Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing...

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

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


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


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.



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


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.



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


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.



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


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.



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

inte

ract

ion

wit

hH

GP

)o

bse

rved

inth

eM

ALD

I-T

OF-M

Ssp

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

w.s

cien

ceg

ate

wa

y.o

rg/t

oo

ls/p

rote

inm

w.h

tm.

a)

Iso

form

pre

do

min

an

tly

ob

serv

ed

on

pro

cess

ing

by

HG

P.

Proteomics 2010, 10, 2845–2857 2855


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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Proteomics 2010, 10, 2845–2857 2857

Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing...

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Transcript of Interaction of recombinant CanPIs with Helicoverpa armigera gut proteases reveals their processing...