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RESEARCH ARTICLE
Interaction of recombinant CanPIs with Helicoverpa
armigera gut proteases reveals their processing
patterns, stability and efficiency
Manasi Mishra, Vaijayanti A. Tamhane, Neha Khandelwal, Mahesh J. KulkarniVidya S. Gupta and Ashok P. Giri
Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune, India
Received: December 31, 2009
Revised: May 10, 2010
Accepted: May 15, 2010
Six diverse representative Capsicum annuum (common name: hot pepper; Solanaceae) protease
inhibitor genes, viz CanPI-5, -7, -13, -15, -19, and 22 comprising 1–4 inhibitory repeat domains
(IRDs), were cloned and expressed in Pichia pastoris. The recombinant proteins were evaluated
for their interactions with bovine trypsin, chymotrypsin, and Helicoverpa armigera gut proteases
(HGP) using electrophoretic (native and denaturing) and mass spectrometric (MALDI-TOF-MS
in combination with intensity fading assays) techniques. These techniques allow qualitative and
semiquantitative analysis of multiple and processed IRDs of purified recombinant Capsicumannuum proteinase inhibitor (rCanPI) proteins. rCanPIs showed over 90% trypsin inhibition,
varying chymotrypsin inhibition depending on the number of respective IRDs and over 60%
inhibition of total HGP. rCanPI-15 that has only one IRD showed exceptionally low inhibition of
these proteases. Interaction studies of rCanPIs with proteases using intensity fading-MALDI-
TOF-MS revealed gradual processing of multi-IRD rCanPIs into single IRD forms by the action
of HGP at the linker region, unlike their interactions with trypsin and chymotrypsin. Intensity
fading-MALDI-TOF-MS assay showed that CanPI-13 and -15, possessing single IRD and
expressed predominantly in stem tissue are degraded by HGP; indicating their function other
than defense. In vitro and in vivo studies on rCanPI-5 and -7 showed maximum inhibition of
HGP isoforms and their processed IRDs were also found to be stable in the presence of HGP.
Even single amino acid variations in IRDs were found to change the HGP specificity like in the
case of HGP-8 inhibited only by IRD-12. The presence of active PI in insect gut might be
responsible for changed HGP profile. rCanPI-5 and -7 enhanced HGP-7, reduced HGP-4, -5, -10
expression and new protease isoforms were induced. These results signify isoform complexity in
plant PIs and insect proteases.
Keywords:
CanPI / H. armigera gut proteases / Intensity fading MALDI-TOF-MS / Pin-II /
Plant–insect interaction / Plant proteomics
1 Introduction
Plants use various strategies to defend themselves against
herbivory with a range of adaptations such as camouflage,
mechanical and chemical defenses. One such mechanism
relies on causing indigestion thereby disrupting the nutrient
acquisition system in infesting insects. Plant proteinaceous
proteinase inhibitors (PIs) are the best example of this type
of postingestive defense [1]. Plant PIs are specific for
each of the mechanistic class of the enzymes and are clas-
sified as the inhibitors of serine, cysteine, aspartic and
Abbreviations: aa, amino acid; AD, artificial diet; CanPI, Capsi-
cum annuum proteinase inhibitor; CI, chymotrypsin inhibition;
HGPI, H. armigera gut protease inhibition; IF-MALDI-TOF-MS,
intensity fading-MALDI-TOF-MS; IRD, inhibitory repeat domain;
PI, proteinase inhibitor; rCanPI, recombinant CanPI; RSL,
reactive site loop; TI, trypsin inhibition
Correspondence: Dr. Ashok P. Giri, Plant Molecular Biology Unit,
Division of Biochemical Sciences, National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune 411 008 (M.S.), India
E-mail: [email protected]
Fax: 191-20-25902648
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2010, 10, 2845–2857 2845DOI 10.1002/pmic.200900853
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metalloproteases. Serine PIs are further categorized as
Kunitz, Bowman-Birk, Squash-family and Wound-inducible
(Pin-II) [2, 3] depending on sequences, biochemical prop-
erties and expression patterns in plant tissue. The useful-
ness of PIs to reduce insect growth and development has
been demonstrated by feeding the larvae on an artificial diet
(AD) containing PIs [4] and their transgenic expression in
plants [5].
Pin-II PIs are of interest because of their large structural
and functional diversity mostly in Solanaceous plants [6].
Most Pin-II precursor proteins show the presence of
N-terminal putative endoplasmic reticulum signal peptide
followed by a variable number (1–8) of inhibitory repeat
domains (IRDs) of around 6 kDa each connected by five
amino acid (aa) linkers and also a C-terminal vacuolar
sorting signal in some cases [7, 8]. Although Pin-II PIs have
a high sequence identity, they also have aa variations in the
reactive site loop (RSL) and other regions of the IRD,
namely the linker region and in signal sequence. These Pin-
II PI precursors may have arisen from a series of gene
duplication or domain duplication events [9]. The mature
Pin-II PIs form a clasped –bracelet-like structure by covalent
joining of partial N and C terminal IRDs as in 6-IRD NaPI
[10]. The structure of unbound form of 2-IRD Pin-II PI of
tomato revealed significant conformational flexibility in the
absence of bound proteases. These PIs can bind to and
simultaneously inhibit two enzyme molecules [11, 12].
NMR-based structures of the preprotein as well as the 6-kDa
mature active inhibitors have been determined in Nicotianaalata and tomato [12–14]. Precursor PIs are processed at the
linker region(s) by plant endogenous proteases to release
IRD(s) [15]. Horn et al[16] showed that upon insect attack
endogenous plant proteinases are upregulated in Nicotianaattenuata and they are probably responsible for processing of
the 7-IRD N. attenuata trypsin proteinase inhibitor. The
variations in the Pin-II PI sequences, followed by their
differential in planta processing can possibly give rise to a
wide range of several IRD(s) (either single or multi-IRD)
which may display different protease specificities.
The digestive complement of Helicoverpa armigeraconsists of endopeptidases such as serine, metallo, cathe-
psin B-like proteinases and exopeptidases. Serine protei-
nases form the dominant mechanistic class (495%) in the
gut environment [17]. In our previous studies, we observed
that diverse Pin-II PIs from Capsicum annuum differentially
influence H. armigera growth and development [18].
Furthermore, C. annuum upon Spodoptera litura and aphid
attack showed strong upregulation of multi-IRD PIs [19].
C. annuum have characteristic CanPI expression pattern in
various tissues. For example, in stem 1- and 2-IRD CanPIsexhibited higher expression while predominance of 2-IRD in
leaves. On the contrary, 3- and 4-IRD Capsicum annuumproteinase inhibitors (CanPIs) showed a higher expression
in fruits [19]. These results indicate significance of CanPIs
for their defensive and endogenous role, which still remains
poorly understood.
Here we attempt to address the questions (i) What is the
fate of recombinant CanPIs (rCanPIs) in insect gut?
(ii) What is the insect’s response to the ingested CanPIs?
(iii) Whether this reaction is different for different CanPIs?
We selected six CanPI genes on the basis of sequence
variation, specificity and number of IRDs and characterized
them with specific reference to their (i) processing by HGP
(ii) stability in proteolytic environment and (iii) inhibitory
activity against HGP. Using intensity fading-MALDI-TOF-
MS (IF-MALDI-TOF-MS), enzyme assays and PI activity
gels, interaction of rCanPIs with HGP was analyzed. As
these studies are based on the product(s) of particular PI
gene(s), it leads to the identification of potential PI(s) or
IRD(s) effective against constitutive and induced insect gut
proteases.
2 Materials and methods
2.1 Sequence analysis of CanPI genes
Out of 21 novel CanPIs identified in our previous study [19],
6 representative genes were selected for their functional
characterization. The criteria for selection included: diversity
in number of IRDs, TI or chymotrypsin inhibition (CI)
specificity of the IRD and aa variation in IRD sequence.
The cDNA and aa sequence analysis was performed using
the DNA star (Laser gene, DNASTAR, Madison, WI, USA)
and Clustal X softwares. CanPI-13 and CanPI-15 having 1-
IRD; CanPI-19 and CanPI-22 having 2-IRDs; CanPI-5having 3-IRDs and CanPI-7 having 4-IRDs were selected
for cloning in Pichia pastoris for recombinant protein
expression.
2.2 Cloning, expression and purification of rCanPIs
The mature peptide region of CanPIs was cloned in
expression vector pPIC9 (Invitrogen, Carlsbad, CA, USA) for
recombinant, extracellular expression in P. pastoris GS115
and purified as described previously [18].
2.3 HGP extraction and protease and PI assays
Gut tissue was dissected from the laboratory-reared 4th
instar larvae of H. armigera and immediately frozen in liquid
nitrogen. Extraction of HGP was performed as detailed in
[17] and the bovine trypsin, chymotrypsin and HGP activity
was determined as given in [20]. rCanPI assays were
performed as detailed in [20] with increasing amount of PIs
(0.3–4 mg) for rCanPI-5, -7, -19, -22 and 8 mg of PIs for
rCanPI-13 and -15. One PI unit is defined as the amount of
inhibitor required for inhibiting one protease activity unit.
The inhibition potential of all six rCanPIs against trypsin,
chymotrypsin and HGP was estimated. Depending on
2846 M. Mishra et al. Proteomics 2010, 10, 2845–2857
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HGP inhibition potential of individual rCanPIs, the ratio
of activities of HGP and inhibition was maintained in
IF-MALDI-TOF-MS assays.
2.4 Activity visualization of proteases and PIs
rCanPIs (30 mg of each) or proteases were resolved in 15 or
8% native-PAGE, respectively and the gel(s) were further
processed for the activity visualization(s) using the gel X-ray
film contact print (GXCT) method [21, 22].
2.5 In vitro and in vivo interactions of rCanPI(s) and
HGP
To study the interaction of PIs with HGP in vitro, 0.5
H. armigera gut protease inhibition (HGPI) units of rCanPIs
(CanPI-5, CanPI-7, CanPI-19, CanPI-22) were incubated
with 1 U HGP for three time points (5 min, 1 h, and 6 h) at
room temperature. For rCanPI-13 and -15, 2 HGPI
units were incubated with the 1 U of HGP. These HGP-
treated PIs were resolved on native-PAGE gels and proces-
sed for TI activity visualization. Remaining HGP activity
was visualized upon incubation with PIs (1PI:2HGP units)
for 1 h.
About 70 mg (amount of inhibitor required for maximum
% inhibition of total HGP activity from a single fourth
instar larva) of rCanPIs (rCanPI-5 and -7) were incorporated
per gram of artificial chickpea flour-based diet [18] to test
their in vivo potential against H. armigera. Larvae were
reared on rCanPI-containing diets and control diet in
separate sets of 30 larvae each. Each larva was maintained
in an individual vial (50 mL). Gut tissue from the 4th
instar larvae (from each set) was dissected and stored
frozen (�801C) in polypropylene tubes (1.7 mL) until further
use. Gut tissues (200 mg) of H. armigera fed with rCanPI-5
and -7 incorporated ADs [18] were extracted in 0.2 M glyci-
ne–NaOH buffer, pH 10, as described earlier. Two-third
portion of this gut extract was heat treated at 701C for
15 min to inactivate and precipitate the active enzymes/
proteases. The heat-treated extracts were clarified by centri-
fugation at 13 000� g for 10 min at 41C and supernatant
resolved by native-PAGE followed by TI activity visualization
by GXCT.
The untreated HGP extracts from the CanPI-5- and
–7-fed H. armigera guts were separated on native-PAGE gels
and processed for protease activity visualization for identi-
fication of CanPI-5 and -7 inhibited/induced HGPs.
2.6 Proteases–PI interaction assays by MALDI-TOF-
MS
rCanPIs and their interactions with HGP and other
proteases were monitored by IF-MALDI-TOF analysis,
where reduction in intensity of a ligand (inhibitor) is
monitored by MALDI-TOF-MS on addition of a target
protease [23]. The mass spectral analysis was done on
Voyager-De-STR MALDI-TOF (Applied Biosystems,
Framingham, MA, USA) equipped with 337-nm pulsed
nitrogen laser. The mode of operation was in a positive
linear mode with an accelerating voltage of 25 kV.
All spectra were acquired by accumulating 50 single laser
shots over each sample spot in the range of 1–30 kDa with
the following settings: delayed ion extraction time of
1100 ns, grid voltage 93% and low-mass ion gate set to
1000 Da. They were processed for advanced baseline
correction and noise removal using Data Explorer software
(Applied Biosystems). The instrument was calibrated using
apomyoglobin and BSA (both Sigma-Aldrich, St. Louis, MO,
USA).
For the analysis of rCanPIs, 3 mg of protein sample
was mixed with 20 mL of freshly prepared sinapinic
acid (Sigma-Aldrich) (30% ACN, 0.1% TFA). About 2 mL
aliquots of this mixture were spotted on the stainless
steel MALDI plate by dried-droplet method and incubated at
371C for 20 min. The MALDI target plate was further
subjected to MALDI-TOF as specified above to get spectral
profiles.
For HGP–rCanPI interaction studies, equal (0.05 U)
HGPI units of each rCanPI were individually incubated
with three dilutions of HGP (0.5, 0.1, 0.01 U or crude, 1:5,
1:50) for various time points (5 min, 30 min, 1, 3 and 6 h).
Total volume of this reaction mixture was 10mL out
of which 5 mL was mixed with 20mL of freshly prepared
sinapinic acid and processed for obtaining their MALDI-
TOF profiles as mentioned above. Interaction of rCanPI-7
with 0.5 U of bovine trypsin and chymotrypsin (both Sigma-
Aldrich) was also monitored by MALDI-TOF as described
above.
3 Results
3.1 Selection of CanPI genes for recombinant
protein characterization
The sequence alignments, dendrograms and nine constitu-
ent IRDs of six CanPI genes selected in this study are
shown in Fig. 1. A typical CanPI consists of a 25 aa
signal peptide followed by 1–4-IRDs coupled by five aa
linker(s). Nine unique IRDs with 2–26% sequence
divergence were identified from these six CanPIsand their multiple sequence alignment revealed major
aa substitutions in RSL (Fig. 1C). Each �50 aa IRD
consists of eight conserved cysteine (C) residues and a single
reactive site (P1), either for trypsin inhibition (TI) or CI.
The presence of arginine (R) or lysine (K) at the P1
position is responsible for TI specificity, whereas the
presence of leucine (L) at the P1 position represents a typical
CI activity.
Proteomics 2010, 10, 2845–2857 2847
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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3.2 MALDI-TOF-MS and electrophoretic analysis of
rCanPIs reveal PI isoforms with variable number
of active IRDs
SDS-PAGE protein profiles of the individually expressed
rCanPIs showed a single�6 kDa protein in 1-IRD CanPIs,�12
and �6 kDa proteins in 2-IRD CanPIs, �19, �12 and �6 kDa
proteins in 3-IRD CanPI and four proteins of size �25, �19,
�12 and �6 kDa in 4-IRD CanPI (Fig. 2 inset). These rCanPIs
were further analyzed by IF-MALDI-TOF-MS to study their
interaction with HGP as well as to determine their accurate
molecular masses. All mass spectra were acquired in the range
of 1–30 kDa. A single peak of 5229 Da was observed in the case
of CanPI-15 (1-IRD); peaks of 12 231 and 5946 Da for CanPI-22
(2-IRD) and 19 214, 12 530 and 6190 Da peaks in CanPI-5
(3-IRD) were observed while CanPI-7 (4-IRD) showed four
expected size peaks of 25 377, 19 245, 12 070 and 6107 Da
(Fig. 2). These results were in accordance with the SDS-PAGE
protein profiles. It is known that the higher molecular mass
proteins exhibit low intensity in the mass spectra due to various
factors, such as: (i) low ion detection efficiency of standard
Micro channel Plate detectors toward higher m/z values,
(ii) poor resolution at higher m/z values and (iii) as they reach
the detector relatively slow. Therefore, low intensity peaks of
high (48 kDa) molecular mass proteins were observed by
zooming the selected region on the X-axis as displayed in insets
in Fig. 2. Mass spectra revealed at least 2–3 sub-peaks (or more
in some cases) of variable intensities for each multiple or
single-IRD isoform (Fig. 2). MALDI-TOF-MS analysis can
qualitatively and semiquantitatively determine the composition
of the processed IRDs in the purified rCanPI pools. Hence, this
technique was further used to monitor the interactions between
rCanPI(s) and various proteases.
3.3 Inhibitory activities of rCanPIs against trypsin,
chymotrypsin and HGP
rCanPIs were resolved on native- and SDS-PAGE and
visualized for TI profiles (Fig. 3A). Multiple TI activity
isoforms were detected for the rCanPIs including 1-IRD
CanPIs indicating the presence of heterogeneity at the
activity level. The number of TI activity isoforms was more
in the case of multi-IRD CanPIs as compared to 1-IRD PIs.
rCanPIs (0.1–2.0mg proteins) were used for the inhibition
studies against trypsin, chymotrypsin and HGP of 4th instar
larvae fed on the AD (AD-HGP) to find out a PI concentration
required for maximum inhibition. These amounts of rCanPIs
showed 70–90% inhibition of bovine trypsin activity; except
for rCanPI-15. CI was in the range of 50–90% by various
rCanPIs. rCanPIs without CI sites namely rCanPI-5, -13 and
-19 exhibited 50–84% CI whereas rCanPI-7 and rCanPI-22,
SP IRD-7 TICanPI-15
SP IRD-12 TI
SP IRD-4 CI IRD-9 TI
CanPI-13
CanPI-22
SP IRD-1 TI IRD-15 TI
SP IRD-1 TI IRD-1 TI IRD-12 TI
CanPI-19
CanPI-5
Li k St dSP Si l tid
SP IRD-14 TI IRD-5 CI IRD-10 TIIRD-4 CI
CanPI 5
CanPI-7
Linker Stop codonSP-Signal peptide
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |N R I C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K A C P R Y C D T R I A Y S K C
CN R I C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K A C P R Y C D T R I A Y S K CE P I C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K P C T L N C D P R I F Y S K CN R I C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K P C T L N C D P R I F Y S K CE P I C T N C C A G L K G C N Y Y N A D G T F I C E G E S D P N H P K A C P K N C D P N I A Y S L CQ P I C T N S S A G L K G C N Y Y N A D G T F I C E G E S D P N H P K A C P K N C D P N I A Y S L C
IRD-7IRD-9
IRD-5
IRD-1IRD-4
TICICITITIQ P I C T N S S A G L K G C N Y Y N A D G T F I C E G E S D P N H P K A C P K N C D P N I A Y S L C
Q P I C T N C C A G L K G C N Y Y N A D G T F I C E G E S D P N H P K A C P K N C D P N I A Y S L CN R L C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K A C P R N C D P N I A Y S L CN R I C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K P C P R N C D T R I A Y S K CN R L C T N C C A G R K G C N Y Y S A D G T F I C E G E S D P N N P K A C P R N C D T R I A Y S L C
IRD-9IRD-10IRD-12IRD-14IRD-15
TITITITITI
: : * * * . . * * * * * * * * . * * * * * * * * * * * * * * : * * . * . * * . . * * * *
P1
CanPI-19/ 2-IRDs; EF136383
CanPI-13/ 1-IRD; EF136387
CanPI-15/ 1-IRD; EF136389
CanPI-5/ 3-IRDs; DQ005912
CanPI-22/ 2-IRDs; EF136386
CanPI-7/ 4-IRDs; DQ005913
BA
Figure 1. (A) Diagrammatic representation highlighting the gene structure of four types of CanPIs found in C. annuum, with their signal
peptide sequence (SP), various IRD(s), linker region(s) and the stop codon. The signal peptide, IRDs and linker regions varying in the aa
sequence are shown in different colors and indicate their positions. (B) Neighbor-joining tree of CanPIs based on deduced aa sequences of
full-length genes, number of IRDs and the accession number. (C) Multiple sequence alignment of deduced aa sequences of unique IRDs
from the CanPIs selected for this study. The IRD numbers are according to the earlier report [22]. The inhibitory active site in the particular
IRD is referred to as TI for trypsin and CI for chymotrypsin inhibition. The reactive site residue P1 is marked by an arrow and the region
close to the active site showing major variation is marked by a box.
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both of which have 2 and 1 CI sites, respectively, showed
more than 90% CI. rCanPIs inhibited 50–60% of HGP
activity (Fig. 3B). rCanPI-15 showed very low TI (10%), HGPI
(6%) and failed to inhibit chymotrypsin activity.
HGPI by all the rCanPIs except rCanPI-15 using BApNA
as a substrate was 80%, whereas it was only 60% by rCanPI-
15. The change in the substrate showed remarkable differ-
ence in the inhibitory activities of rCanPIs.
3.4 rCanPI–HGP interactions detected by IF-MALDI-
TOF-MS
The interactions of rCanPIs with different proteases were
studied by IF-MALDI-TOF-MS as described previously [24].
The interaction assays between rCanPIs and HGP were opti-
mized with three HGP concentrations (undiluted, 1:5 and 1:50
dilutions) and intensities of the rCanPIs were monitored from
5 min to 6 h (Figs. 4–6). In these interaction studies, a peak of
1062 Da was considered as an internal standard for relative
quantification of CanPI peak because of its consistent signal
intensity (RSD 5 7.46%) for all CanPI sample preparations
and second there was no other peak that was closer to
5000–6000 Da mass range. As shown in Fig. 4 relative inten-
sity of rCanPI-15 peak at 5229 Da was progressively decreased
up to 6 h after its interaction with 1:5 HGP.
HGP and multi-IRD CanPIs interaction showed different
patterns in addition to the IF phenomena. In the case of
rCanPI-22 having 2-IRDs, the intensity of both 11 895 (2-IRD)
and 5950 Da (1-IRD) peaks reduced at 5 min of incubation.
However, at later time points (3 and 6 h) 2-IRD peak was not
detectable, whereas 1-IRD peak (5778 Da) intensity increased.
This enrichment of the 1-IRD peak indicated the processing of
2-IRD into 1-IRD by HGP (Supporting Information Fig. 1A).
Similar interactions were observed between rCanPI-5
and diluted 1:5 HGP (Supporting Information Fig. 1B).
A difference of �181 Da was noted in the processed 1-IRD
peak with respect to control, after 6 h of 1:5 HGP treatment.
Figure 2. Characterization of rCanPIs having either 1-, 2-, 3- or 4-IRDs each by SDS-PAGE and MALDI-TOF-MS. Proteins stained with
Coomassie Blue and mass peaks of 25 kDa (4-IRDs), 19 kDa (3-IRDs), 12 kDa (2-IRDs) and 6 kDa (1-IRD) were detected depending on the
number of IRDs present in the rCanPIs. The proteins of increasingly higher molecular mass appear as low intensity peaks in the mass
spectra because of the drop-off of the detection efficiency with increasing mass. These peaks were approximately four times magnified in
the insets.
Proteomics 2010, 10, 2845–2857 2849
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The interaction of rCanPI-7 with two representative
concentrations of HGP at four time points is represented in
Fig. 5. The IF of the four peaks in rCanPI-7 was prominent
with higher concentration of HGP (1:5), whereas dilute
HGP (1:50 HGP) demonstrated the sequential conversion of
4-IRD to 3-, 2- and finally 1-IRD forms. After 6 h, the
intensity of 1-IRD peak (5767 Da) was much higher than the
untreated rCanPI-7 peak (6107 Da). A transient increase in
the molecular mass diversity of the lower IRD forms
followed by the appearance of stable and intense 1-IRD peak
at 5767 Da with both the HGP concentrations was evident
(Fig. 5). There was no further processing and/or degrada-
tion of this peak (5767 Da) up to 6 h of interaction.
Comparison of the observed and calculated molecular
masses of processed repeats was then used for determining
the probable sites within the linkers where proteases would
have acted (Table 1).
3.5 Comparing interactions of rCanPI-7 with trypsin,
chymotrypsin and HGP
Bovine trypsin, chymotrypsin and HGP interactions were
monitored by IF-MALDI-TOF-MS in the time interval of
5 min to 6 h. The representative spectra are shown in Fig. 6.
In the rCanPI-7 and trypsin interaction, decrease in the
1-IRD peak intensity was evident at 3 h with a slight
reduction in the 2-, 3- and 4-IRD intensities. With chymo-
trypsin there was a decrease in the intensities of 1-, 2- and
3-IRD peaks up to 3 h. The interesting feature of the rCanPI-
7–chymotrypsin interaction was the appearance of peaks at
10 and 15 kDa and increase in 4-IRD peak intensity. rCanPI-
7 and HGP interaction is as detailed above in Section 3.4.
3.6 In vitro and in vivo stability of CanPIs to HGP
In vitro stability of rCanPIs treated with HGP was analyzed
by native in-gel TI activity visualization of rCanPI isoforms
(Fig. 7). 1-IRD PIs, rCanPI-13 and rCanPI-15 displayed four
or five TI isoforms of which only one or two remained stable
after HGP treatment for 6 h. rCanPI-22 (2-IRD PI) exhibited
six TI isoforms, out of which two prominent higher mobility
isoforms were stable to HGP. Out of the five TI isoforms of
rCanPI-19 (2-IRD PI), three remained stable up to 1 h in the
presence of HGP and showed partial degradation at 6 h.
Most of the TI isoforms of rCanPI-5 (3-IRD PI) and
-7 (4-IRD PI) were found to be stable in the presence of
HGP even up to 6 h. However, HGP altered the mobility of
TI isoforms as in the case of rCanPI-7 where few new lower
A B
Figure 3. (A) Trypsin inhibitory activity (TI) visualization of rCanPIs. rCanPIs were resolved in 15% SDS-PAGE (upper) and 12% native-
PAGE (lower) and subjected to in-gel TI activity visualization using GXCT. Multiple rCanPI TI activity was detected in both the gels. The
first TI activity protein in the SDS-PAGE was corresponding to the apparent molecular mass of the predicted mature rCanPI of the
respective sequence. (B) Enzyme inhibition by rCanPIs. Maximum percent inhibition of bovine trypsin, chymotrypsin and HGP by
minimum amounts (mg) of rCanPIs in azo-caseinolytic assays is represented in the bar graph. Each value is an average of six replicates
with bars indicating standard error. Gut proteases from the AD-fed H. armigera (AD-HGP) are inhibited maximally (50–60%) by at least
1.33-mg rCanPIs. The minimum amount of rCanPIs was individually used to check the inhibition of standard trypsin and chymotrypsin.
rCanPI-15 required higher amounts (2.67mg) for trypsin inhibition. HGP inhibition by rCanPIs was also estimated by using BApNA as a
substrate. The change in substrate showed significant difference in the activities of the rCanPIs.
2850 M. Mishra et al. Proteomics 2010, 10, 2845–2857
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mobility TIs appeared within 5 min of HGP treatment and
isoforms exhibiting low intensity became stronger. TI
activity profiles indicated the processing and partial proteo-
lysis of rCanPIs. The stability of individual rCanPI varied in
the proteolytic environment.
HGP of 4th instar larvae displayed at least 11 protease
isoforms (HGP-1–HGP-11 in Fig. 8(A)). Inhibition of HGP
isoforms by rCanPIs was studied by performing protease
activity visualization of HGP pretreated with rCanPI
(Fig. 8(A)). HGP-3–HGP-6 were inhibited by all rCanPIs,
whereas HGP-2, HGP-7 and HGP-10 remained active in
each case. HGP-1 was inhibited by all rCanPIs except 1-IRD
rCanPIs, which unexpectedly showed even higher protease
activity of this HGP isoform. HGP-9 was differentially
inhibited by rCanPI-5, -7 and -19. It was interesting to note
that HGP-8 was inhibited effectively only by rCanPI-5 and -
13, which share one unique IRD sequence (IRD-12). Overall,
most of the isoforms of HGP were inhibited by rCanPI-5
and -7 (Fig. 8(A)-lanes 5, 6).
Gut extracts of the 4th instar H. armigera larvae fed on
AD with rCanPI-5 and -7 were visualized for protease
activity (Fig. 8(B)). A major reduction in the activity of
protease isoforms HGP-4, -5, -9 and -10 was detected in the
rCanPI-5 and –7-fed gut extracts as compared with the
control (larvae fed on AD). HGP-7 activity was seen to be
enhanced in inhibitor-fed gut extracts. The appearance of
novel protease isoforms, three in case of CanPI-7 and one in
CanPI-5-fed insect gut, was detected (indicated by arrows in
Fig. 8(B)).
The rCanPI-fed H. armigera gut extracts were visualized
for the presence of PIs. Three and two prominent PI
isoforms were found in rCanPI-5 and –7-fed H. armigera gut
tissue, respectively (Fig. 8(C)). These TIs correlated with
in vitro profiles of HGP-treated rCanPI-5 and -7 (Fig. 7).
4 Discussion
4.1 MALDI-TOF analysis can be used to precisely
determine the CanPI–protease interaction
P. pastoris-expressed rCanPIs revealed multiple active
isoforms formed due to the processing of the mature
precursor protein [18]. MALDI-TOF-MS of 1-, 2-, 3- and
4-IRD rCanPIs showed 1, 2, 3 and 4 number of peaks,
respectively, corresponding to the processed precursors. The
proteolytic processing of precursor is a characteristic feature
of Pin-II PIs. Plant endogenous proteinases efficiently
process the 6-IRD N. alata precursor into 1-IRD form as
indicated by weak 3- and 4-IRD isoforms and strong 1-IRD
isoform [25]. The five aa linker regions in Pin-II PIs and also
in CanPIs (QRNAK, EENAE, EASAE, EGNAE, EETQK)
form a hydrophilic loop that presents the protease proces-
sing site on the surface of the molecule [26]. Further
protease processing of CanPIs toward termini enhances
their molecular diversity [16].
In this study, rCanPIs interacting with different proteases
over a time period were analyzed by MALDI-TOF. The mass
spectra revealed the molecular mass of each of the precur-
sors, processed IRDs and also the molecular diversity before
and after rCanPI–protease interaction. On interaction with
trypsin, no major variation in the diversity of mass peaks was
observed, whereas interaction with chymotrypsin showed
additional peaks of 10 and 15 kDa, which might be because of
cleavage within the IRDs. In the case of rCanPI-7 and HGP
interaction, the 2-, 3-, and 4-IRD isoforms were efficiently
Figure 4. IF-MALDI-TOF-MS analysis of rCanPI-15. The decrease
in the relative intensity of rCanPI-15 (6 kDa) upon addition of
target protease, HGP was evident. The internal control (1062 Da)
has been used as the reference for relative quantification.
Proteomics 2010, 10, 2845–2857 2851
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processed into single IRDs indicating the processing of the
precursor PIs at the linker regions followed by trimming of aa
at either or at both the termini. For multi-domain Pin-II PIs,
it has been postulated that it is very difficult for each domain
to bind to a protease without a steric hindrance [10]. In the
EGNAE linker, NkA was the most commonly processed
proteolytic cleavage site (Table 1). Several-fold processing of
PIs in suitable proteolytic environment, like in plants and
insect gut, leads to increase in its IRD diversity which may
have functional significance with respect to modification of
its inhibitory potential.
4.2 Sequence variation in the CanPIs influences
their interactions with different proteases
The eight fully conserved cysteines work as a structural
scaffold to hold the RSL in a relatively rigid conformation
that helps to prevent proteolytic cleavage of the inhibitor
upon interaction with proteases [11]. The strength of the
protease–PI interaction is determined by the compatibility
of all aa residues (P4–P40). It is interesting that the residues
outside this loop, referred to as adventitious contacts, can
also significantly affect the affinities of the inhibitor for
closely related target proteases [27].
rCanPIs display variation in terms of their inhibition
potential against proteases such as trypsin and chymo-
trypsin, which correlates with the number of its TI and CI
domains. Over 90% inhibition of these was attained by
rCanPI-7 (4-IRD), which has a combination of two TI and
two CI IRDs. rCanPIs with only TI IRDs (rCanPI-5, -13 and
-19) also showed �84% CI, probably because of the cross
reactivity as reported in earlier studies [28]. With equal
rCanPI protein HGPI was 6–60%; highest by rCanPI-5 and
-7 and lowest by rCanPI-15. Higher HGPI by rCanPI-7
(4-IRD PI) at a lower protein concentration than rCanPI-13
(1-IRD PI) could be attributed to multiple and diverse IRDs
in rCanPI-7. As an exception, rCanPI-15 (IRD-7; TI)
Figure 5. IF-MALDI-TOF-MS analysis of rCanPI-7–HGP interaction. Different concentrations of HGP (0.5, 0.1, 0.01 U) were incubated with
rCanPI-7 (0.05 HGPI unit) for 5 min, 1, 3 and 6 h at 241C. The reaction assay setup and MALDI sample preparation is described in Section 2.
Due to the interactions between the PI and HGP, change in the intensity and diversity of the CanPI peaks was detected and is indicated by
arrows. The higher molecular mass range from 8 kDa onwards is enlarged to enhance visibility of peak. All the spectra were processed for
advanced baseline correction and noise removal.
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revealed low inhibition efficiency against all proteases
tested, when azocasein was used as a substrate. Interest-
ingly, it showed about 60% HGPI and other rCanPIs also
showed around 80% HGPI when synthetic substrate
BApNA was used. This shows that inhibitor efficiency is
strongly influenced by the nature of substrates present, and
thus among other factors might show further differences in
natural conditions.
Gut extract of H. armigera is a complex of several
enzymes and various proteases [17]. Differential inhibition
of the total 11 HGP activity isoforms (HGP-1–HGP-11,
Fig. 8(A)) was observed by various rCanPIs with multi-IRD
PIs showing higher suppression of HGP isoforms. Specific
inhibition of HGP-8 only by rCanPI-5 and rCanPI-13 was
noted which may be because of IRD-12. IRD-12 is not
present in any other rCanPIs and has high similarity with
IRD-14 and IRD-15 (present in CanPI-7 and CanPI-22,
respectively) with a difference of only single aa close
to the reactive site (Fig. 1(C)). Even then CanPI-7 and -22
did not show inhibition of HGP-8; highlighting the
functional significance of single aa changes in IRDs.
Similarly, HGP-9 was strongly suppressed by rCanPI-5
and rCanPI-7 compared with other rCanPIs. This
implies that one of the component IRDs of these rCanPIs
(IRD-1, -12, -4, -14, -10, -5) might be specific for inhibition of
HGP-9. HGP-2 remained uninhibited by any of the rCanPIs
indicating its insensitivity to any of the IRDs tested.
Differential inhibition potential of Pin-II PIs can also be
attributed to the orientation of inhibitory domains in space
relative to each other [14]. The diversity in CanPIs with
respect to the number of IRDs per gene and sequence
variations in IRDs themselves points toward a more
complex interaction of CanPIs with endogenous and/or pest
gut proteases.
Figure 6. IF-MALDI-TOF-MS analysis of the interaction(s) between rCanPI-7 and bovine trypsin (a), chymotrypsin (b) and HGP (c). 0.5, 0.5,
0.1 U of trypsin, chymotrypsin, HGP, respectively were incubated with 0.05 HGPI units of inhibitor for 5 min and 3 h and analyzed by
MALDI-TOF to detect the changes in the four normal peaks of rCanPI. Bovine trypsin and chymotrypsin do not act on the linker regions in
the rCanPI-7, whereas HGP cleaves on the linkers in turn processing the multi-IRD forms to lower IRD forms.
CanPI-15 CanPI-13 CanPI-22
+HGP
5min. 1 h 6 h 5min. 5min.1 h 1 h6 h
5min. 1 h 6 h5min. 1 h 6 h 5min. 1 h 6 h
6 h+HGP +HGP
CanPI-7CanPI-5CanPI-19
+HGP +HGP +HGP
Figure 7. In vitro stability of CanPIs toward HGP. Equal HGPI
units (0.5) each of CanPI-15, -13, -22, -19, -5, -7 were incubated
with 1 HGP unit at 241C for 5 min (lane 2), 1 h (lane 3), 6 h (lane 4)
and the reaction mixtures were resolved on 12% native-PAGE
gel. Each rCanPI without HGP treatment (lane 1) was loaded as
control. The gels were processed for TI activity visualization by
GXCT. 1-IRD CanPIs show reduced intensities of isoforms as
compared with multi-IRD PIs in the presence of HGP.
Proteomics 2010, 10, 2845–2857 2853
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4.3 In vivo stability of CanPIs: significance against
H. armigera
In this study, the in vitro experiments to check the stability
of each rCanPI indicated that HGP caused processing and
in some cases (rCanPI-15) degradation of the PIs. On the
contrary, the processed IRDs from rCanPI-5 and -7
remained stable even after a prolonged incubation with
HGP. Experiments demonstrated the low efficiency of
single IRD CanPIs against HGP; indicating the importance
of presence of multiple IRD genes in planta for defense.
This could be well correlated to the induced upregulation
of higher IRD PI transcripts upon aphid and S. liturainfestation [19]. In vivo studies to determine the fate of
CanPIs in insect precisely displayed an active PI in the gut
(Fig. 8(C)). Host PIs are degraded by the pest gut enzymes
and such PIs are therefore ineffective as pest control
molecules [22]. Harsulkar et al. [29] have reported that PIs
from non-host plants can effectively inhibit gut proteases
and the larval growth. Stability in the insect gut environ-
ment is an important feature of PIs to be used in insect
defense.
rCanPI-5 and -7-fed gut extracts showed inhibition of
HGP-4, -5 and -10 and HGP-7 was seen to be overexpressed.
Additionally, induction of novel proteases (marked by
arrows in Fig. 8(C)) that might be insensitive to rCanPIs was
observed. The change in the expressions of gut protease
isoforms in response to rCanPIs signifies their dynamic
interactions.
C. annuum possess an array of Pin-II PI genes ranging
from 1- to 4-IRD CanPIs with differential patterns of
expression in various tissues. The 1- and 2-IRD PIs were
predominantly found to be expressed in the stem tissue [19].
Our study shows their degradation in the presence of HGP,
which infers that they might not have role in defense against
insect; however, these PIs may have a physiological role
in planta. The inhibition potential of multi-IRD CanPIs due
to their efficient processing by gut proteolytic machinery to
produce a wide spectrum of structurally and functionally
divergent IRDs can be attributed to their involvement
in defense. The upregulation of diverse multi-repeat
CanPIs proposes their molecular coevolution in response to
pest attack [14]. Further divergence within the single
genes facilitates the targeting of diverse proteases. Thus,
(1:2
)
:2)
1:2)
(1:2
)
(1:2
)
(1:2
)
-15
+ H
GP
(
-7 +
HG
P(1
-5 +
HG
P(1
-19
+ H
GP
(
-22
+ H
GP
(
-13
+ H
GP
(
Can
PI-
HG
P
Can
PI-
Can
PI-
Can
PI-
Can
PI-
Can
PI-
PI-
5 fe
d
PI-
7 fe
d
PI-
5
PI-
7
HGP-1HGP-2
HGP-3HGP-4HGP 5
P P Co
ntr
ol g
ut
Co
ntr
ol g
ut
P P
HGP-1
HGP-5
HGP-6
HGP-7
G 8HGP-5
HGP-4
HGP-8
HGP-9
HGP-10 HGP-8
HGP-7
HGP-6
HGP-10
HGP-11
HGP-10
HGP-9
In vitro Protease activity Protease activity PI activity
In vivo
A B C
Figure 8. Stability of CanPIs to HGP. (A) In vitro: Comparative inhibition of HGP isoforms by different rCanPIs. Equal HGPI units of CanPI-
15, -13, -22, -19, -5 and -7 were incubated with HGP for 30 min at 241C. The above reaction mixtures were then resolved on 8% native-
PAGE. The gels were processed for protease activity visualization by GXCT. rCanPI-5 and -7 show inhibition of maximum HGP isoforms.
(B) In vivo: Inhibition of HGP isoforms by rCanPIs. Equal amounts of gut tissues of H. armigera fed on rCanPI-5 and -7 containing AD and
those fed on control diets were extracted in 1:1 (weight: volume) in 0.2 M glycine–NaOH pH 10.0 buffer. Equal volumes of these gut
extracts were resolved on 8% native-PAGE. The gel was processed for protease activity visualization. CanPI-7-fed H. armigera gut extract
shows an overall reduced protease activity as compared to the controls. Appearance of new protease isoforms is evident from this
analysis. (C) In vivo: Stability of rCanPIs in H. armigera gut. The rCanPI-5 and –7-fed H. armigera gut extracts were analyzed for TI activity
visualization by GXCT after inactivation of the proteases by heat treatment at 701C for 15 min. TI activity of both rCanPI-5 and -7 was
detected in these extracts indicating their in vivo stability in H. armigera gut.
2854 M. Mishra et al. Proteomics 2010, 10, 2845–2857
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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Tab
le1.
Mo
lecu
lar
mass
of
pro
cess
ed
Can
PI-
7IR
Ds
an
dp
red
icte
dsi
teo
fp
roce
ssin
g
Iso
form
so
fC
an
PI-
7g
en
era
ted
aft
er
pro
teo
lyti
cp
roce
ssin
gO
bse
rved
mo
lecu
lar
mass
(kD
a)
Fra
gm
en
teq
uiv
ale
nt
too
bse
rved
mo
lecu
lar
mass
Calc
ula
ted
mo
lecu
lar
mass
(kD
a)
Pre
dic
ted
site
of
pro
cess
ing
at
the
lin
kers
1R
form
s6.1
11
E-I
RD
-14-E
6.1
1E
GN
Ak
E-I
RD
-14-Ek
GN
AE
6.1
06
AE
-IR
D-1
4-E
6.1
8E
GNk
AE
-IR
D-1
4-Ek
GN
AE
5.9
46
K-I
RD
-45.9
7Q
RN
Ak
K-I
RD
-45.7
67
a)
AE
-IR
D-1
45.7
1E
GNk
AE
-IR
D-1
45.7
67
a)
AK
-IR
D-4
5.7
QR
Nk
AK
-IR
D-4
5.7
67
a)
AE
-IR
D-5
5.7
4E
GNk
AE
-IR
D-5
5.7
67
a)
AS
AE
-IR
D-1
05.7
2Ek
AS
AE
-IR
D-1
02R
form
s12.2
70
IRD
-14-I
RD
-512.2
2E
GN
AEk
-IR
D-1
4-I
RD
-5-k
EA
SA
E12.0
96
IRD
-4-I
RD
-14
12.1
8Q
RN
AKk
-IR
D-4
-IR
D-1
4-k
EG
NA
E12.0
59
GN
AE
-IR
D-5
-IR
D-1
012.0
8Ek
GN
AE
-IR
D-5
-IR
D-1
03R
form
s19.2
45
NA
K-I
RD
-4-I
RD
-14-I
RD
-5-E
AS
A19.2
QRk
NA
K-
IRD
-4-I
RD
-14-I
RD
-5-
EA
SAk
E18.2
46
AE
-IR
D-1
4-I
RD
-5-I
RD
-10
18.2
5E
GNk
AE
-IR
D-1
4-I
RD
-5-I
RD
-10
4R
form
s25.8
19
KA
CS
QR
NA
K-I
RD
-4-I
RD
-14-I
RD
-5-I
RD
-10-L
YE
K25.8
9S
P-k
KA
CS
QR
NA
K-I
RD
-4-I
RD
-14-I
RD
-5-I
RD
-10-L
YE
K25.3
77
KA
CS
QR
NA
K-I
RD
-4-I
RD
-14-I
RD
-5-I
RD
-10
25.3
6S
P-k
KA
CS
QR
NA
K-I
RD
-4-I
RD
-14-I
RD
-5-I
RD
-10
25.0
51
SQ
RN
AK
-IR
D-4
-IR
D-1
4-
IRD
-5-I
RD
-10
25.0
6S
P-K
ACk
SQ
RN
AK
-IR
D-4
-IR
D-
14-I
RD
-5-I
RD
-10
Th
em
ole
cula
rm
ass
es
of
1-,
2-,
3-
an
d4-I
RD
iso
form
so
frC
an
PI-
7(m
ajo
rp
eaks
inn
ati
ve
rCan
PIp
oo
las
well
as
on
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ract
ion
wit
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GP
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rved
inth
eM
ALD
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ect
raw
ere
use
dto
com
pare
wit
hth
eca
lcu
late
dm
ole
cula
rm
ass
es
of
the
pro
cess
ed
IRD
san
dth
us
pre
dic
tth
esi
teo
fp
roce
ssin
gat/
wit
hin
the
lin
ker
reg
ion
.T
he
lin
kg
iven
belo
ww
as
use
dfo
rca
lcu
lati
ng
mass
es
of
pro
cess
ed
IRD
sb
yre
mo
vin
gaa
on
eb
yo
ne.
htt
p:/
/ww
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cien
ceg
ate
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y.o
rg/t
oo
ls/p
rote
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tm.
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form
pre
do
min
an
tly
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serv
ed
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pro
cess
ing
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HG
P.
Proteomics 2010, 10, 2845–2857 2855
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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multi-domain protein(s) with various PI specificities is the
plant’s answer to the gut protease variants expressed by
insects.
The Council of Scientific and Industrial Research (CSIR),Government of India, New Delhi supported this project undernetwork project grants to National Chemical Laboratory, Pune(NWP0003). M. M. and V. A. T. acknowledge CSIR for theresearch fellowships. They acknowledge Bhagyashree Swarge forhelp in cloning and expression of CanPI genes.
The authors have declared no conflict of interest.
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