Heat-induced Conformational Changes of Ara h 1, a
Major Peanut Allergen, Do Not Affect Its Allergenic Properties*
Stef J.
Koppelman
§,
Carla A. F. M.
Bruijnzeel-Koomen¶,
Martin
Hessing
, and
Harmen
H. J.
de Jongh
**
From the
TNO Nutrition and Food Research Institute,
3700 AJ Zeist, ¶ Department of Dermatology/Allergology, University
Medical Center Utrecht, 3508 GA Utrecht, and the
Centre for
Protein Technology TNO-WAU, 6700 EV Wageningen, The Netherlands
 |
ABSTRACT |
Ara h 1, a major peanut
allergen was isolated, and its structure on secondary, tertiary, and
quaternary level at ambient temperature was investigated using
spectroscopic and biochemical techniques. Ara h 1 appeared
to be a highly structured protein on a secondary level, possesses a
clear tertiary fold, and is present as a trimeric complex. Heat
treatment of purified Ara h 1 results in an endothermic, irreversible transition between 80 and 90 °C, leading to an increase in
-structures and a concomitant aggregation of the protein. Ara h 1 from peanuts that were heat-treated prior to the
purification procedure exhibited a similar denatured state with an
increased secondary folding and a decreased solubility. The effect of
heat treatment on the in vitro allergenic properties of
Ara h 1 was investigated by means of a fluid-phase IgE
binding assay using serum from patients with a clinically proven peanut
allergy. Ara h 1 purified from peanuts heated at different
temperatures exhibited IgE binding properties similar to those found
for native Ara h 1, indicating that the allergenicity of
Ara h 1 is heat-stable. We conclude that the allergenicity
of Ara h 1 is unaffected by heating, although native
Ara h 1 undergoes a significant heat-induced denaturation
on a molecular level, indicating that the recognition of conformational
epitopes of Ara h 1 by IgE either is not a dominant mechanism or is restricted to parts of the protein that are not sensitive to heat denaturation.
 |
INTRODUCTION |
Peanut allergy is one of the most severe food allergies due
to its persistency and the life-threatening character (1). The
prevalence of peanut allergy in the Western world has been estimated at
1 in 10,000 up to 1 in 200 (2) and seems to be increasing during the
last decades. An explanation for this increased prevalence is
controversial. The fact that sensitization routes are not always
obvious (3) confuses this phenomenon even more. Doses as low as 100 µg may provoke symptoms (4), indicating that accidental ingestion of
minute traces of peanut endanger the life of subjects with peanut
allergy. The nature of the allergenic compounds in peanuts has been
studied extensively in recent years (5, 6), and two major peanut
allergens, Ara h 1 (7, 8) and Ara h 2 (8, 9),
have been identified. Purified Ara h 1 has been subjected to
some biochemical and immunological studies, and it appeared to be a
63-kDa glycoprotein (7) with distinct IgE binding sites both on the
protein part (10-12) and on the carbohydrate moiety of the molecule
(13). The gene encoding for Ara h 1 was cloned (14), and the
gene product resembled similar biochemical and immunochemical
properties (15), although the molecular weight appeared to be somewhat
higher (16) possibly due to incorrect processing of a pro-peptide
sequence (8). Sequence analysis of Ara h 1 showed a
significant homology with the vicilin seed storage protein family (12),
and, remarkably, only one cysteine residue was found in the entire
protein (14). Several isoforms of Ara h 1 with different
iso-electric points and slightly different molecular weights have been
described (17), and it has been postulated that Ara h 1 is
assembled in di- and trimeric complexes (12), even in the presence of
surfactants (17).
Peanuts are widely used in the food industry owing to their nutritive
value and to their taste. Consumer product diversification led to an
increase in recipes containing peanuts, resulting in an increased risk
for inadvertent ingestion of peanuts by allergic individuals.
Additionally, contamination of intended peanut-free products with
traces of peanuts led to several fatal and near-fatal allergic
reactions. Test kits to establish the presence of peanut protein in
finished foods are currently commercially available (18, 19), but the
lack of legislation concerning labeling of food allergens hampers a
systematic control of suspected products (20). The origin of peanut
proteins in foods is not always obvious. In most cases, roasted or
fried peanuts are used because of their improved flavor and taste
compared with their raw counterparts. Foods containing vegetable oil,
however, may contain peanut proteins (10, 21) that are not heated
during processing. Therefore, peanut allergic individuals are exposed
to both native and heat-treated peanut proteins and both can provoke
allergic reactions. It is generally accepted that peanuts preserve
their allergenic character upon heating, as binding properties of IgE
and IgG to a crude peanut extract are neither diminished nor enhanced
by heating (22, 23).
However, it is not known whether in these complex systems heat
treatment leads to denaturation of the peanut allergens on a molecular
level; consequently, the effect of heat denaturation on the allergenic
properties of the peanut allergens is not known. The aim of this study
was to investigate the heat-induced conformational changes of Ara
h 1, and to study the coinciding effects on its allergenic
properties. We found that native Ara h 1 is a highly structured protein, on secondary, tertiary, and quaternary folding levels. Both heat-treated purified Ara h 1 and Ara h
1 isolated from heated peanuts show conformational changes,
whereas the in vitro allergenic potential is hardly
affected. This study clearly shows that the allergenic character of
Ara h 1 is heat-stable, although the structural organization
of this major peanut allergen is changed significantly upon heating.
 |
MATERIALS AND METHODS |
Peanut Pretreatments
Peanuts (Arachis hypogea) from the Runner cultivar
(Cargill, Dawson, GA) were generously provided by Imko Gelria
(Doetinchem, The Netherlands) and were stored at 10 °C until use.
Peanuts were ground and heat-treated at 50, 80, 90, 110, 140, 155, 170, or 200 °C for 15 min in a thermostated prewarmed hot air oven. Heat treatment at 140 °C and 150 °C resulted in a light brown
coloration of the ground peanuts and the release of a typical roasted
peanut flavor. At higher temperatures, ground peanuts appeared brown (170 °C) or dark brown (200 °C) under the release of a burning smell. Heat treatment up to 110 °C did not give rise to coloration or scent. After heat treatment, the ground peanuts were stored at
4 °C until use. Peanut protein extracts were made by mixing 20 g of ground peanut with 100 ml of 20 mM
bis-Tris-propane1 buffer (pH
7.2). After 2 h of stirring at room temperature, the aqueous
fraction was collected by centrifugation (3,000 × g at room temperature for 30 min). The aqueous phase was subsequently centrifuged (10,000 × g at room temperature for 30 min) to remove residual traces of fat and insoluble particles. The
clear extracts were extensively dialyzed against 20 mM
bis-Tris-propane buffer (pH 7.2) at 4 °C. Protein concentrations
were determined using Bradford analysis with BSA as a standard.
Reducing SDS-PAGE from extracts from ground peanuts heated up to
140 °C showed similar patterns where Ara h 1 migrated as
a single band with an apparent molecular mass of 63 kDa, making up
approximately 10% of the total extracted protein based on densitometer
analysis of the gel. In extracts from ground peanuts heated at
155 °C and higher temperatures some high molecular mass protein
bands were absent. Extracts were stored at
20 °C.
Purification of Ara h 1
Ara h 1 was purified generally as described
previously (7, 8) with minor modifications. In short, dialyzed extracts from heat-treated and non-heat-treated ground peanuts were applied on
an 8-ml Source Q column (FPLC protein purification system, Pharmacia,
Uppsala, Sweden) previously equilibrated with 20 mM bis-Tris-propane of pH 7.2 (loading buffer) at room temperature. The
column was washed with loading buffer until the
A280 of the effluent was less than 0.02. Proteins were eluted using a linear sodium chloride gradient in loading
buffer (up to 1 M in 200 ml at a flow of 4 ml/min).
Fractions were collected and analyzed on SDS-PAGE. Ara h 1 eluted from 290 to 310 mM sodium chloride and appeared to
be essentially pure (>95%) as judged from a densitometer scan of an
SDS-PAGE gel stained with Coomassie Brilliant Blue. Comparison of
non-reduced and reduced SDS-PAGE gels showed that approximately 10% of
Ara h 1 was present as di- and trimers. Further purification
steps were omitted in order to maintain the native character of
Ara h 1. The N-terminal sequence was determined according to
the Edman degradation procedure on an Applied Biosystems Protein sequencing system (SeCU, Utrecht, The Netherlands) and appeared to be
identical to the earlier published sequence of Ara h 1 (8). Purified Ara h 1 was stored at
80 °C until use. If not
mentioned otherwise, samples were desalted on a PD-10 column
(Pharmacia, Uppsala, Sweden), previously equilibrated with a 10 mM phosphate buffer (pH 6.7) containing 50 mM
sodium chloride. Concentrations of Ara h 1 were determined
by absorbance measurement at 280 nm using a molar extinction
coefficient of 36130 M
1 cm
1
(A280 0.1% (1 mg/ml) = 0.59) calculated based
on the amino acid composition of Ara h 1 (16).
Patient Sera
Serum from 8 adult patients with a documented peanut allergy was
used for studying the interaction of Ara h 1 with IgE. Each of these individuals had a positive skin prick test to peanuts and a
convincing history of peanut anaphylaxis. The presence of IgE in the
serum specific for Ara h 1 was demonstrated by SDS-PAGE and
subsequent Western blotting. Both a non-allergic and an allergic, but
not peanut-allergic, individual were used as controls. Venous blood was
withdrawn from the individuals and allowed to clot. Serum was collected
by centrifugation and stored in aliquots at
20 °C until use. All
studies were approved by the Medical and Ethical Committee of the
University Medical Center of Utrecht (Utrecht, The Netherlands).
Spectroscopic Measurements
Far-UV CD--
Far-UV CD spectra of 0.10 mg/ml Ara h
1 in 10 mM sodium phosphate buffer (pH 7.4) were
recorded as averages of 10 spectra on a Jasco J-715 spectropolarimeter
(Jasco Corp.) at temperatures ranging from 20 to 90 ± 0.5 °C
with intervals of 10 °C. Quartz cells with an optical path length of
0.1 cm were used. The temperature in these cells was measured using a
thermocouple wire. The scan range was 260-185 nm, the scan speed 50 nm/min, the spectral resolution 0.2 nm, bandwidth 1.0 nm, and the
response time 0.125 s. Spectra were corrected for a protein-free
spectrum obtained under identical conditions, and subsequent noise
reduction was applied according to the Jasco software. The spectra were
analyzed from 240 to 190 nm with a 1-nm resolution to estimate the
secondary structure content of the proteins. Spectra were fitted using
a non-linear regression procedure with reference spectra of polylysine
in the
-helix,
-strand, and random coil conformation (24) and the spectrum of
-turn structures, extracted from spectra of 24 proteins with known x-ray structure (25). Such a fitting procedure gives the
relative contributions of the reference spectra that make up the best
fit of the measured spectrum and from which the secondary structure can
be calculated. A problem with the interpretation of the far-UV CD data
at different temperatures could be the unknown effect of elevated
temperatures on the spectra used as reference in the analysis. However,
far-UV CD measurements of polylysine in the random coil conformation
showed no spectral changes at elevated temperatures and the
root-mean-square of the fits remained below 8 over the whole
temperature range.2
Near-UV CD--
Near-UV CD spectra of 1.0 mg/ml Ara
h 1 in 10 mM sodium phosphate buffer
(pH 7.4) were recorded as averages of 25 spectra at temperatures
ranging from 20 °C to 90 ± 0.5 °C at intervals of 10 °C.
Quartz cells with an optical path length of 0.1 cm were used. The
temperature in these cells was measured using a thermocouple wire. The
scan range was 350-250 nm, the scan speed 50 nm/min, the data interval
0.5 nm, bandwidth 1.0 nm, and the response time 0.25 s.
Temperature resolved experiments were performed monitoring the
ellipticity at defined wavelengths both in the far- and near-UV region
by heating samples with a rate of 0.5 °C/min and averaging the CD
signal over 16 s.
Fluorescence Spectra--
Fluorescence spectra of 0.1 mg/ml
Ara h 1 in 10 mM sodium phosphate buffer (pH
7.4) were recorded as averages of three spectra on a Perkin Elmer
Luminescence Spectrometer LS 50 B with pulsed xenon source. Spectra
were recorded at temperatures ranging from 20 to 70 ± 0.5 °C
at intervals of 10 °C. Excitation was at 295 nm, and the resulting
emission was measured from 305 to 405 nm with a scan speed of 120 nm/min. Both the excitation and emission slit were 3.5 nm. Spectra were
corrected for a protein-free spectrum obtained under identical
conditions, and the spectra were subsequently smoothed using the
software supplied by Perkin Elmer.
FTIR--
FTIR measurements were performed on an ATR ZnSe
crystal after evaporation of the solvent of a 70-µl sample of 1 mg/ml
Ara h 1 to such an extent that the peptide backbone remained
at least fully hydrated, based on the shape of the
H2O/amide A band at 3400-3100 cm
1. Spectra
were recorded as averages of 16 scans on a Bio-Rad FTS 6000 Spectrometer equipped with a KBr beamsplitter, a deuterated triglycin
sulfate detector with an Eurotherm automatic temperature controller.
Spectra were recorded from 400 to 6000 cm
1 and stored
from 1200 to 2000
1, with a nominal resolution of 2 cm
1. The spectral resolution was enhanced to 1 cm
1 by zero filling prior to Fourier transformation. The
interferograms were symmetrized, and the contribution of atmospheric
water was eliminated by subtraction of the appropriate spectrum.
Turbidity Experiments
A stock solution of 5 mg/ml Ara h 1 in 10 mM phosphate buffer (pH 6.7) in the presence of 50 mM sodium chloride was heated to 60 °C. Aliquots of this
prewarmed stock solution were added to the same buffer in a cuvette
with a 1-cm path length, equilibrated at 85 °C making final
protein-concentrations from 0.05 to 1.0 mg/ml. Next, the absorbance at
400 nm of the sample was monitored on a Hitachi U-3000
spectrophotometer at 85 °C for 1 h under continuous stirring of
the sample.
Ultracentrifugation Experiments
To determine sedimentation coefficients of the Ara h
1 samples 5-20% sucrose step gradients (12 ml total volume) were
prepared in 10 mM phosphate buffers (pH 6.7). Prior to the
experiments, the gradients were allowed to diffuse to linearity during
24 h at 4 °C, and 0.3-ml aliquots of 4 mg/ml Ara h 1 were loaded on top of the gradient. Next, the tubes were centrifuged in
a Beckman L60 centrifuge at 186,000 × g for 16 h
at 20 °C. After centrifugation, the gradient was fractionated in
0.5-ml aliquots, of which the absorbance at 280 nm was determined.
Sedimentation coefficients were estimated after calibration of the
gradient in a separate experiment using proteins with known S values
(
-globulin (11.2 S), catalase (7 S), BSA (4.4 S), trypsin (2.5 S),
and ribonuclease (1.78 S)).
Size Exclusion Chromatography
Chromatographic analysis of samples containing 0.1 mg/ml
Ara h 1 was performed using a Pharmacia Smart System on a
Superdex 200 column (3.2 × 300 mm; Pharmacia Biotech, Uppsala,
Sweden), equilibrated and run at 20 °C at 80 µl/min in a 10 mM sodium phosphate buffer (pH 7.0) filtered through
0.2-µm filters (Schleicher & Schuell, Dassel, Germany). Prior to
analysis, the Superdex 200 column was calibrated using blue dextran
(2.000 kDa), thyroglobulin (667 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), and sodium ascorbate (176 Da). Samples were loaded
onto the column using a 50-µl loop.
Differential Scanning Calorimetry (DSC)
DSC was performed on a Micro-DSC III (Setaram, Caluire, France)
using 0.9-ml vessels and a detection limit for transitions of minimal
84 µJ g
1 °C
1. A 4 mg/ml Ara h
1 solution in 10 mM sodium phosphate buffer (pH 7.4)
was heated from 20 °C to 100 °C with a scan rate of
0.5 °C/min, cooled to 20 °C with 3 °C/min, and subsequently
reheated to 100 °C with 0.5 °C/min.
IgE Binding Experiments
Affinities of Ara h 1 for IgE were measured using IgE
binding experiments generally according to Burks et al.
(23). Dilutions of Ara h 1 purified from peanuts treated at
different temperatures (final concentrations: 0.003-100 µg/ml,
calculated based on the A280 and the molar
extinction coefficient) were incubated with a 1:30 dilution of patient
serum in phosphate-buffered saline (PBS) containing 1% BSA and 0.1%
Tween 20. In this fluid phase, Ara h 1 was allowed to bind
to IgE for 1 h at room temperature under gently shaking
conditions. In order to determine the non-bound IgE fraction, the
incubation mixtures were transferred to the 96-well plates pretreated
as follows. 96-well plates were coated with 10 µg/ml Ara h
1 purified from non-heat-treated peanuts in PBS and subsequently
blocked with BSA (1% in PBS containing 0.1% Tween 20) to diminish the
nonspecific binding. IgE bound to the Ara h 1-coated wells
was detected using an anti-human IgE antibody conjugated to horseradish
peroxidase. Between each step, plates were washed five times with PBS
containing 0.1% Tween 20. The inhibition of IgE binding as a function
of the amount of Ara h 1 present in the preincubation sample
reflects the affinity of Ara h 1 for IgE. The concentrations
needed for half-maximal inhibition were calculated using a
semi-logarithmic equation and were used to compare the affinities of
the different forms of Ara h 1 for IgE.
 |
RESULTS |
Structural Properties of Ara h 1 at Ambient Temperatures--
Fig.
1A displays the far-UV CD spectrum of
Ara h 1 at 20 °C. The spectrum has a negative extreme
around 209 nm, with a small shoulder around 222 nm, and crosses zero
ellipticity at 199 nm. Such a spectrum is characteristic for a protein
with a high degree of structures at a secondary level (25). Spectral
analysis to obtain an estimation of the secondary structure content
using non-linear least square regression procedures reveals 31%
-helices, 36%
-structures, and 33% random coil. This secondary
structure content is qualitatively confirmed by the shape of the amide
I band in the IR spectrum of Ara h 1 (Fig.
2A, solid
line) where a maximum is observed at 1638 cm
1,
indicative for a high degree of
-structures (26, 27). Also, a clear
shoulder around 1660 cm
1 is apparent, indicating a
comparable amount of helical structures (28).

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Fig. 1.
A, far-UV CD spectrum of 0.1 mg/ml
Ara h 1 in 10 mM phosphate
buffer (pH 7.4) using an optical path length of 1 mm. B, the
ellipticity at 200.8 (solid circles) and 207 nm
(open circles) of a sample of 0.1 mg/ml Ara
h 1 in 10 mM phosphate buffer (pH 7.4) as a function
of temperature. The heating rate was 0.5 °C/min.
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Fig. 2.
A, amide I regions of ATR-FTIR spectra
of Ara h 1 at 20 °C prior to
(solid line) and after (dashed
line) heating a 1 mg/ml Ara h
1 solution in 10 mM phosphate buffer (pH 7.4)
for 60 min at 90 °C. B, amide I regions of ATR-FTIR
spectra of Ara h 1 at 20 °C
isolated from peanuts heated for 15 min at 20 (solid line),
50 (long dash), 80 (medium
dash), 90 (short dash), 110 (dotted line) and 140 °C (dotted/dashed
line).
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To get insight in the tertiary folding level of Ara h 1,
near-UV CD spectra have been recorded (Fig.
3A), which can serve as a
measure for the existence of tertiary interactions. Two major spectral
bands with a positive ellipticity can be observed, one centered around
280 nm, and one around 312 nm. Ara h 1 contains 22 phenylalanines, 6 tyrosines, and 5 tryptophans, which generally absorb
in the 260-290 nm, 280-300 nm, and 300-320 nm region, respectively, when these residues are involved in a tertiary interaction network (27). From this spectrum, a distinct tertiary fold can be ascribed to
the protein, based on the CD intensities observed, comparable to those
found for other plant storage proteins like patatin (29) or
glycinin.3 An alternative for studying tertiary
interactions is by monitoring the tryptophan fluorescence of Ara
h 1 (spectrum not shown). The observed fluorescence maximum of 348 nm, however, is close to that of free tryptophan in an aqueous
environment (353 nm), whereas a solvent-buried local environment of the
tryptophans would have maxima around 330-335 nm (31). This indicates
that the tryptophans of Ara h 1 are relatively on the
outside of the protein.

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Fig. 3.
A, near-UV CD spectrum of 1.0 mg/ml
Ara h 1 in 10 mM phosphate
buffer (pH 7.4) at 20 °C using an optical path length of 1 mm.
B, the ellipticity at 288 and 313 nm of a sample of 1.0 mg/ml Ara h 1 in 10 mM phosphate buffer (pH 7.4)
as a function of temperature. The heating rate was 0.5 °C/min.
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To investigate the conformational state of Ara h 1 at a
quaternary level, ultracentrifugation experiments have been performed to determine the S value. Fig.
4A shows the sucrose-gradient
profile, where it can be seen that the protein distribution displays a symmetric band of approximately 8 S, based on calibration of the gradient with various proteins with known S values. 8 S would correspond to a protein-complex of 180-200 kDa, indicative for a
trimer form of Ara h 1. Another indication for a trimer
organization of this protein is provided by size exclusion
chromatography on a calibrated Superose 6 column, which elution profile
is displayed in Fig. 4B. The peak observed at 1.25 ml
corresponds to a mass of 180 kDa (for globular proteins).

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Fig. 4.
A, elution profile of Ara h 1 in 10 mM phosphate buffer (pH 7.4) on a 5-20% sucrose
density gradient after ultracentrifugation at 186,000 × g for 16 h at 20 °C. The top and bottom of the
gradient and the positions of reference proteins with known S values
are indicated in the plot. B, elution profile of Ara h
1 in 10 mM phosphate buffer (pH 7.4) on a Superdex 200 column at 20 °C. The excluded and included volume of the column are
indicated.
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Structural Changes of Isolated Ara h 1 during Heat
Treatment--
To study the heat denaturation of Ara h 1,
DSC experiments have been performed as presented in Fig.
5. A clear endothermic transition can be
observed with an onset temperature of 83 °C and a maximum at
87 °C. The energy content of this transition is 30 kcal/mol.
Lowering the heating rate did not affect the position of the
transition, indicating that the sample was always in thermodynamic equilibrium under the conditions used (results not shown). Upon cooling
of the sample, no transition was observed, demonstrating that the
denaturation was not reversible, and complete, as indicated by the
second heating scan. These latter two traces are also displayed in Fig.
5, but are shifted vertically to improve the clarity of the figure.

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Fig. 5.
Differential calorimetric scan of 0.9 ml of 4 mg/ml Ara h 1 in 10 mM phosphate buffer
(pH 7.4) with a heating rate of 0.5 °C/min and a cooling rate of
3 °C/min. The arrows indicate whether a trace is a
heating or cooling scan, and the traces are vertically displaced to
improve clarity of the figure.
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To get insight in what the effects are of heat treatment on the
conformation of the protein at the distinct folding levels, we
monitored on-line the changes in CD ellipticity in the far-UV region at
200.8 and at 207 nm as a function of the temperature with a heating
rate similar to that used for the DSC experiments (Fig. 1B).
These wavelengths are chosen because at these wavelengths the
ellipticity of, respectively, the
-helix and
-strand are negligible and, consequently, any change in the
-helix or
-strand content would be detected. This dual wavelength approach enabled us to
detect changes in secondary structure accurately even if helices are
converted in strands or vice versa. It can be clearly seen
that up to 50 °C no spectral changes occur. Above this temperature at both wavelengths chosen, an increase in negative ellipticity can be
observed. However, this change in ellipticity does not reflect changes
in the secondary structure content of the protein, as the shape of the
CD-spectra does not change up to 80 °C (not shown), but only the
overall intensity increases. This could be caused by a reduced
absorption flattening of the spectra at higher temperatures, due to
dissociation of the quaternary complex of the protein. Above 80 °C a
decrease in negative ellipticity is apparent, but this is accompanied
by an increased level of optical density of the sample caused by
extensive aggregation of the material. Apparently, denaturation of
Ara h 1 results in an immediate aggregation behavior. That
this denaturation of Ara h 1 does affect the secondary structure content is shown by ATR-FTIR measurements of the heated material after cooling to 20 °C, as shown in Fig. 2A
(dashed line). Clearly the shape of the amide I
band is affected by the heating step, resulting in a more pronounced
shoulder at 1630 cm
1, indicative for the formation of
extended
-structure most probably related to the aggregation of the
material (32), and an increased intensity around 1658 cm
1. Apparently, denaturation of Ara h 1 leads
to a secondary more structured conformation of the protein.
When monitoring the changes in ellipticity in the near-UV CD spectra of
Ara h 1 as a function of temperature, it can be seen that
whereas the intensity at 288 nm is at 80 °C only reduced by
approximately 25%, the ellipticity at 313 nm is reduced by 65% of its
original intensity (Fig. 3B). This indicates that the tryptophan residues present gain upon heating more rotationally mobility due to reduced local packing, than the phenylalanine residues.
When the tryptophan fluorescence intensity was monitored as a function
of temperature an almost linear decrease of intensity was observed
(results not shown), which is an intrinsic property of tryptophan
fluorescence. No shift of the fluorescence maximum could be observed,
indicating that the tryptophan residues, which are readily
solvent-exposed at ambient temperatures, preserve this exposed
character upon heating.
To investigate the nature of the aggregation phenomenon upon
denaturation of the protein, we studied the kinetics of aggregation by
monitoring the turbidity of the sample at 85 °C as a function of
time (Fig. 6A). It can clearly
be seen that the kinetics of aggregation increases with increasing
protein concentration. In a sample with a protein concentration of 1 mg/ml 700-800 s are required to obtain a maximal turbidity. Pelleting
of the material by centrifugation revealed that all protein was
complexed into water-insoluble aggregates, as demonstrated by
determining the protein concentration in the supernatant in the cases
studied (results not shown). When the initial slope of the change in
turbidity as a function of time is plotted versus the
protein concentration (Fig. 6B), a relation is found that
can be described by a simple squared function of the protein
concentration (dashed line). These results
indicate that the aggregation phenomenon of Ara h 1 is not a
cooperative process, but follows a particle collision type of
mechanism. Analysis of the heated Ara h 1 by SDS-PAGE
demonstrates that, whereas native Ara h 1 has an apparent
molecular mass of approximately 63 kDa, the heated material forms
stable dimers, trimers, and larger complexes (results not shown).

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Fig. 6.
A, the turbidity recorded at 400 nm of
Ara h 1 solutions in 10 mM phosphate buffer (pH
7.4) in a 1-cm cell at 85 °C as a function of time for different
concentrations of Ara h 1. The protein-stock solution was
equilibrated at 60 °C and subsequently diluted at least 20-fold. The
final protein concentrations were: 0.05 mg/ml (solid
line), 0.10 (long dash), 0.15 (medium dash), 0.25 (short
dash), 0.35 mg/ml (dotted line) or are
indicated in the figure. B, the initial rate constant of the
increase of turbidity at 400 nm observed in panel
A is plotted as a function of the protein concentration. The
dashed line represents the theoretical
relationship when the aggregation mechanism is a diffusion-controlled
process.
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Structural Properties of Ara h 1 Isolated from Heated
Peanuts--
To test whether a similar behavior as described above for
temperature-induced denaturation of isolated Ara h 1 is also
apparent when this protein is heated in peanuts prior to isolation, we heated peanuts for 15 min at various temperatures, cooled them to
20 °C, and isolated Ara h 1 from the peanut as described
under "Materials and Methods." Extraction from peanuts heated at
20, 50, 80, and 90 °C led to similar yields, whereas the extraction yields of peanuts heat-treated at 110 and 140 °C were lower (75% and 32%, respectively). Incubation at temperatures higher than 155 °C resulted in no extraction of protein at all from the peanut. Whether this is due to aggregation of the protein, chemical reactions, a reduced accessibility for extraction or another mechanism is unknown
at the present. Up to 140 °C, Ara h 1 was purified
successfully and the protein pattern as analyzed by SDS-PAGE was
similar for all samples (not shown). Investigation of the secondary
structure of these proteins on ATR-FTIR, as demonstrated in Fig.
2B by analysis of the shape of the amide I band reveals that
these are comparable for Ara h 1 isolated from peanuts
heated at 20, 50, 80, and 90 °C. The spectrum of the protein from
peanuts heated at 110 °C clearly shows an increased intensity around
1630 cm
1, indicative for increased content of extended
-structures. This is even more pronounced for the protein heated at
140 °C in the peanut. From these results and from comparison with
the IR spectrum of the isolated protein heated in aqueous solution
(Fig. 2A, dashed line), we suggest
that in peanuts denaturation of Ara h 1 also can take place,
but requires slightly higher temperatures (90-110 °C) compared with
that of the isolated protein. The obtained denatured state shows a
great resemblance to that of denatured isolated Ara h 1, in
that an increased secondary folding is adopted, with similar IR
spectral features.
Recognition of Ara h 1 by IgE from Human Sera--
Binding of IgE
present in sera from patients with a peanut allergy to Ara h
1 was studied in a binding assay with a fluid phase character,
similar to previously described assays (22, 23) in order to maintain
the native structure of Ara h 1 optimally. The specificity
of this approach was tested using either another major peanut allergen,
Ara h 2, or a soy protein extract instead of Ara h
1. Both preparations did not show a dose-dependent
effect as was observed for Ara h 1, whereas a peanut protein
extract bound IgE in the fluid phase completely (not shown). As a
quantitative determination for IgE binding affinity in the fluid phase,
the concentration Ara h 1 at the half-maximal signal was
used (see "Materials and Methods"). The value for native, not
heat-treated Ara h 1 1.41 µg/ml. Affinities of Ara h
1 isolated from peanuts heated at different temperatures were
determined in the same way and are shown in Table
I. Although it might appear that, from 50 to 140 °C, a small decrease in affinity can be observed, no correlation between the native state of the protein and its IgE-binding affinity is present.
View this table:
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|
Table I
Affinity between IgE and Ara h 1 isolated from peanuts heated at
different temperatures
The concentration of Ara h 1 required for half-maximal
inhibition of IgE binding is used as a quantitative determination for
the affinity between Ara h 1 and Ara h 1-specific
IgE.
|
|
 |
DISCUSSION |
Structural Properties of Ara h 1 at Ambient Temperatures--
From
the results presented in this work on the structural features of
Ara h 1 at the secondary (Figs. 1 and 2), tertiary (Fig. 3),
and quaternary (Fig. 4) folding level, we can conclude that this
protein is highly structured. Screening of the primary sequences of the
Swiss Protein Data bank reveals a 46% sequence identity and a 52%
sequence weighted similarity with phaseolin, a seed storage protein
from the French bean also belonging to the legume family. This latter
protein has been crystallized, and the structure is resolved to a 2.2 Å resolution (33, 34) and comprises a
-barrel with a so-called
"jelly-roll" folding motif with a solvent-buried character,
followed by a helical domain. Interestingly, comparison of the
predicted secondary structure of Ara h 1 (35) with the known
structure of phaseolin shows a full match of the predicted with the
observed
-strands, respectively. Recently, a successful structural
alignment between Ara h 1 and phaseolin was presented (12).
Whereas in phaseolin (397 amino acids) 172 residues adopt a
-structure, a comparable number is found for Ara h 1 (541 amino acids) based on the 36% found to be
-structured according to the far-UV spectral analysis (Fig. 1). It is also interesting to note
that four of the five tryptophans of Ara h 1 are located at
residues 30, 44, 51, and 73, an N-terminal region that shows no overlap
with the primary sequence of phaseolin (alignment is from residue 77 onward). It can be suggested that this N-terminal domain does not
participate in the buried core of the protein, explaining the
relatively solvent-exposed character of these tryptophans as
observed in their fluorescence characteristics described above. On a
quaternary level, Ara h 1 adopts a trimeric complex (Fig. 4), in agreement with recent observations (12, 17).
Thermal Denaturation of Ara h 1--
From the description of the
conformational changes of Ara h 1 studied at the different
folding levels upon heat-denaturation, the following picture emerges.
The suggested core of the protein comprising
-stranded folding
motifs provides the protein with a large stability against heat
denaturation, as observed for various other plant storage proteins like
soy glycinin.4 At
a secondary level, up to the denaturation temperature no changes in the
secondary structure content are observed (Fig. 1B). The larger relative reduction of near-UV CD intensity in the 313 nm region
(± 65%) compared with the 288 nm region (± 25%, Fig. 3B) indicates that especially the domains where the tryptophans reside (N-terminal) have a low stability and can gain more flexibility at
higher temperatures. The near-UV CD of the phenylalanines, however, is
hardly affected by temperatures up to 80 °C, in agreement with the
model that they reside in the core of the protein as suggested by the
comparison with the aligned structure of phaseolin. The increase of
far-UV ellipticity at temperatures higher than 50 °C may reflect a
dissociation from the trimer to the monomeric or dimeric form, which
gives rise to a lower absorption flattening.
The enthalpy content of 30 kcal/mol for the irreversible denaturation
occurring around 85 °C (Fig. 5) is relatively small compared with
those reported for complete unfolding of globular proteins, like
-lactoglobulin (105 kcal/mol at 85 °C) (36), lysozyme (129 kcal/mol at 78 °C), or metmyoglobin (135 kcal/mol at 83 °C) (37).
This suggest that only a limited part of the protein adopts a different
conformation upon heat denaturation and that the majority of the
structure elements are preserved. Indeed, it is found that the
heat-denatured protein possesses an even higher degree of secondary
structure, with an increased content of extended
-structures. The
appearance of extended
-structures often reflects the formation of
large protein complexes, as indeed observed for Ara h 1 (Figs. 1B, 3B, and 6). For this protein we have
not been able to detect whether, prior to the formation of these large
aggregates, a limited unfolding of the protein has to take place, as
could be observed for example for a potato storage protein (29), but as
the aggregation kinetics points to a particle collision model (Fig. 6),
the possible exposure of hydrophobic sites on the partial unfolded
protein is expected to be relatively small and only sufficient to
result in stable protein-protein interactions if the proteins are
already at close range.
Ara h 1 isolated from peanuts heated at 110 °C and
140 °C exhibits a denatured state on secondary level similar to that
for Ara h 1 heated in an aqueous environment, although the
denaturation temperature is higher. This can be explained by the
presence of other peanut components like fats and carbohydrates,
resulting in a low hydration. This condition is known to stabilize
protein structures by dehydration of the polypeptide backbone, shifting the denaturation temperatures to higher values (38). As there is a
close structural resemblance between the heat-denatured isolated Ara h 1 and that from heated peanuts, it is suggested that
comparable processes are taking place. This hypothesis is further
supported by the solubility behavior of Ara h 1 at different
temperatures, as shown by the diminished extractability from peanuts
heated at 110 °C or higher. Whereas the extractability of Ara
h1 from peanuts heated for example at 140 °C is 3 times smaller
than that from non-heated peanuts, the extracted and thus water-soluble protein is denatured, but apparently was not able to form insoluble complexes. Consequently, Ara h 1 may play a physiological
role as soluble allergen, even after extensive heating of the material.
Implications of Structural Changes of Ara h 1 on Its Allergenic
Properties--
This is the first report on the effect of heat
treatment on the allergenic properties of an isolated peanut allergen.
Peanut allergy is a type I or immediate type allergy, with IgE playing a key role in the allergic reaction. Therefore, an assay was developed to study the interaction between native or heat-treated Ara h 1 and IgE. We found that native Ara h 1 binds with a
high affinity to IgE from serum obtained from peanut-allergic patients,
and that the IgE binding affinity of Ara h 1 is not strongly
affected when Ara h 1 isolated from peanuts heated at
various temperatures is used (Table I). This is in accordance with the
observation that heat treatment of peanuts did not affect the IgE
binding (22, 23). The difference between heat-labile and heat-stable allergens is usually based on clinical data like the patient's experiences, skin prick testing, and oral challenges (39). In some
cases, the difference is further underscored by animal studies and
immunological and biochemical experiments. Typically, the affinity of
heat-labile allergens like Der p I and Der f I
for IgE as studied using enzyme-linked immunosorbent assay techniques decreases up to 100- or 1000-fold (40) upon heating, whereas this
affinity for heat-stabile allergens like Der p II and
Der f II decreases only 2-fold (40). The minor differences
in affinity that we have found for native Ara h 1 and
heat-treated Ara h 1 indicate that Ara h 1 is a
heat-stable allergen although the molecular structure is affected upon
heating. As we applied a pool of sera from patients with peanut
allergy, we cannot exclude differences in IgE-Ara h 1 interaction among different patients. Skin prick testing, oral
challenges, and in vitro basophil degranulation with the
different forms of Ara h 1 are experiments to be undertaken in order to ultimately prove the heat-stable allergenicity of Ara
h 1.
The observation that Ara h 1 denatures upon heat treatment
while the IgE binding remains unaltered allows to speculate on the
nature of the IgE epitopes on Ara h 1. IgE epitopes on
Ara h 1 have been mapped using synthetic overlapping
peptides (11). 23 epitopes were found, while four immunodominant
epitopes were identified. In a recent study, these 23 IgE-binding
epitopes have been structurally analyzed by a homology-based molecular
model with phaseolin (12). It was shown in that work that the assigned epitopes are located at diverse sites of the protein, residing at both
secondary structured and non-structured parts. Using point mutations in
these epitope regions, it was also demonstrated that especially
hydrophobic residues are of importance for IgE binding (12). These
results complement very well to the findings presented in this work, as
the limited unfolding of (parts of) Ara h 1 upon heat
denaturation where only a limited number of hydrophobic groups become
solvent-exposed, based on the small enthalpy change involved and the
diffusion controlled aggregation, does not affect its IgE binding
affinity. In addition to these extensive studies on linear IgE epitopes
on Ara h 1 using synthetic peptides, our work demonstrates
that conformational IgE epitopes are either not present on Ara h
1 or restricted to those parts of the protein that are not
sensitive to heat denaturation. Our work contributes to the understanding of the exceptional allergenicity of peanuts by describing the thermal denaturation of Ara h 1 and explaining its
heat-stable allergenic nature.
 |
ACKNOWLEDGEMENTS |
We thank Dr. W. J. Koers
(University Medical Center Utrecht, Department of
Dermatology/Allergology, Utrecht, The Netherlands) for the patient
sera, Dr. E. F. Knol (University Medical Center Utrecht,
Department of Dermatology/Allergology, Utrecht, The Netherlands), and
Dr. A. J. Vlot (University Medical Center Utrecht, Department of
Internal Medicine, Utrecht, The Netherlands) for critically reading the
manuscript, and C. M. M. Lakemond (Center for Protein Technology TNO-WAU, Wageningen, The Netherlands) for assisting with the
ultracentrifugation experiments.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Protein
Technology, TNO Nutrition and Food Research Institute, P. O. Box 360, 3700 AJ Zeist, The Netherlands. Tel.: 31-30-6944296; Fax:
31-30-6957224; E-mail: koppelman{at}voeding.tno.nl.
**
Present address: Wageningen Centre for Food Sciences, 6700 EV
Wageningen, The Netherlands.
 |
ABBREVIATIONS |
The abbreviations used are:
bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
PAGE, polyacrylamide gel electrophoresis;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
DSC, differential scanning calorimetry;
FTIR, Fourier transform infrared;
ATR, attenuated total
reflection.
2
S. J. Koppelman and H. H. J. de
Jongh, unpublished results.
3
Lakemond, C. M. M., de Jongh, H. H.-J., Hessing,
M., Gruppen, H., and Voragen, A. G. J., submitted for publication.
4
C. M. M. Lakemond, personal communication.
 |
REFERENCES |
-
Sampson, H. A.,
and McCaskill, C., C.
(1985)
J. Pediatr.
107,
669-675[Medline]
[Order article via Infotrieve]
-
Tariq, S. M.,
Stevens, M.,
Matthews, S.,
Ridout, S.,
Twiselton, R.,
and Hide, D. W.
(1996)
Br. Med. J.
313,
514-517[Medline]
[Order article via Infotrieve]
-
van Reijsen, F. C.,
Felius, A.,
Wauters, E. A.,
Bruijnzeel-Koomen, C. A.,
and Koppelman, S. J.
(1998)
J. Allergy Clin. Immunol.
101,
207-209[Medline]
[Order article via Infotrieve]
-
Hourihane, J. O.,
Kilburn, S. A.,
Nordlee, J. A.,
Hefle, S. L.,
Taylor, S. L.,
and Warner, J. O.
(1997)
J. Allergy Clin. Immunol.
100,
596-600[Medline]
[Order article via Infotrieve]
-
Sachs, M. I.,
Jones, R. T.,
and Yunginger, J. W.
(1981)
J. Allergy Clin. Immunol.
67,
27-34[Medline]
[Order article via Infotrieve]
-
Barnett, D.,
and Howden, M. E.
(1986)
Biochim. Biophys. Acta
882,
97-105[Medline]
[Order article via Infotrieve]
-
Burks, A. W.,
Williams, L. W.,
Helm, R. M.,
Connaughton, C.,
Cockrell, G.,
and O'Brien, T. J.
(1991)
J. Allergy Clin. Immunol.
88,
172-179[Medline]
[Order article via Infotrieve]
-
de Jong, E. C.,
van Zijverden, M.,
Spanhaak, S.,
Koppelman, S. J.,
Pellegrom, H.,
and Penninks, A. H.
(1998)
Clin. Exp. Allergy
28,
743-751[CrossRef][Medline]
[Order article via Infotrieve]
-
Burks, A. W.,
Williams, L. W.,
Connaughton, C.,
Cockrell, G.,
O'Brien, T. J.,
and Helm, R. M.
(1992)
J. Allergy Clin. Immunol.
90,
962-969[Medline]
[Order article via Infotrieve]
-
Hourihane, J. O.,
Bedwani, S. J.,
Dean, T. P.,
and Warner, J. O.
(1997)
Br. Med. J.
314,
1084-1088[Abstract/Free Full Text]
-
Burks, A. W.,
Shin, D.,
Cockrell, G.,
Stanley, J. S.,
Helm, R. M.,
and Bannon, G. A.
(1997)
Eur. J. Biochem.
245,
334-339[Abstract]
-
Shin, D. S.,
Compadre, C. M.,
Maleki, S. J.,
Kopper, R. A.,
Sampson, H.,
Huang, S. K.,
Burks, A. W.,
and Bannon, G. A.
(1998)
J. Biol. Chem.
273,
13753-13759[Abstract/Free Full Text]
-
van der Veen, M. J.,
van Ree, R.,
Aalberse, R. C.,
Akkerdaas, J.,
Koppelman, S. J.,
Jansen, H. M.,
and van der Zee, J. S.
(1997)
J. Allergy Clin. Immunol.
100,
327-334[Medline]
[Order article via Infotrieve]
-
Burks, A. W.,
Cockrell, G.,
Stanley, J. S.,
Helm, R. M.,
and Bannon, G. A.
(1995)
Int. Arch. Allergy Immunol.
107,
248-250[Medline]
[Order article via Infotrieve]
-
Stanley, J. S.,
Helm, R. M.,
Cockrell, G.,
Burks, A. W.,
and Bannon, G. A.
(1996)
Adv. Exp. Med. Biol.
409,
213-216[Medline]
[Order article via Infotrieve]
-
Burks, A. W.,
Cockrell, G.,
Stanley, J. S.,
Helm, R. M.,
and Bannon, G. A.
(1995)
J. Clin. Invest.
96,
1715-1721[Medline]
[Order article via Infotrieve]
-
Buschmann, L.,
Petersen, A.,
Schlaak, M.,
and Becker, W. M.
(1996)
Monogr. Allergy
32,
92-98[Medline]
[Order article via Infotrieve]
-
Koppelman, S. J.,
Bleeker Marcelis, H.,
van Duijn, G.,
and Hessing, M.
(1996)
World Ingredients
12,
35-38
-
Yeung, J. M.,
and Collins, P. G.
(1996)
J. AOAC Int.
79,
1411-1416[Medline]
[Order article via Infotrieve]
-
Smith, J. (1997) Int. Food Ingredients 13-14
-
Phillips, R. D.,
and Beuchat, L. R.
(1981)
ACS Symp. Ser.
147,
275-298
-
Nordlee, J. A.,
Taylor, S. L.,
Jones, R. T.,
and Yunginger, J. W.
(1981)
J. Allergy Clin. Immunol.
68,
376-382[Medline]
[Order article via Infotrieve]
-
Burks, A. W.,
Williams, L. W.,
Thresher, W.,
Connaughton, C.,
Cockrell, G.,
and Helm, R. M.
(1992)
J. Allergy Clin. Immunol.
90,
889-897[Medline]
[Order article via Infotrieve]
-
Greenfield, N.,
and Fasman, G. D.
(1969)
Biochemistry
8,
4108-4116[Medline]
[Order article via Infotrieve]
-
Chang, C. T.,
Wu, C.-S. C.,
and Yang, Y. T.
(1978)
Anal. Biochem.
91,
13-31[Medline]
[Order article via Infotrieve]
-
Kalnin, N. N.,
Baikalov, I. A.,
and Venyaminov, S. Y.
(1998)
Biopolymers
30,
1273-1280
-
Vuilleumier, S.,
Sancho, J.,
Loewenthal, R.,
and Fersht, A. D.
(1993)
Biochemistry
32,
10303-10313[Medline]
[Order article via Infotrieve]
-
de Jongh, H. H. J.,
Goormaghtigh, E.,
and Ruysschaert, J.
(1996)
Anal. Biochem.
242,
95-103[CrossRef][Medline]
[Order article via Infotrieve]
-
Pots, A. M.,
de Jongh, H. H. J.,
Gruppen, H.,
Hamer, R. J.,
and Voragen, A. G. J.
(1998)
Eur. J. Biochem.
252,
66-72[Abstract]
-
Deleted in proof
-
Berkhout, T. A.,
Rietveld, A.,
and de Kruijff, B.
(1987)
Biochim. Biophys. Acta
897,
1-4[Medline]
[Order article via Infotrieve]
-
Goormaghtigh, E.,
Cabiaux, V.,
and Ruysschaert, J.
(1994)
Subcell. Biochem.
23,
405-450[Medline]
[Order article via Infotrieve]
-
Lawrence, M. C.,
Suzuki, E.,
Varghese, J. N.,
Davis, P. C.,
Van Donkelaar, A.,
Tulloch, P. A.,
and Colman, P. M.
(1990)
EMBO J.
9,
9-15[Abstract]
-
Lawrence, M. C.,
Izard, T.,
Beuchat, M.,
Blagrove, R. J.,
and Colman, P. M.
(1994)
J. Mol. Biol.
238,
748-776[CrossRef][Medline]
[Order article via Infotrieve]
-
Rost, B.,
and Sander, C.
(1993)
J. Mol. Biol.
232,
584-599[CrossRef][Medline]
[Order article via Infotrieve]
-
Lapanje, S.,
and Poklar, N.
(1989)
Biophys. Chem.
34,
155-162[CrossRef][Medline]
[Order article via Infotrieve]
-
Privalov, P. L.,
and Khechinashvili, N. N.
(1974)
J. Mol. Biol.
86,
665-684[Medline]
[Order article via Infotrieve]
-
Gekko, K.,
and Timasheff, S. N.
(1981)
Biochemistry
20,
4677-4686[Medline]
[Order article via Infotrieve]
-
Sampson, H. A.,
and Burks, A. W.
(1996)
Annu. Rev. Nutri.
16,
161-177[CrossRef][Medline]
[Order article via Infotrieve]
-
Lombardero, M.,
Heymann, P. W.,
Platts-Mills, T. A.,
Fox, J. W.,
and Chapman, M. D.
(1990)
J. Immunol.
144,
1353-1360[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.