases in the pro-form [19–23] reveal that N-terminal propeptide part adopts
a specific globular structure which is conserved among the family despite a relatively low homology in their amino acid sequences Previous reports suggest that an acidic pH induces a conformational change in the N-terminal propeptide domain, resulting in a molten globule state and thereby the activation process is trig-gered [24] The molten globule state of the N-terminal propeptide domain results in a reduction in the associ-ation affinity of the propeptide towards the protease domain and cleavage of the propeptide occurs leading
to a mature active enzyme To date, there has been practically no detailed report available in the literature
on the role of the CT-extension (or CT-propeptide) in the maturation process for proteases in this family There is one study on kiwifruit cysteine protease, actinidin, which shows that its C-terminal propeptide
is required for correct processing [7] In our studies,
we have observed that for the precursor
rproErv-C+CT, in vitro autoactivation essentially removes the N-terminal propeptide part leading to a mature active protease (rmErv-C+CT) with a molecular mass of
27 kDa (Fig 2) with the CT-extension remaining attached Moreover, this autocatalytic processing does not occur at acidic pH, instead autoactivation is found
to occur at a basic pH of 8.0 By contrast, in
rproErv-CDCT, in vitro activation to mature enzyme
(rmErv-CDCT) occurs at an acidic pH of 4.5, like in other members of the family [17] The 24-amino-acid CT-extension contains six negatively charged residues (one aspartate and five glutamates) (Fig 1A) and our previous molecular modeling studies [16] indicated that this negatively charged region of the C-terminus could
A
B
Fig 8 (A) The structure of the catalytic cleft and unprimed
sub-sites region of mature Erv-C with modeled CT-extension The
mature catalytic domain is represented as an electrostatic potential
surface and the CT-extension as a stick model with C atoms in
green The inhibitor E-64 (taken from the structure of Erv-C and
E-64 complex; Protein Data Bank ID 2PRE ) is also docked for
com-parison and is represented as a stick model with C atoms in brown.
The catalytic cysteine residue (C25) of Erv-C is represented as a
ball and stick model (light blue) and the S2 subsite residues are
rep-resented as a stick model (light pink) The minimum distance
(1.9 A ˚ ) between P2 (Leu23) of the CT-extension and P2(Leu) of
E-64 are indicated by the dotted line (B) The last four residues,
F21P22L23A24, of the CT-extension domain (C atoms are shown in
magenta) and the neighboring residues of Erv-C mature domain
within 4.2 A ˚ (C atoms are shown in deep bottle green) Interdomain
distances within 4.2 A ˚ are shown in green.
Trang 8be positioned structurally in such a way that it could
interact with a positively charged zone of the
N-termi-nal propeptide in the zymogen Therefore, one may
postulate that at acidic pH, this electrostatic
inter-action between the N- and C-terminal propeptides⁄
extensions may be strong enough to restrict the
confor-mational change in the N-terminal propeptide,
required for the activation process But at basic pH,
this interaction may become weak, leading to a free
and flexible C-terminus, and the presence of the six
acidic residues in this region may locally mimic an
acidic environment to trigger the activation process
This may be the explanation for autocatalytic
activa-tion at a higher pH for rproErv-C+CT which is not
observed when the protein is expressed without the
extension part in rproErv-CDCT
Because the C-terminal extension of 24 residues
remains intact in the mature form of the rmErv-C+CT
molecule (Fig 2) after in vitro processing, autocatalytic
trimming does not occur in this case It should be
mentioned, however, that in some vacuolar papain-like
cysteine proteases the CT-extension (propeptide),
car-rying a vacuolar sorting signal, is cleaved by
intermo-lecular proteolysis by a different vacuolar protease
in vivo [9,10] So, in this study, we performed an
in vitro transactivation experiment using three latex
enzymes Erv-A, -B and -C from the same plant, and
papain from papaya latex and two other serine
(tryp-sin and chymotryp(tryp-sin) and one aspartic (pep(tryp-sin)
prote-ases The results showed that this extension is also not
cleaved by any of these proteases in their optimum
condition of activity However, because no experiment
has been carried out in vivo, the possibility of
intermo-lecular cleavage of the CT-extension by a different
vacuolar protease under specific in vivo conditions can
not be ruled out
The results of enzyme kinetic studies show that the
proteolytic activities of the two recombinant forms
(rproErv-C+CTand rproErv-CDCT) are not of the same
order (Table 1) In optimum temperature
determina-tion using azocasein as a substrate, rmErv-C+CT does
not show any proteolytic activity when activity is
assayed below 45C, but activity increases suddenly
beyond 60C and sufficient activity is retained in the
temperature range 65–75C (Fig 3) with the highest
being at 65C This activity profile is different for
rmErv-CDCT where activity increases systematically
with temperature, although the highest activity in this
case is also at 65C (Fig 3) However, the specific
activity of rmErv-CDCT is almost twice as high as that
of rmErv-C+CTat their optimum temperature of
activ-ity (65C) (Table 1) When the activity is measured
with a small peptide like N-benzoyl-Phe-Val-Arg-pNA,
both forms, rmErv-C+CT and rmErv-CDCT, show activity at room temperature although the former has
a 10 times lower Kcat⁄ Km value (Table 1) Thus, for a small peptide, enzymatic activity is observed at room temperature for rmErv-C+CT (around 25C), which is totally absent when this form is assayed with azocaesin (Table 1) These data suggest that the CT-extension interferes with a protein substrate at a lower tempera-ture and the enzyme active site is not accessible to the protein substrate But at a higher temperature (beyond
60C), this interference is partly removed and the enzyme can show proteolytic activity to the protein substrate This behavior of the enzyme differs for a smaller peptide substrate, where the enzyme can work
on this peptide even at lower temperatures, although
to a lesser extent Molecular modeling studies show that the C-terminal tail blocks the unprimed subsites beyond S2 of the enzyme thus inhibiting the endopep-tidase activity at temperatures below 55C for azocae-sin Perhaps, at a higher temperature, a reduction of the association affinity for this tail towards the unprimed subsites occurs leaving the subsites partially free for substrate binding This is in conformity with a report [25] that the CT-extension sometimes has an inhibitory property towards some enzymes Blocking
of unprimed subsites is also found in other papain-like proteases by different mechanisms either by a mini-chain like cathepsin-H [26] or an exclusion domain in cathepsin-C [27] or by C-terminal extension in bleomy-cin hydrolase [12,13] In all these cases, each protease loses its endo-peptidase activity and functions as an aminopeptidase because their unprimed subsites are blocked as found here A superposition of the struc-tures of cathepsin-H and bleomycin hydrolase on the modeled structure of rmErv-C+CT revealed that although the blocking strategy of these peptides is sim-ilar, in the first two proteases the blocking peptides extend closer to the active site than in rmErv-C+CT (Fig 9)
We also note from this study that the catalytic activ-ity and maturation kinetics (both pH of activation and time of activation) of the two forms vary (Fig 2A) Alternative splicing in plant systems has been reported [28] to affect the stability and translatability at the RNA level and produce truncated or extended proteins with altered (increased, decreased or loss of) activity, cellular localization, regulation and⁄ or stability Here,
we used the clone of Erv-C which was constructed from the cDNA (mRNA) of the leaf of the plant [16] and the deletion mutant of that has been generated by eliminating the last 24 amino acids at the C-terminus The mature Erv-C isolated from the latex of the plant also does not have the CT-extension [15] Because both
Trang 9enzymes, latex Erv-C and recombinant mature
rmErv-CDCT without a CT-extension, appear to be the same,
having a similar molecular mass (Fig 2C) and similar
enzymatic properties [29], we cannot ignore the
possi-bility that Erv-C may exist in two isoforms (splice
vari-ants) in different plant tissues, and that the activation
requirements of the two isoforms are different
depend-ing on the presence or absence of a 24-amino-acid
neg-atively charged CT-extension
We have noted that both recombinant protease
forms can fold efficiently in vitro So the CT-extension
does not appear to have any noticeable effect on proper
folding of the protein in vitro However, our
observa-tions also indicate that this extension has some effects
in the maturation process and activity of the protease
It suggests two possibilities for the presence of the
CT-extension in the enzyme: either it carries the signal for
vacuolar sorting and after that is cleaved by another
enzyme in vivo, or an mRNA (cDNA) exists in the leaf
of the plant that essentially encodes an isoform of
Erv-C which is present in the latex of the plant
Materials and methods
Cloning, expression, purification and refolding of
rproErv-C+CT
The entire open reading frame of the Erv-C precursor had
vector [16] A fragment of the original cDNA encoding the prodomain, the mature domain and CT-extension of Erv-C was PCR-amplified from this clone using primers (Forward: 5¢-CCCGGATCCATGGACATATCTACC-3¢ and Reverse: 5¢-GGTCTCGAGTTAAGCAAGTGGAAAAGCT-3¢) desig-ned to delete the pre-peptide (the signal peptide) and to include the restriction sites for BamHI and Xho1 (under-lined) to facilitate cloning into pET-28a(+) expression vec-tor (Novagen, Madison, WI, USA) The amplified product was then subcloned into respective restriction sites of pET-28a(+) expression vector The resultant plasmid was trans-formed in E coli strain DH10B Insertion of the correct gene⁄ transcript was confirmed by DNA sequencing, restric-tion digesrestric-tion and colony PCR with gene⁄ vector-specific primers This subcloned transcript was named
rproErv-C+CTand it was expressed using E coli strain BL21(DE3) Hexa-His-tagged recombinant protein expression was car-ried out as described earlier [29] except that the cells were grown for 5 h after induction with 0.5 mM isopropyl b-D -thiogalactoside instead of overnight
The overexpressed recombinant rproErv-C+CTwas puri-fied by Ni-NTA affinity chromatography (Qiagen, Hilden, Germany) under a denaturing condition and refolding of the eluted purified protein was done as for rproErv-CDCT
by dialysis method [29] The refolded protein was concen-trated by Amicon Ultra-4 (10 kDa cut-off) for further stud-ies The generation of rproErv-CDCclone and its expression and purification was done as described previously [29]
Conversion of rproErv-C+CTto its mature form Autocatalytic processing
In vitro conversion of the purified and refolded
optimizing several parameters like proper activator (reduc-ing agent), concentration of the activator, pH, temperature and time At a designated time interval, aliquots of the sample were collected from the reaction mixture and mixed
sam-ple buffer containing 2–3 mM irreversible inhibitor E-64
required for complete maturation Proenzyme processing of
SDS⁄ PAGE analysis Purity of the mature forms of
blot analysis using rabbit antiserum raised against pure and mature Erv-C from the plant latex as primary antibody (Bangalore Genei, Bangalore, India) using the protocol described earlier [29] Wild-type Erv-C from plant latex was also used for comparison
Effect of external proteases on C-terminal processing
can be cleaved by external proteases in trans mode, four
Fig 9 Superposition of the catalytic cleft and the unprimed subsite
region of cysteine proteases blocked by different peptides like the
mini-chain in cathepsin H (Protein Data Bank ID 8PCH ; sky blue),
CT-extension in bleomycin hydrolase (Protein Data Bank ID 1CB5 ;
green) and the modeled CT-extension in Erv-C (magenta) The E-64
molecule (brown) in complex with Erv-C (Protein Data Bank ID
2PRE ) is also used in the superposition for comparison The
respec-tive catalytic domains of the proteases are shown in Ca
presenta-tion in corresponding colors The Ca atoms of the catalytic dyad
residues Cys and His are shown in yellow and blue, respectively.
Trang 10cysteine proteases [Erv-A, -B and –C (isolated from the
latex of Ervatamia coronaria in our laboratory) and papain
from Carica papaya (Merck, Kenilworth, NJ, USA)], two
serine proteases [trypsin and chymotrypsin from bovine
pancreas (Sigma-Aldrich, St Louis, MO, USA)], one
aspar-tic protease [pepsin from porcine stomach mucosa (SRL,
was digested using the abovementioned proteases (90 lg) at
the optimum pH and temperature of activity of each
prote-ase In brief, digestion by Erv-A, Erv-B, Erv-C were carried
by E-64; trypsinization was carried out at pH 8.0 and
cocktail inhibitor (Roche, Mannheim, Germany); digestion
by chymotrypsin was carried out at pH 8.0 at 50C for 1 h
with 2 mMCaCl2and blocked by complete mini EDTA free
cocktail inhibitor; pepsinization was carried out at pH 4.0
and 37C for 2 h and blocked by E-64 and pepstatin
abovemen-tioned enzymes separately at 20C for 24 h under the same
conditions The proteolytic digestion in each experiment
was checked by SDS⁄ PAGE analysis
Measurement of proteolytic activity
Substrate gel zymography using 0.1% gelatin as a substrate
was used to demonstrate the protease activity of
recombi-nant refolded proteases (rproErv-CDCT and rproErv-C+CT)
using a protocol described previously [29]
The specific activity of the recombinant proteases
sub-strate azocasein The specific activity of the native mature
Erv-C isolated from the plant latex was also determined
using the same protocol for comparison (Table 1) For this
assay, a reaction mixture containing 0.5 mL of 0.2%
azoca-sein in Tris⁄ HCl buffer (pH 8.0), 0.5 mL of recombinant
(pH 8.0) and incubated for 30 min The reaction was then
terminated with 5% trichloroacetic acid The mixture was
centrifuged at 9300 g for 5 min to remove precipitate and
the absorbance of the supernatant was measured at 366 nm
to determine the amount of released azopeptides using the
specific absorption coefficient (A1%366= 40) for azocasein
solution [30] One enzyme unit was defined as the amount
of soluble protease required to release 1 lg of soluble
azo-peptidesÆmin)1 The specific activity was the number of
units of activity per milligram of protein For specific
activ-ity measurements the concentration of pro-protease has
been used in the calculations because once the proteases are
autocatalytically activated as described above, it is difficult
to isolate the active mature enzyme from degraded
propep-tide parts
Determination of optimum temperature for enzymatic activity of rproErv-CDCTand rproErv-C+CT
Topt or ‘temperature optima’ is the temperature at which the enzyme shows maximum activity Proteolytic activity of both the recombinant proteins (rproErv-CDCTand
rproErv-C+CT) was measured in the range 30–90 at 5C intervals
to determine the optimum temperature of activity (Topt) for both recombinant proteins The pro-proteases were first converted to mature enzymes and the proteolytic activity was then measured using azocasein as a substrate as described above at specific temperatures
Kinetic measurements using a chromogenic peptide
Erv-C-specific chromogenic substrate N-benzoyl-Phe-Val-Arg-pNA (Sigma-Aldrich) [17] was used in this study for kinetic measurements of the recombinant proteins Liber-ated pNA was monitored for 15 min at 410 nm on a
Thermo Electron Corporation, Rockville, MD, USA) Conditions for the measurement of the kinetic parameters
Vmaxvalues of rproErv-C+CTwere calculated by nonlinear fitting of the Michaelis–Menten saturation curve using the
prism) The kcat value was calculated by using the equa-tion kcat=Vmax⁄ [E]Twhere [E]Tis the total concentration
of the active enzyme, the values of which were mea-sured by active-site titration with the irreversible inhibitor, E-64 using the above mentioned pNA containing
calculations
Measurement of IC50value of E-64
rproErv-CDCT) were converted into their respective mature forms The irreversible inhibitor, E-64 was added in increasing concentrations to the aliquots until the residual activity reached 0 The residual activity (DA410 nmÆmin)1) was deter-mined with respect to the activity of the enzyme (carried out without any inhibitor) as described in the previous sec-tion, against the peptide substrate (N-benzoyl-Phe-Val-Arg-pNA) for both the proteases This residual activity of the enzyme was plotted against the inhibitor concentration The inhibitor concentration required for half-maximal inhi-bition (IC50) of E-64 for these enzymes were determined from these plots