The amino acid sequence is closely related 76% identical to that of a trypsin, HzTrypsin-C, which was purified and identified in a similar way from insects raised on a diet without additio
Trang 1Properties of purified gut trypsin from Helicoverpa zea ,
adapted to proteinase inhibitors
Mariateresa Volpicella1, Luigi R Ceci2, Jan Cordewener3, Twan America3, Raffaele Gallerani1,
Wolfram Bode4, Maarten A Jongsma3and Jules Beekwilder3
1
Dipartimento di Biochimica e Biologia Molecolare, Universita` di Bari, Italy;2Centro di Studio sui Mitocondri e Metabolismo Energetico, C.N.R., Sezione di Trani, Italy;3Plant Research International, Wageningen, the Netherlands;
4
Max-Planck-Institut fu¨r Biochemie, Mu¨nchen, Germany
Pest insects such as Helicoverpa spp frequently feed on
plants expressing protease inhibitors Apparently, their
digestive system can adapt to the presence of protease
inhibitors To study this, a trypsin enzyme was purified from
the gut of insects that were raised on an inhibitor-containing
diet The amino-acid sequence of this enzyme was analysed
by tandem MS, which allowed assignment of 66% of the
mature protein amino acid sequence This trypsin, called
HzTrypsin-S, corresponded to a known cDNA sequence
from Helicoverpa The amino acid sequence is closely related
(76% identical) to that of a trypsin, HzTrypsin-C, which was
purified and identified in a similar way from insects raised on
a diet without additional inhibitor The digestive properties
of HzTrypsin-S and HzTrypsin-C were compared Both trypsins appeared to be equally efficient in degrading pro-tein Four typical plant inhibitors were tested in enzymatic measurements HzTrypsin-S could not be inhibited by
> 1000-fold molar excess of any of these The same inhibi-tors inhibited HzTrypsin-C with apparent equilibrium dis-sociation constants ranging from 1 nM to 30 nM Thus, HzTrypsin-S seems to allow the insect to overcome different defensive proteinase inhibitors in plants
Keywords: gut; Helicoverpa; inhibitor; insect; trypsin
Larvae of the lepidopteran insect species Helicoverpa are a
pest in Asia, Australia and the Americas They cause yield
losses on many important crops, like cotton, chickpea, corn,
and tomato For instance, of the total cotton area in China
(4.7 million hectares), 30% was lost to H armigera in the
mid nineties [1] Chemical control of Helicoverpa insects is
often not effective, as they are notorious for development of
resistance to chemicals such as DDT, organophosphates
and pyrethroids [2]
One form of natural defence of plants against insects is
mediated by protease inhibitors [3] The inhibitors are
thought to have coevolved with insect herbivory, and to
function by blocking the digestive proteases in the larval gut,
thereby limiting the release of amino acids from food
protein As a consequence, the larvae are arrested in development, and eventually die Genes encoding protease inhibitors have been used to produce resistant transgenic plants as a crop-protection strategy This has met with initial success [4–6], but disappointing results have been reported for Helicoverpa spp., and a few other pest insects Although several groups have shown that a major part of Helicoverpa gut protease activity can be blocked by a number of inhibitors [7,8], even the most efficient inhibitor (soybean Kunitztrypsin inhibitor, SKTI), which inhibits 95% of trypsin activity in gut extracts, does not affect the larval development of this insect on artificial diet [9] or transgenic plants [8]
The lack of effect on larval development is caused by the adaptation of Helicoverpa spp to protease inhibitors, which
is mediated by their ability to alter the complement of proteolytic activity in their gut In response to inhibitor ingestion, the arsenal of gut proteinases is switched to enzymes that are insensitive to the plant inhibitors [10,11] The adaptation of gut proteolysis to protease inhibitors is accompanied by changes in transcription of protease genes
A number of trypsin and chymotrypsin cDNA clones have been isolated from Helicoverpa mid-guts [9,12] Reported gene expression data provide correlations to the changes in proteolysis in the insect gut However, due to lack of a suitable expression system, the protease genes have not yet been linked to their function in terms of sensitivity to various inhibitors, substrate specificity or relative contribu-tion to protein digescontribu-tion
In this report, for the first time enzymes directly involved
in resistance to plant defence were purified from Helicoverpa gut Individual enzymes were sequenced and their interac-tion with substrates and plant protease inhibitors analysed
Correspondence to M J Beekwilder, Plant Research International,
Postbus 16, 6700 AA Wageningen, the Netherlands.
Fax: + 31 317 418094, Tel.: + 31 317 477164,
E-mail: M.J.Beekwilder@plant.wag-ur.nl
Abbreviations: SKTI, soybean Kunitztrypsin inhibitor; MTI-2,
mustard trypsin inhibitor II; BApNA, Na-Benzoyl- L
-Arg-p-nitroani-lide; ZRRpNA, Z-Arg-Arg-p-nitroani-Arg-p-nitroani-lide; ZFRpNA,
Z-Phe-Arg-p-nitroanilide; ZRpNA, Z-Arg-Z-Phe-Arg-p-nitroanilide; RpNA, L
-Arg-p-nitro-anilide; SAAPLpNA, N-Succinyl-Ala-Ala-Pro-Leu-p-nitro-Arg-p-nitro-anilide;
TLCK, N-tosyl- L -lysine-chloromethyl ketone; SBBI, soybean
Bow-man–Birk inhibitor; CID, collision-induced dissociation; PI-2, potato
inhibitor II; HzTrypsin-C, Helicoverpa zea trypsin from insects raised
on control diet; HzTrypsin-S, Helicoverpa zea trypsin from insects
raised on SKTI-containing diet.
(Received 12 July 2002, revised 5 November 2002,
accepted 6 November 2002)
Trang 2Materials and methods
Insects
H zea eggs were purchased from French Agriculture
(Lamberton, MN, USA), and hatched at 28C on artificial
diet as described [13] The diet contains per litre: 160 g
cornmeal, 80 g wheat germs, 80 g yeast flakes, 8 g ascorbic
acid, 2 g sorbic acid, 1 g p-hydroxybenzoic acid, 0.1 g
streptomycin and 30 g agar Each individual first instar
larva was sealed into a chamber containing 5 mL artificial
diet In the final stages of the fourth instar, 50 larvae that
were about to molt were transferred to artificial diet
supplemented with 0.5% (w/v) (250 lM) SKTI soybean
trypsin inhibitor (type II-S, Sigma), while another 50 larvae
remained on artificial diet without inhibitor After 48 h,
insects were chilled on ice, and guts with contents were
excised, aliquoted and frozen at)80 C Frozen guts were
thawed on ice, and mixed 1 : 3 with 50 mMTris/HCl pH 8
containing 1% polyvinylpolypyrrolidone with 0.5MNaCl,
leading to about 10 mL per 50 guts Guts were
homogen-ized three times using an S541 potter tube at 60 r.p.m., and
centrifuged for 15 min at 10 000 g, 4C to remove solid
particles The supernatant was filtered through a 0.22 lm
filter
Affinity chromatography
Mustard trypsin inhibitor II (MTI-2) was produced in
Pichia pastoris as described [14] Fifteen mg MTI-2 was
coupled overnight at 4C to 1.5 g CNBr-activated
seph-arose 4B (Amersham Pharmacia Biotech AB) in 20 mL
1 mM HCl according to the manufacturer’s instructions
The material was used to pour a 5-mL MTI-2 column,
which was equilibrated with E-buffer (50 mM Tris/HCl
pH 8, 0.5MNaCl) Four mL gut content supernatant of
H zea(4 mg total protein) was loaded on the column, after
which it was washed with 35 mL E-buffer and 35 mL
E-buffer without salt MTI-2-bound protein was eluted
stepwise using 5 mL of G-buffer (0.1M HCl/glycine)
pH 3.2; 5 mL G-buffer pH 2.2; 5 mL G-buffer pH 1.5
and 5 mL G-buffer pH 1.5 + 20% dimethylsulfoxide
Eluted fractions were neutralized to pH 8 using 2M Tris
pH 10.5 and stored at +4C
Protease assays
Protein fractions were normalized for protein content Two
lg protein were mixed with 150 lL assay buffer (25 mM
glycine/NaOH pH 10; 0.1 mgÆmL)1BSA; 2.5 mMCaCl2)
After incubation at 22C for 30 min, 50 lL of substrates in
assay buffer containing 10% dimethylsulfoxide were added
to a final concentration of 1 mM, and substrate breakdown
was monitored at 405 nm Substrates were
Na-Benzoyl-L-Arg-p-nitroanilide (BApNA), Z-Arg-Arg-p-nitroanilide
(ZRRpNA), Z-Phe-Arg-p-nitroanilide (ZFRpNA),
Z-Arg-p-nitroanilide (ZRpNA) andL-Arg-p-nitroanilide (RpNA)
for trypsin, and N-Succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
(SAAPLpNA) for chymotrypsin activity BApNA and
RpNA were from Sigma, SAAPLpNA, ZRRpNA,
ZFRpNA and ZRpNA were from Bachem (Bubensdorf)
Assays for inhibitor specificity were carried out in the presence of N-tosyl-L-lysine-chloromethyl ketone (TLCK), SKTI, Soybean Bowman–Birk Inhibitor (SBBI; Sigma), MTI-2 [14] or potato trypsin/chymotrypsin inhibitor PI-2 [15] The concentration of reactive sites of these inhibitors was determined on bovine trypsin as described previously [15] Inhibitors were added to the protease and buffer, and preincubated for 30 min before adding the substrate Dietary protein breakdown was measured as follows: 1 g artificial diet was frozen, ground into fine powder with a mortar, and extracted with 10 mL acetone and six 10-mL portions of hexane Soluble protein was extracted from the residual pellet by vigorous stirring with 10 mL water for 2 h
at 4C Insoluble matter was removed by centrifugation, and small soluble peptides by precipitation with 3 vols acetone The final acetone pellet (360 lg protein) was dissolved in 1 mL buffer (50 mMglycine/NaOH, pH 10) to
a clear solution Aliquots of 100 lL soluble protein were mixed with 100 lL insect enzyme and 100 lL buffer and incubated at 37C After 30 min 100 lL 40% trichloro-acetic acid was added, incubated for 5 min at room temperature and centrifuged for 5 min The supernatant was used to measure absorption of light at 280 nm Absorption of soluble dietary protein that had not been incubated with enzyme, and enzyme that had not been incubated with soluble dietary protein were used as controls Control values were constant over a number of experiments Azocasein assays were performed as described previously [13]
SDS/PAGE and IEF For SDS PAGE, 2.5 lg total gut proteins and 0.5 lg (15 lL) of the fractions were diluted with 5 lL sample buffer (20% glycerol, 20 mM Tris pH 6.8, 0.4% SDS, 0.001% Bromophenol blue), and kept on ice Protein staining was performed by using silver nitrate [16] Activity staining with casein as described [13]
For IEF, 300 lL of the fractions (10 lg protein) were precipitated with 10% trichloroacetic acid, washed with ice-cold acetone and resuspended in 125 lL rehydration so-lution (8M urea, 2M thio-urea, 2% Chaps, 2 mM dithio-threitol, 2 mM EDTA) Immobilized pH gradient (IPG) buffer (0.5%, pH 6–11, Amersham) was added, mixed and the sample was allowed to enter an Immobiline DryStrip pH 6–11 (7 cm; Amersham) overnight Focusing was performed for 6 h from 500 to 8000 V The strip was subsequently equilibrated in a solution containing 1% dithiothreitol,
50 mMTris/HCl pH 8.8, 6MUrea, 30% glycerol, 2% SDS, and stained with Coomassie brilliant blue
MS Protein bands were excised from the gel, dried, and digested
in gel with trypsin [17] Protein was extracted, and loaded onto a C18 PepMap column (15 cm· 75 lm) Peptides were eluted by a 30-min gradient from 0.5% formic acid in water to 0.5% formic acid in 50% acetonitril at a speed of 0.2 lLÆmin)1 The C18 column was connected to the electro-electro-spray of a Q-Tof-2 Mass spectrometer (Micromass) by a PicoTip (New Objective) The Qtof mass
Trang 3spectrometer was instructed to determine charge of the
eluting peptides, and, if appropriate (i.e 2 + or 3 +), the
QtofMS switched to the MS/MS mode applying
collision-induced dissociation (CID) The resulting CID spectrum
contains the sequence information for a single peptide
TheMASS-LYNXpackage V4.3 (MicroMass) was used to
process MS data First theMAXENT3 module was used to
deconvolute the data MS/MS spectra containing CID
products were selected for further processing The BioLynx
PepSeq module was used to interpret MS/MS spectra and
to generate peptide sequences The MS/MS spectra (usually
around 25 per peptide) were further scrutinized manually by
using the ManSeq mode
MS results were compared to 34 database accessions with
the following numbers: AF045138 (H armigera trypsin),
AF233731–AF233734 (H zea chymotrypsins), AF261980–
AF261989 (H zea trypsins) and Y12269-Y12287 (H
armi-geraserine proteases)
Results
H zea larvae adapt to the presence of SKTI in the diet
To obtain gut proteases that were resistant to protease
inhibitors, larvae were adapted to SKTI Two populations
of H zea larvae were raised in parallel One population of
larvae was reared during its entire larval development on a
control diet, consisting of corn materials When these insects
were in their fifth instar, guts were isolated Trypsin activity
of gut extracts was tested, and appeared to be 95%
inhibitable by 0.5 lMSKTI Guts from the other
popula-tion of larvae were isolated at the same time, but the insects
had been transferred to corn diet supplemented with SKTI
48 h previously Extracts from this population have a
completely different trypsin activity (BApNAse): only 2% is
inhibitable by 0.5 lM SKTI These results confirm those
published for H armigera and for H zea [9,12]
Insect trypsins can be efficiently purified by affinity
chromatography
The H zea crude gut extracts were used for purification of
the Trypsin-like enzymes by affinity chromatography
Mustard trypsin inhibitor MTI-2, a proteinaceous trypsin
inhibitor, was cross-linked to Sepharose, and used as affinity
ligand MTI-2 is known to be a very potent trypsin
inhibitor, but to have a low affinity for chymotrypsin [18] In
a pilot experiment we established that both active trypsin
and chymotrypsin (bovine) can be sequestered by this
material, and can be separated by eluting at different pH
values (data not shown) Apparently, the relatively low
affinity of MTI-2 on this column for enzymes like
chymo-trypsin is still sufficient to isolate them Fig 1 shows the
activity of the eluted fractions from the H zea samples
Trypsins (Fig 1, bold lines) and chymotrypsins (Fig 1, grey
dotted lines) were eluted from the MTI-2 column after
stepwise lowering of the pH In this paper we focus on
trypsins
The MTI-2 column concentrates tryptic activity (as
measured by BApNA degradation) from H zea guts on
both diets in a limited number of fractions In the case of the
control guts, hardly any trypsin activity is detected in the
flow-through and washes of the column (Fig 1A) All eluted tryptic activity ( 40% of the input) is concentrated
in fractions of pH 1.5 with 15% dimethylsulfoxide, while most chymotrypsin activity is released at pH 3.2 and 2.2 For the SKTI guts, no such harsh treatment was needed to recover all protease activity (Fig 1B) Around 33% of the tryptic activity did not bind to the column Treatment at
pH 3.2 released 14% of trypsin (mixed with chymotrypsin activity), while 43% was eluted at pH 2.2 (with hardly any chymotrypsin activity) This latter fraction was not found to
be contaminated with SKTI from the diet by the IEF and
MS analysis (below) The control diet trypsin eluting at
pH 1.5 with 15% dimethylsulfoxide is hereafter referred to
as HzTrypsin-C, and the SKTI diet trypsin eluting at
pH 2.2 as HzTrypsin-S
Trypsin fractions are functionally pure The purity of trypsin fractions was tested using activity gels, SDS/PAGE and IEF Fig 2C (first lane) shows that the control gut has four caseinolytic proteins (bands C1–C4) One of the major caseinolytic bands (C1, at 23 kDa) was concentrated by the affinity chromatography, and was highly pure as judged by silverstaining (Fig 2A) Notably, band C1 was dominant both in the chymotrypsin fraction (pH 2.2), and in the Hz Trypsin-C fraction (pH 1.5 + dimethylsulfoxide) Apparently the mobility of both trypsin and chymotrypsin in this semi-denaturing gel system is similar
Fig 1 Elution profile from affinity column of control-diet gut content (A) and SKTI diet gut content (B) The bold line indicates trypsin activity as measured with substrate ZRRpNA The regular line indi-cates protein concentration as absorption measured at 260 nm The grey dotted line indicates chymotrypsin activity, as measured with substrate SAAPLpNA.
Trang 4The SKTI diet gut contained five caseinolytic bands (S1–
S5; Fig 2D) Caseinolytic bands S2, S3 (both in
chymo-trypsin fractions; pH 3.2) and S4 (in the HzTrypsin-S
fraction; pH 2.2) were concentrated by the affinity
chroma-tography They appeared as strong bands on the
silver-stained gel (Fig 2B) The chymotrypsin fraction contains
some additional proteins that did not display proteolytic
activity in the casein gel The HzTrypsin-S fraction contains
a single dominant band (at 26 kDa) in the silver-stained
gel, which comigrates with the bottom of a smear of activity
Therefore, we conclude that both HzTrypsin-C and
HzTrypsin-S are functionally pure, and contain no
signifi-cant contaminant nonprotease protein
Trypsin fractions were separated further on IEF gels
(Fig 2E) Denaturing SDS/PAGE, which is frequently
conducted following IEF in two-dimensional gel systems, did not improve the separation, as all proteins in the fractions run at about the same molecular size (23.5 kDa) The second dimension gel was therefore omitted HzTryp-sin-C (Fig 2E, top strip) shows three dominant bands and a fourth minor band: the major bands focus around pI 9 (M1, M2 and M3), and the minor around pI 7 HzTrypsin-S does not have the pI 7 band, and has three major species (M11, M12, M13) and a minor band in the pI 9–10 area, but at a different position than in the HzTrypsin-C (Fig 2E, lower strip) IEF strips covering a different pH range did not show additional bands, and we did not observe any protein of a pI corresponding to SKTI (which is around 5 for different isoforms of SKTI)
Identity assignment of trypsins by MS
To link the isolated digestive enzymes to a protein and gene sequence, the major IEF bands from HzTrypsin-C and HzTrypsin-S were sequenced by MS Bands M1, M2, M3, M11, M12 and M13 were excised from the focussing gel (Fig 2E) and digested by bovine trypsin Tryptic peptide fragments were analysed by MS and tandem MS Masses of peptides were matched to the full Swissprot database, and automatically sequenced For all IEF bands between two and five peptides were identified whose masses and peptide sequences related to one of the 29 available Helicoverpa proteinase genes (Table 1, bold figures) More information than the exactly matching peptides was sought, to identify the trypsins more accurately By manual checking of MS/
MS spectra, amino acid sequences for tryptic fragments were identified that almost completely matched the identi-fied Helicoverpa proteases (Table 1, underlined)
HzTrypsin-S corresponds to HzT15 and HzTrypsin-C
to HaY12269 Although HzTrypsin-S focuses at three different isoelectric points, it appears to relate to a single trypsin gene IEF bands M11, M12 and M13 clearly relate to trypsin cDNA HzT15 (accession AF261980; Table 1, upper panel), and not to any of the other 33 Helicoverpa protease genes in the database Nine out of 11 predicted tryptic fragments of HzT15 are found in the spectra of M11, M12 and M13 (Table 1, last column) In Fig 3, amino acids that were identified by the MS and MS/MS analyses are underlined The analysed sequences cover 66% of mature HzT15
In addition, the HzTrypsin-C peptide sequences relate it
to a single trypsin All three major IEF bands (M1, M2 and M3; Fig 2E) represent two Helicoverpa virtually identical trypsin genes, HaY12269 (accession Y12269; Table 1, lower panel) and Hz42 As the cDNA sequence Hz42 in the database is incomplete, we refer to the HaY12269 sequence Seven out of 11 predicted tryptic fragments of HaY12269 are found in the spectra of M1, M2 and M3 (Fig 3) The peptides with a sequence that matches HaY12269 cover 47% of the total mature sequence of HaY12269 The covered percentage is lower than for HzT15 (66%), as fewer high-mass peptides were analysed No peptides of M1 and M2 could be identified that relate to any other protease gene, while M3 contains some additional peptides that
Fig 2 Detection of affinity-purified H zea proteins and protease
activity after semidenaturing SDS/PAGE (13%) by silver staining
(A and B) and a Coomassie-stained casein overlay (C and D) The left
two gels are from insects reared on control diet, and the right two gels
are from insects reared on SKTI diet On each gel total gut content and
pH 3.2 and pH 2.2 fractions are loaded: for the control diet the pH 1.5
with 15% dimethylsulfoxide (lane 1.5+) is loaded also Positions of
molecular size markers are indicated (E) IEF of HzTrypsin-C (top
strip, fraction 1.5+ from A and C) and HzTrypsin-S (bottom strip,
fraction 2.2 from B and D) in the range from pH 11 (left) to pH 6
(right) Bands analysed by MS are indicated.
Trang 5match a H zea chymotrypsin, HaY12273 (not shown).
HzTrypsin-C apparently contains a few minor
contamina-tions, such as the HaY12273 chymotrypsin In addition, the
minor band that focuses at pI 7 is not a trypsin (data not
shown)
Manual scrutiny of MS/MS spectra revealed some
additional data Many peptides have peptide sequences
that almost match HzT15 or HaY12269, but have a mass
that is different from the predicted tryptic fragments
(Table 1, top panel fragments 2, 3, 7 and 9; lower panel
fragments 2, 6 and 8) In M11 and M12, we identified a
modified amino acid at the position of Arg62 (porcine
trypsin numbering, Fig 3) of the HzT15 cDNA sequence
In the peptides with a matching sequence here, a larger
mass difference (234 and 280 Da) than accounted for by
Arg (156 Da) is found (Table 1, top panel fragment 3)
The molecular weight of 280 Da corresponds to that of
Arg-pyrimidine, a methylglyoxal modification of Arg [19]
In other fragments, a larger mass difference than would be
predicted is found at the position of the Cys residues in the cDNA We assume that such differences arise from incomplete reduction and modification of the Cys resi-dues, after recovery from the IEF strip Apart from the Cys-based artifacts, all identified peptides exactly match the sequence of the cDNAs Only one position, the Trp in the predicted peptide 2 of HzT15 (Table 1, top panel fragment 2) is not identified in HzTrypsin-S, but instead
an amino acid of the same mass as Leu and Ile is found (Fig 4) Such an amino acid is indeed found in all available Helicoverpa protease cDNAs, except HzT15, indicating a possible cDNA sequence determination artifact or genetic variation
It was quite unexpected to identify only a single protease in three IEF bands that focus on clearly distinct
pH values, both for M1, M2 and M3 and M11, M12 and M13 We would have expected some of the other Helicoverpa trypsin genes to be represented This was investigated more closely by comparing single-dimension
Table 1 Fragments, amino acid sequences and observed and predicted masses from trypsin HzT15 in M11, M12 and M13 (upper panel) and HaY12269
in M1, M2 and M3 (lower panel) Bold underlined masses have a protein sequence and mass exactly as predicted Underlined masses have an almost completely matching sequence after manual analysis, except for nonunderlined residues Masses in italics have been found in MS spectra, but were not analysed by MS/MS to analyse their sequence.
HzT15
1441
1459 1459 1459
948
902 948
824
4 VGSTWANSGGVVHNVNQNIIHPQFNPNNLNNDVAILR 4022 4022 4022
6 AGSIAGPNYNVADNQVVWAAGWGDTFSGSNQGSEQLR 3822 3822 3822 3822
HaY12269
1645 1645
1722 1722 1722
1740 1740 1740
1163 1163 1163
1667 1667 1667
a Predicted masses are tryptic fragments predicted by the program PEPTIDE MASS [31].
Trang 6MS spectra of M1, M2, M3, M11, M12 and M13 Hardly
any difference between the spectra of M1, M2 and M3, or
difference between M11, M12 and M13 was observed
Possibly there are minor differences in single amino acids
in fragments not tested by the MS (e.g fragments 1, 7 and
9 in Table 1, lower panel), or differences in protein
modifications (either natural or artefacts of sample
preparation), but the available protein and cDNA data
do not provide an explanation for this phenomenon
Therefore it is concluded that both HzTrypsin-C and
HzTrypsin-S have unexpected purity: all major protein
bands of a fraction have the same peptide sequence
Sequence comparison of sensitive and resistant trypsins
To obtain some insight into the differences between
HzTrypsin-C and HzTrypsin-S, the amino acid sequences
of HaY12269 (HzTrypsin-C) and HzT15 (HzTrypsin-S)
were aligned to each other and to porcine trypsin using
standard methods (Fig 3) The mature HaY12269 and
HzT15 amino acid sequences are 76% identical Five
regions can be identified in which differences between
HzT15 and HaY12269 are concentrated, but where also
both Helicoverpa trypsins differ most from porcine trypsin
(Fig 3, indicated by X) These regions are annotated 37, 60,
99, 145 and 175, with reference to their position in the
sequence of porcine trypsin according to the
chymotrypsi-nogen numbering Remarkably, all five regions differing
between HzT15 and HaY12269 overlap with contact
residues of the enzyme with SKTI [20] Notably, in loops
60 and 99, one additional amino acid is present in HzT15 Four out of five regions are covered by the MS analysis of HzTrypsin-S
Trypsin activity on protease substrates The apparent purity of the HzTrypsin-C and HzTrypsin-S fractions provided the opportunity to compare specific enzymatic activities of the two proteins First, TLCK was used as inhibitor to compare the number of active sites The concentration of TLCK needed per lg protein to inhibit trypsin activity was found to be comparable for control trypsin and SKTI trypsin To characterize proteolytic activity, breakdown of four substrates was compared The soluble protein from the corn-based insect diet was equally well cleaved by HzTrypsin-C and HzTrypsin-S (the activity ratio S/C was 98%; Table 2) Also the activity towards azocasein, ZRRpNA, ZFRpNA, ZRpNA and RpNA was similar for both enzymes (ratios S/C were 72%, 124%, 135%, 76% and 106%; Table 2) Remarkably, the substrate BApNA differentiates clearly between the trypsins Break-down of this substrate by HzTrypsin-S is less efficient (11%; Table 2) than by the control trypsin BApNA differs from the other substrates in the residue that binds the S2 substrate-binding pocket on the surface of trypsin These observations suggest that the HzTrypsin-S can work efficiently when a natural amino -acid is in the P2 position (as in ZFRpNA and ZRRpNA, and in proteins) HzTryp-sin-S is much less efficient with the N-substituted benzoyl group carried by BApNA at that position, which is clearly less flexible at the Ca position of the P2 residue than an amino acid
Inhibition ofHelicoverpa trypsins by four plant protease inhibitors
The effect of plant protease inhibitors on the isolated trypsins was tested We anticipated that inhibitors with
a different architecture would have different inhibitory
Fig 4 Inhibition curves of 6 n M HzTrypsin-C (C-SKTI, C-PI2, C-BBI and C-MTI2: open symbols) and 6 n M HzTrypsin-S (S-SKTI, S-PI2, S-BBI and S-MTI2: filled symbols) with four different inhibitors Plotted
is the molar inhibitor concentration (horizontally, logarithmic scale)
vs the measured residual tryptic activity in percentage of uninhibited activity.
Fig 3 Alignment of HaY12269 (HzTrypsin-C) and HzT15
(HzTryp-sin-S) deduced amino acid sequences Stretches of X on top of the
alignment indicate highly diverging regions between HaY12269 and
HzT15 Underlined amino acids were identified by MS Most Cys
residues were not identified due to partial alkylation Porcine trypsin is
shown for comparison Dots have been inserted to maximize
homo-logy The numbering is according to the chymotrypsinogen
nomen-clature.
Trang 7properties towards the insect proteases For that purpose,
inhibitors SKTI (representative of the Kunitzfamily [20],
occurring in most plant species), SBBI (of the Bowman–
Birk family [21], primarily present in legumes), PI-2 (of the
potato inhibitor II family [22], only found in solanacaeae)
and MTI-2 (of the mustard inhibitor family, only found in
cruciferae) were chosen To quantify inhibition, low
concentrations of enzyme were mixed with calibrated
con-centrations of inhibitors HzTrypsin-C and HzTrypsin-S
were taken to be pure, which is 80% accurate as judged by
the MS analysis Equal amounts of protein from different
protease fractions were mixed with a range of
concentra-tions of each of the protease inhibitors, and residual
activities to degrade substrate ZRRpNA were measured
(Fig 4)
HzTrypsin-C was inhibited strongly by SKTI and PI-2
This allowed titration of the concentration of active sites of
this enz yme to be 5 nM This value corresponds to the
measured protein concentration, and the apparent
equilib-rium dissociation constant Ki of SKTI and PI-2 to this
enzyme at 1 nM MTI-2 and SBBI are needed in higher
concentration (around 0.1 lM; 20-fold molar excess) to
achieve full inhibition of the HzTrypsin-C (Fig 4) For
medium-affinity interactions like this, the 50% inhibitory
concentration (IC50) roughly corresponds with the Ki
Hence, we calculate the Kiof MTI-2 and SBBI towards
HzTrypsin-C to be 30 nM The inhibition constants for
SKTI and SBBI towards HzTrypsin-C are in the same
range as those reported by Johnston et al [7]
The HzTrypsin-S can hardly be inhibited by any of the
concentrations of inhibitor tested At 10 lM inhibitor,
SKTI and PI-2 confer about 50% reduction in activity,
while MTI-2 and SBBI still have almost no effect on
HzTrypsin-S activity (Fig 4) The concentration of
trypsin active sites per lg protein is similar to that
of HzTrypsin-C, as indicated by the TLCK inhibition (see
above) Therefore the HzTrypsin-S concentration in the
assay was 5 nM This means that at least a 2000-fold
molar excess of either of the inhibitors is insufficient to
inhibit this enzyme The Kiof all four inhibitors towards
HzTrypsin-S is therefore > 1000 nM
Discussion
Linking enzymatic properties toH zea trypsin genes The aim of this paper was to characterize trypsins involved
in the coevolution of plant protease inhibitors and insect digestive proteases The plant side of this coevolution has been well characterized: inhibitor genes have been shown to
be induced by wounding, insect feeding and defence-signalling hormones [3], and also biochemical properties of isolated or recombinantly expressed inhibitors, and their effect on proteolysis in the insect gut have been extensively studied (e.g [14,15]) On the other hand, the insect side of the coevolution (i.e adaptation to plant defensive inhibitors), was first noted 7 years ago [10,11], but not much progress has been made towards understanding the biochemical properties of the proteases since then This is mainly due to lack of an appropriate recombinant expression system Regulation of Helicoverpa protease genes upon inhibitor ingestion has been studied [9,12] Helicoverpa responds to plant protease inhibitors with an intricate change in the expression of protease genes Among five H zea trypsin genes tested, three are up-regulated, among which is HzT15, and two are slightly down-regulated, among which is Hz42 (which is 98% identical to HaY12269) in response to SKTI [12] Apparently the adaptation does not involve a single up-regulated protease gene Therefore, it has been difficult
to establish a conclusive link between gene expression and gene function One may presume that at least a subset of the up-regulated genes represent the inhibitor-insensitive trypsins in the adapted gut, and, vice versa, a subset of the slightly down-regulated genes represent the inhibitor-sensi-tive, nonadapted protease species However, this is obscured
by a number of factors, including the presence of the inhibitor in the gut While transcripts by which inhibitor-sensitive trypsin is encoded (HaY12269 and Hz42) have been reported to be still quite abundant under these circumstances [9,12], we could not isolate such activity from the inhibitor-adapted gut Most likely, the HzTrypsin-C is tightly bound by SKTI from the diet, and therefore does not contribute to proteolytic activity in the gut Hence, inter-pretation of gene expression data towards a physiological model is confused by the fact that not all expressed proteases are active To fully appreciate what happens when the insect gut adapts to inhibitors, a link to enzymological data by protein identification is required Usually the link between gene and function is established
by recombinant expression of proteases So far, insect serine proteases could not be expressed as active enzymes in a variety of hosts tested (Escherichia coli, yeast, insect cells; unpublished data), so no definite assignment of these trypsin genes to their function has been possible A few lepidop-teran proteases have been purified, but have not been analysed and compared with respect to adaptation to plant protease inhibitors [7,23,24] The link to corresponding trypsin genes was made by limited N-terminal sequencing, which can be quite inaccurate for a highly homologous gene family such as insect trypsins Modern protein biochemistry provides a number of very sensitive tools (collectively referred to as proteomics) to isolate, identify and characterize proteins in a secreted body fluid, such as
Table 2 Specific activity of gut trypsin fractions towards different
substrates.
HzTrypsin-C HzTrypsin-S Ratio S/C (%)
Dietary protein 0.045 b 0.044 b 98
Azocasein 0.329c 0.238c 72
a Activity is assayed in 200 lL using 2 lg trypsin protein of each
fraction, and is expressed as pNA release (change in absorption at
405 nm) per minute.bActivity is assayed as absorbance at 280 nm
per lg trypsin protein after 30 min incubation and trichloroacetic
acid precipitation c Activity is assayed as absorbance at 340 nm
per lg trypsin protein after 30 min incubation and trichloroacetic
acid precipitation.
Trang 8the content of a gut In the given case of the Helicoverpa
trypsins, these tools substituted for the use of heterologous
expression and established reliable links between sequence
and function
Inhibition and physiological role of trypsins
HzTrypsin-C is one of the major trypsins (> 40%) that
H zeadeploys to digest plant material without inhibitors
This trypsin could strongly be inhibited by SKTI and PI-2
(Ki¼ 1 nM), but less strongly by SBBI and MTI-2
(Ki¼ 0.03 ı`M) All four inhibitors are probably effective
against HzTrypsin-C at physiological concentrations,
because both gut enzymes and plant inhibitors occur at
approximately 10 lMconcentration in the insect gut It was
calculated that 10 lMof inhibitor with Ki¼ 0.1 lMis able
to inhibit > 90% of the activity of 10lMtrypsin [25] To
overcome the loss of protease activity due to the dietary
inhibitors, a novel trypsin, HzTrypsin-S, is synthesized,
which is highly insensitive to all plant inhibitors tested
It is puzzling why these insects do not constitutively
express the inhibitor-insensitive trypsin genes, but instead
perform an induced, time and energy-consuming change in
gene expression of protease genes Synthesis of proteases is
an important metabolic activity of the gut cells, as protease
mRNAs make up 20% of gut cDNAs [9] One suggested
answer was that there may be no such thing as a protease
insensitive to all types of inhibitors encountered by a
polyphagous insect, so that flexibility of regulation would be
an asset allowing appropriate subsets of genes to be
expressed depending on the host plant [26] Now it appears
that a trypsin insensitive to a very wide range of plant
protease inhibitors does exist (HzTrypsin-S) Remarkably,
the advantageous property is not compromised by a lower
efficiency in plant protein degradation compared to a
sensitive enzyme like HzTrypsin-C This is in keeping with
the observation that dietary inhibitors do not affect larval
growth rates of Helicoverpa [9,12] The question why
PI-insensitive proteases are not constitutively expressed
remains unanswered The protease properties may affect
other fitness parameters (e.g progeny numbers) which may
only become obvious in complex ecological circumstances
(e.g direct competition) that have not been tested
What determines insensitivity to plant protease
inhibitors?
HzTrypsin-S is fully adapted to the defensive protease
inhibitors of plants The adaptation must have biophysical
and protein-structural reasons Firstly, those reasons may be
revealed by analysing amino acid differences between
HzTrypsin-C and HzTrypsin-S Extensive hypotheses based
on sequence comparison have been formulated [12] Others
have concluded that the Helicoverpa protease sequences do
not contain an obvious clue to the mechanism of resistance
to inhibitors [9] There are 57 differing amino acids between
the two trypsins described in this paper (Fig 3) In Fig 5A,
these residues are shown in yellow, superimposed on the
porcine trypsin crystal structure It can be clearly seen that
these amino acids preferentially map in loops of the porcine
trypsin structure that border the active site groove In
Fig 5B, the differing residues are combined with those that are in contact with inhibitors (Fig 5B, blue) There is clearly overlap (Fig 5B, green) between contact residues and residues that differ between the two H zea trypsins Differ-ing residues seem to form a rDiffer-ing around the active site of the trypsins, rather than affecting the active site itself However,
as all contact-loops are affected by multiple mutations, it is difficult to estimate the importance of individual regions
Fig 5 Spacefilling representation of the structure of the porcine trypsin component of the Trypsin-SKTI complex [20] as generated by RAS-MOL, viewed toward the substrate-binding site (A) In black sticks the P3 to P2¢ residues of SKTI are represented, as analogue of a substrate.
In (B) the same view as in (A) is shown, but relevant residues are now indicated according to the chymotrypsinogen numbering In (A) and (B) yellow residues are at positions in the alignment where HzTrypsin-C and HzTrypsin-S are different Blue residues are con-tacting SKTI Green residues are the overlap between contact residues and different residues.
Trang 9Secondly, the inhibition data may serve to assign function
to regions of the trypsin The four inhibitors tested differ in
their contact residues with surface loops of porcine trypsin
(or homologous enzymes) Generally the contact loops of
Trypsin-like enzymes are referred to as the 37, 60, 99, 145
and 170 loops [27] (Figs 3 and 5B, green and blue) SKTI
has very little contact with the 175 loop, and very extensive
contacts with the 99 loop [20], while PI-2 has hardly any
contacts to the 60 loop, 99 loop and 145 loop [22], and SBBI
has very few contacts with the 37, 60, 145 and 175 loops [21]
Because HzTrypsin-S is resistant to inhibitors MTI-2, PI-2,
SKTI and SBBI, one may conclude either that there is no
single feature that impairs inhibitor binding, or that such a
feature is close to the active site, where all inhibitors bind
Thirdly, clues to the mechanism of adaptation of
HzTrypsin-S may be inferred from the difference in
substrate specificity of both trypsins (Table 2) The chemical
substrate BApNA clearly distinguishes between the two
enzymes This substrate, which carries a benzoyl group at
the P2 position, is degraded by HzTrypsin-S relatively
poorly, as compared with HzTrypsin-C, whereas substrates
like ZRRpNA, ZFRpNA, azocasein and dietary plant
protein, which carry an amino acid at the P2position, and
RpNA, without a P2residue, do not distinguish the enzymes
Apparently, the S2 pocket of HzTrypsin-S (Fig 5B) is
functionally different from that of HzTrypsin-C, resulting in
reduced accommodation of, e.g the benzoyl group carried
by BApNA The same difference may possibly be at the root
of the ability of HzTrypsin-S to avoid inhibitor binding
Properties of the purified proteins as reported here are
essential to our understanding of the way successful insects
deal with plant defence For full protein structural
under-standing, regions differing between HzTrypsin-S and
HzTrypsin-C that may contribute to occlusion of inhibitors
should be addressed by a series of targeted mutations and
recombinant expression However, to our knowledge no
suitable expression system has as yet been identified for
insect trypsins, despite extensive efforts Alternatively,
crystal structures of the complexes of purified enzymes with
MTI-2 could help to narrow further the structural
determi-nants of insensitivity to protease inhibitors Such
informa-tion would provide valuable insight into the molecular basis
of the adaptations of generalist pests Also, it may lead to
design of novel, improved inhibitors It will be a challenge
to use the purified enzymes now obtained to improve
inhibitors through methods such as phage display and
rational design [18,28–30] Similarly, ecologists will find
challenges in determining the true costs and benefits of the
deployment of these enzymes for these insects
Acknowledgements
This research was conducted as part of an EU RTD project M V was
supported by EMBO fellowship ASTF 9601 We are grateful to B Oliva
for helpful advise, and R de Maagd, R Bino, D Reverter and J van
Loon for careful reading of the manuscript.
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