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Departments of
*
Pediatrics and
Biochemistry and Molecular Biology and
Biomedical Visualization Center, University of Arkansas for Medical Sciences, Arkansas Children’s Hospital Research Institute, Little Rock, AR 72205; and
§
Department of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are a number of characteristics that increases the capacity of a
food allergen to provoke a dangerous systemic allergic reaction. These
include its ability to stimulate high titers of IgE and to resist
gastrointestinal degradation sufficiently to produce fragments
containing multiple IgE binding epitopes. The more degraded an allergen
becomes, the more fragments are produced that contain single
IgE-binding epitopes. Protein fragments containing single IgE-binding
sites are incapable of cross-linking IgE-bound Fc
R1 receptors and
therefore of causing mast cell degranulation. Thus, the biochemical and
structural aspects of allergens play a critical role in the disease
process.
The general biochemical characteristics of most food allergens indicate that they are low m.w. glycoproteins (<70 kDa) with acidic isoelectric points that are highly abundant in food. These proteins are usually resistant to proteases, heat, and denaturants, allowing them to resist degradation during food preparation and digestion (6, 7). Several studies have shown that the most allergenic portion of the peanut is the protein fraction of the cotyledon (8, 10). The peanut allergen Ara h 1 is a vicilin-like seed storage protein found in the cotyledon. This protein is one of the main storage proteins of the seed and is utilized as a nitrogen and amino acid source during development of a new peanut plant. In addition to its importance to the developing plant, Ara h 1 is recognized by serum IgE from >90% of peanut-sensitive patients, thus establishing it as an important allergen in the etiology of this disease (11, 12). The linear IgE-binding epitopes of this allergen have been mapped and shown to consist of 23 independent binding sites (13). Individual patients with IgE Abs to Ara h 1 have been shown to have IgE that recognizes multiple epitopes on the Ara h 1 protein (13). These sites are evenly distributed along the linear sequence of the molecule. However, a molecular model of the tertiary structure of the Ara h 1 protein shows that the IgE-binding sites were clustered into two main regions. In addition, Ara h 1 forms homotrimers, a physical characteristic that may be important in establishing it as an allergen (14).
Using fluorescence anisotropy, we have shown that the formation of a
highly stable Ara h 1 homotrimer is mediated primarily through
hydrophobic interactions. A molecular model of the Ara h 1 trimer
indicates that hydrophobic residues on 
-helical bundles located on
the ends of each monomer contribute to the stability of the structure.
The IgE-binding epitopes are clustered in the regions of
monomer-monomer contact. The importance of the trimer structure to the
overall allergenicity of the molecule was investigated using assays
designed to determine the stability of Ara h 1 to digestion with
proteases. In this report, when Ara h 1 was incubated with digestive
enzymes, various protease-resistant fragments containing IgE-binding
sites were obtained, indicating that the quaternary structure of Ara h
1 may play an important role in protecting these epitopes from
digestive enzymes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty grams of crude peanut extract were added to 500 ml extraction buffer (50 mM Tris-Cl (pH 8.3), 5 mM DTT, 1 mM EDTA, 1 mM PMSF) containing 200 mM NaCl. In this report, the extraction buffer contains the chemicals indicated within the parentheses, but the salt concentration is varied as indicated for different experiments. The solution was stirred gently at room temperature, cleared by centrifugation at 13,000 x g for 30 min at 4°C, and subjected to ammonium sulfate precipitation (15). Ammonium sulfate was added to 70% saturation. The remaining supernatant was then taken to 100% ammonium sulfate saturation and the Ara h 1 protein collected by centrifugation. The pellet was resolubilized in extraction buffer (pH 8.3) by sonication on ice at 40% power using a Heat Systems Disruptor (Fischer Scientific, Atlanta, GA). After sonication, the solubilized proteins were desalted on disposable PD-10 gel filtration columns (Pharmacia Biotech, Piscataway, NJ) and loaded onto a High Prep S, cation exchange resin column (2.5 x 12 cm, Bio-Rad Laboratories, Hercules, CA). A linear salt gradient (200–800 mM NaCl) was used to elute Ara h 1 from the column and 2.5-ml fractions were assayed for Ara h 1 content by a 12% SDS-polyacrylamide gel (Novex, San Diego, CA) and Coomassie Brilliant Blue staining. Fractions containing Ara h 1 were pooled and desalted into desired buffers on PD-10 columns just before use in all experiments. The desired buffers used were identical with extraction buffer containing 100 mM NaCl with varying pH values (pH 8.3 for tryptic and chymotryptic digestion reactions and pH 2 for pepsin digestion for the digestion reactions). Protein concentrations were monitored using the Bio-Rad protein assay reagent kit (Bio-Rad Laboratories). At each stage, samples were subject to electrophoresis on 12% SDS-polyacrylamide gels (Novex), and the purity of the Ara h 1 fractions was assessed by Coomassie staining and densitometry of the resulting protein bands. Purified Ara h 1 was stored in aliquots at -80°C.
Serum IgE
Serum from 15 patients with documented peanut hypersensitivity reactions (mean age, 25 yr) was used to identify Ara h 1 during purification. The patients had either a positive double-blind, placebo-controlled food challenge or a convincing history of peanut anaphylaxis (laryngeal edema, severe wheezing, and/or hypotension (16). Equal aliquots of IgE-containing serum from 12 to 15 patients was pooled and used for our experiments. Each patient’s serum contained IgE that recognized Ara h 1. All studies were approved by the Human Use Advisory Committee at the University of Arkansas for Medical Sciences.
Cross-linking reactions
Purified Ara h 1 was desalted into PBS, and two identical samples were diluted to a final concentration of 3 µM. The pH was adjusted with HCl to either pH 2 or pH 7.6, and the solution was allowed to incubate at room temperature for 1 h. After incubation, the Ara h 1 sample at pH 2 was adjusted to pH 7.6, and both samples were then subjected to cross-linking. The process of pH adjustment and cross-linking was minimized (<2 min) to prevent reequilibration of Ara h 1 monomers and trimers before cross-linking. The protein cross-linking reagent, dithiobis(succinimidylpropionate) (Pierce, Rockford, IL) was used. Dithiobis(succinimidylpropionate) was dissolved in N,N-dimethylformamide as a 74 mM stock solution. A 1-µl sample of cross-linker stock solution was added to 50 µl of the Ara h 1 samples. The reactions were quenched after 80 s by addition of SDS sample buffer (lacking DTT), heated to 100°C for 5 min, and subjected to SDS-PAGE. Limited cross-linking was performed to minimize the formation of nonspecific complexes.
Protease digestions
Purified Ara h 1 was desalted and diluted to a final concentration of 8 µM in extraction buffer containing 100 mM NaCl adjusted with HCl to pH 8.3 for tryptic and chymotryptic digestions and pH 2.0 for pepsin digestion (all proteases were purchased from Sigma (St. Louis, MO)). The diluted Ara h 1 was incubated in the presence of 0.5 µg/ml of trypsin, chymotrypsin, or pepsin at 37°C, and aliquots were taken at 0 min, 10 s, and 1, 2, 4, 8, 15, 30, 60, 120, and 180 min. The digestion reaction in each aliquot was quenched by the addition of SDS-sample buffer. Samples were then subjected to SDS-PAGE and either stained or transferred to nitrocellulose for immunoblot analysis using pooled serum from peanut allergic individuals.
Immunoblot analysis
For the detection of Ara h 1 or IgE-binding fragments of Ara h 1, immunoblot analysis was performed using serum IgE from a 15-person pool of peanut-allergic individuals. SDS-PAGE (12%)-resolved proteins were transferred to nitrocellulose membrane (0.45 µm, Schleicher and Schuell, Keene, NH) electrophoretically. The membranes were blocked in Tris-buffered saline with Tween 20 (TBST) plus 1% BSA for 2 h at room temperature. The membrane was then washed in TBST and incubated with a 1:10 dilution of pooled human sera for 1 h. Detection of the bound IgE was accomplished using 125I-labeled anti-human IgE secondary Ab (Sanofi, Chaska, MN) and subsequent exposure to x-ray film.
Fluorescein labeling
Ara h 1 was desalted into NaHPO4 buffer, pH 8, and labeled with FITC according to the methods described by Fernando and Royer (17) for dansyl labeling. Briefly, the FITC was dissolved in N,N-dimethylformamide at 250 mg/ml to make the stock solution, and 10 µl of the solution were added to 1 ml of Ara h 1 solution. This mixture was then incubated at room temperature for 10 min. The free FITC was separated from the labeled protein using a desalting column (described above) that was preequilibrated in binding buffer (10 mM HEPES/KOH (pH 7.9), 1 mM EDTA, 1 mM DTT, 5% glycerol).
Fluorescence anisotropy measurements of Ara h 1
All fluorescence measurements were made using a Beacon
fluorescence polarization spectrometer (Pan Vera, Madison, WI) with
fixed excitation (490 nm) and emission (530 nm) wavelengths.
Fluorescence measurements were done at room temperature (24°C) in
previously described binding buffer containing different salt
concentrations from 0 to 1.8 M NaCl, in a final volume of 1.1 ml.
Fluorescence anisotropy is described in detail by Fernando and Royer
(17). A constant amount of fluorescein-labeled Ara h 1
protein (10 nM) in binding buffer was mixed with serial dilutions (by
0.5 or 0.8 increments) of unlabeled Ara h 1 to analyze oligomer
formation at room temperature. Each data point is an average of three
independent measurements, and each curve is fitted to a sigmoidal
function using Origin (Microcal Software, Northampton, MA). Analysis of
the slope of the binding curves and midway point between top and bottom
plateaus allowed the calculation of the cooperativity values (
) and
apparent dissociation constants (Kapp)
for Ara h 1 oligomer formation (18). In the equation used
by Origin program for fitting a sigmoidal curve, the
Kapp is defined as the midway point
between the lower and upper plateaus and the slope of the curve (
value) defines the cooperativity of the interaction. This equation is
described in detail by Czernik et al. (18). The intensity
of fluorescence remained constant throughout the anisotropy
measurements.
Homology-based model of Ara h 1 trimer
A homology-based model of an Ara h 1 monomer (14)
was used to construct the trimeric model. Several small areas
representing Ara h 1 amino acid residues
Leu166-Val193,
Pro226-Phe240,
Asp249-Asp259, and
Arg300-Arg327 were omitted
during trimer construction because of structural uncertainty. The
coordinates of the C atoms, of Asn48,
Ile117, Phe174,
Glu202, Ala312, and
Gly362 from the x-ray crystal structure of
phaseolin4 (Protein
Data Bank code 2PHL) were used as reference points to fit the C
atoms of Asn37, Ile100,
Phe158, Glu204,
Ala35, and Gly396 from Ara
h 1 to form a trimer. The Fit Monomers program from SYBYL (version 6.3,
Tripos, St. Louis MO) was used to create the initial framework. The
energy of the Ara h 1 trimer was minimized with a harmonic force
constraint of 100 using the Charmm force field and the adopted basis
Newton-Raphson method for 500 iterations using the Charmm Minimization
program resident in QUANTA (version 96, Molecular Simulations,
Burlington, MA). The stereochemical quality of the model was assessed
by using PROCHECK (version 2.1.4, Oxford Molecular, Palo Alto, CA). The
computations were performed on a Silicon Graphics workstation running
IRIX 6.4.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fluorescence anisotropy and molecular modeling methods were used
to gain a better understanding of the structural properties of Ara h 1
that may contribute to its stability and allergenicity. The Ara h 1
protein is known to form a homotrimer at relatively low concentrations,
and this structure has been suggested to be important to the overall
allergenicity of the molecule (14). To determine the
stability and types of monomer interactions that mediate the formation
of a homotrimer, fluorescence anisotropy measurements were performed in
the presence of increasing salt concentrations (0–1.8 M NaCl).
Purified, fluorescein-labeled Ara h 1 (10 nM) was mixed with various
concentrations of unlabeled Ara h 1. The anisotropy of fluorescence
observed at each concentration was determined and plotted as
milli-anisotropy U vs the concentration of Ara h 1 (Fig. 1
). The ability of the trimer to form at
NaCl concentrations as high as 1.8 M is indicated by the presence of
the plateau observed at Ara h 1 concentrations of 0.1–0.3 µM. There
is an increase in the dissociation constant as indicated by the
increase in Kapp from 0.065 to 0.170
µM as the NaCl concentration is increased from 0.1 M to 1.8 M. The
cooperativity () of trimer formation also decreased from 2.4 to 2.1
over the same salt concentrations (Table I). Minimal transition is seen in the
anisotropy curve on dilution of Ara h 1 in the absence of NaCl. This
result is most likely due to accentuated oligomerization of the protein
in the absence of counterions in the solution; therefore, only minimal
dissociation is seen on dilution of the protein. Thus, due to the
minute change in anisotropy the Kapp
and values cannot be obtained with the fitting program used (see
Materials and Methods). Collectively, these results indicate
that although some ionic interactions are involved in the cooperative
interaction of monomers, the formation and stability of the Ara h 1
trimer are primarily due to hydrophobic interactions.
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Because we had demonstrated that hydrophobic interactions were
primarily responsible for Ara h 1 trimer formation, we examined the
location of any surface-accessible hydrophobic amino acid residues
present on the Ara h 1 monomer that may contribute to trimer formation.
To accomplish this, a homology-based model of Ara h 1 tertiary
structure, representing aa 172–586 (14), was utilized.
The tertiary structure of the molecule consists of four domains: an
helical bundle on one end, two sets of opposing anti-parallel ß
sheets and a helical bundle on the opposite end. The space-filled
model of the Ara h 1 molecule and the position of the hydrophobic amino
acid residues (alanine, isoleucine, leucine, methionine, phenylalanine,
proline, tryptophan, valine) are shown in Fig. 2. The distal regions of the molecule
contain the majority of the surface-accessible hydrophobic amino
acids.
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-1 and -2 angles, were also tested in the Ara h
1 trimer. The majority of the side chains are within the ideal 2.5 SD
range where the percentage of residues outside the criteria is 19.3%
for the Ara h 1 trimer and 16.9% for the phaseolin trimer. In addition
to these criteria, a variety of main chain parameters (Omega angles,
Van der Waals contacts, 
angles, and H-bond energy) were tested for
stereochemical quality and were comparable with that of the phaseolin
x-ray crystal structure. Taken together, these data indicate that the
homology-based model of the Ara h 1 trimer is reasonable and similar to
the phaseolin trimer. The global fold of the Ara h 1 trimer indicates
that formation of this structure is due to the helical bundles on
the ends of one Ara h 1 monomer overlapping with those of the adjacent
monomer (Fig. 3, top). The
hydrophobic residues depicted in Fig. 2
are those that form the
interface between the monomers, similar to the structure of the
phaseolin trimer (19).
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, bottom). Epitopes 11 and 12 on the helical bundle of one monomer
and epitopes 20 and 21 on the helical bundle of another monomer
contact one another when the trimer is formed. Whereas most of the
amino acid residues contained within these epitopes are surface
accessible in the Ara h 1 monomer, 
40% of the residues within these
epitopes lose surface accessibility when the trimer is formed as
calculated by the QUANTA molecular simulation program. Digestion-resistant fragments containing intact IgE epitopes
To determine whether quaternary structure played any role in
protecting the Ara h 1 molecule from proteolytic digestion it was
essential to determine whether the Ara h 1 trimer would remain intact
when exposed to the environment of the stomach. Therefore, purified Ara
h 1 was exposed to acidic pH. At the end of this incubation period, a
cross-linking reaction was performed, and the amount of Ara h 1 trimer
formed under these conditions was visualized by SDS-PAGE analysis (Fig. 4). Even though effects of acid
hydrolysis can be seen on the integrity of the protein, it is clear
that the Ara h 1 oligomer was found to be stable even after incubation
at pH 2 and could still bind IgE (data not shown).
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). A
representative digestion of Ara h 1 with chymotrypsin has been shown in
Fig. 5 as an example. The data from
digestions with pepsin, trypsin, and chymotrypsin have been summarized
in Table II. These peptide fragments ranged in size from 16 to 61 kDa
during 3 h of digestion. To determine whether the long lived
peptides contained IgE binding epitopes, the Ara h 1 digestion products
were subjected to immunoblot analysis using serum IgE from a pool of
peanut-sensitive patients. In each case, many peptides contained intact
binding sites that could be recognized by serum IgE (Table II). Knowing
that Ara h 1 contains 23 IgE binding sites that are evenly distributed
along the linear sequence of the molecule (13), these
results suggest that large proteolytic fragments of Ara h1 contain
multiple IgE-binding epitopes and survive digestion by the
gastrointestinal enzymes tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The only therapeutic option presently available for the prevention of a food hypersensitivity reaction is food avoidance. Unfortunately, for a ubiquitous food such as peanut, the possibility of inadvertent ingestion is great (26, 27, 28). Because of the potential severity of the allergic reaction, it has been suggested that a hypoallergenic Ara h 1 gene could be developed to replace its allergenic homologue in the peanut genome, thus blunting allergic reactions in sensitive individuals who inadvertently ingest this food (14). Because the Ara h 1 gene product is such an abundant and integral seed storage protein, it would be necessary for the altered vicilin to retain as much of its native function, properties, and three-dimensional structure as possible. The data presented here indicate that most of the IgE-binding epitopes are located at the contact points between the monomers in the Ara h 1 homotrimer. Amino acid substitutions designed to reduce the allergenicity of this protein may have deleterious effects on trimer formation and protein function. On the other hand, weakly destabilizing mutations may be desirable in making IgE-binding sites available to digestive enzymes.
It has been shown that Ara h 1 is capable of forming trimeric complexes
in vitro similar to that of other vicilins (14). This
multimeric form is also observed in the phaseolin x-ray crystal
structure (19) and within the initial multiple isomorphous
replacement electron density maps of
canavalin5
(29). For phaseolin, the primary site of monomer contact
was overlap of the terminal helical bundles. A trimeric model of
Ara h 1 based on the phaseolin structure revealed that the helical
bundles of the Ara h 1 monomers also appear to be the primary site for
monomer contact. In addition, fluorescence anisotropy experiments
clearly indicate that trimer formation is primarily mediated through
hydrophobic interactions; this is confirmed by the location of
hydrophobic residues in the areas of monomer-monomer contact. As
previously mentioned, the location of the intramolecular hydrophobic
contacts coincides with the location of preponderance of the
IgE-binding epitopes. Also, when examining all of the available
protease recognition sites located on the Ara h 1 primary sequence, it
is clear that several of these sites are protected from digestion.
Considering that Ara h 1 contains 23 IgE epitopes that, when the
protein is denatured, are somewhat evenly distributed along the primary
amino acid sequence of the molecule, and the majority of the
peanut-allergic individuals are known to recognize a minimum of five
epitopes, it becomes obvious that any fragment larger than 10 kDa must
contain more than one IgE-binding site. Collectively, these results
suggest that the formation of a trimeric complex may afford the
molecule some protection from protease digestion and denaturation,
allowing passage of large fragments of Ara h 1 containing several
intact IgE-binding epitopes across the small intestine, therefore
contributing to its allergenicity.
Studies designed to develop hypoallergenic alternatives to replace native allergens in plants must take into account not only those amino acid substitutions that result in IgE binding but also those that will not disrupt the native structure of larger protein complexes. Thus, the development of an assay system that allows us to measure trimer formation and stability will permit us to test the integrity of recombinant proteins before plant transformation. Also, mutations in the recombinant protein that may allow trimer formation while having a destabilizing effect may increase the susceptibility of Ara h 1 to acid hydrolysis and digestion, therefore rendering it less likely to cause sensitization. Given the widespread use of peanuts in consumer foods and the potential risk this poses to individuals genetically predisposed to developing peanut allergy and to the health of individuals already peanut sensitive, these approaches are currently being explored in our laboratories.
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Footnotes |
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2 Current address: U.S. Department of Agriculture-Agricultural Research Service Southern Regional Research Center, New Orleans, LA 70179.
3 Address correspondence and reprint requests to Dr. Gary A. Bannon, UAMS Slot 516, 4301 West Markham, Little Rock, AR 72205.
4 The atomic coordinates for the crystal structure of phaseolin can be accessed through the Brookhaven Protein Data Bank under PDB 2PHL (19 ).
5 The atomic coordinates for the crystal structure of canavalin can be accessed through the Brookhaven Protein Data Bank under PDB 1CAU (29 ).
Received for publication November 18, 1999. Accepted for publication March 10, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 0 mM NaCl; •, 100 mM NaCl; 
, 300 mM NaCl; 
, 500 mM NaCl;
, 900 mM NaCl; +, 1.4 M NaCl; x, 1.8 M NaCl.
