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INTRODUCTION |
Epidemiologic studies suggest that between 10 and 20% of the
world population exhibits some form of IgE-mediated hypersensitivity, which is manifested as asthma, atopic dermatitis, or allergic rhinitis
(1). A number of studies have shown that sensitivity to house dust mite
allergens is the most important risk factor for asthma (1, 2). More
than 10 mite allergens have been defined, and the 14-kDa Group 2 allergens (Der p 21 and Der f 2) are considered major
allergens because of the fact that 80-90% of patients have specific
IgE Ab to these allergens (3). Previously
we reported that Der p 2 is structurally a member of the immunoglobulin
superfamily, although the function of the allergen remains unknown
(4).
Therapy for allergic disease includes allergen avoidance,
pharmacotherapy, and allergen-specific immunotherapy. Recently, new
strategies for immunotherapy have been proposed with the aim of
improving efficacy, patient compliance, and associated risks (5). Our
studies have focused on the generation of hypoallergenic variants; the
underlying hypothesis is that reducing IgE reactivity will reduce
IgE-mediated side effects (6). The mapping of epitopes on Der p 2 and
Der f 2 is an important step toward the development of hypoallergenic
variants. Using murine mAb and sera from mite-allergic subjects,
we have shown that the epitopes on the Group 2 allergens are
conformational and that the three disulfide bonds stabilize this
structure (6, 7). Mutational analysis of surface residues found that
substitution of threonine for lysine at position 100 had reduced
avidity for mAb 7A1, and mutations of residues 44-46 affected the
avidity of a second mAb,
DpX (8). A third mAb, 6D6, belongs to a
group of mAb that recognize the different naturally occurring isoforms
of Der p 2 at residue 1142
(9, 10). Although the above studies have been informative, they provide
an incomplete map of the epitopes and do not necessarily identify those
residues on Der p 2 that contribute significantly to the binding energy.
Epitopes can also be mapped by measuring changes in amide hydrogen
exchange rates of the antigen that occur as a result of the formation
of an immune complex (11-13). These rates can be conveniently measured
using NMR techniques. This method was first used by Patterson
et al. (11) to localize the epitope of an anti-cytochrome
c mAb. We have subsequently measured similar effects in a
number of anti-lysozyme antibodies (13). In general, those residues
that are in the structural epitope (those that either contact the Ab or
are buried by it) show the largest reduction in exchange rates.
Once the location of the epitope has been obtained using amide exchange
measurements the residues, which are important for mAb binding, can be
systematically identified using scanning alanine mutagenesis (14-16).
The mutation to alanine reduces the side chain to as small as possible
without substantially altering the secondary structure. Usually,
glycine and proline are not altered, because both of these residues
strongly influence the configurational entropy of the peptide chain.
This approach has been used to map B-cell epitopes in a number of
different model systems. Jin et al. (14) studied 43 different mutants of human growth hormone in combination with 21 different monoclonal antibodies. A study by Dall'Acqua et
al. (15) introduced alanine mutations into both the antibody and
antigen (lysozyme) and reported the structure of a complex between the
antibody and one of the lysozyme mutants. Benjamin and Perdue (16)
characterized the interaction of 70 mutants of staphylococcal nuclease
with 10 different mAb. All of these studies found that an average of 3 to 4 residues were energetically important to mAb binding. In this
study, a residue was classified as being energetically or functionally
important if the free energy of binding of the mutant to mAb binding
was 1.0 kcal/mol or greater. The studies of growth hormone and
staphylococcal nuclease examined virtually the entire surface of the
respective proteins, and the study of lysozyme involved residues known
to be in contact with Ab from crystallographic studies. In this study, the hydrogen exchange data were used to provide a starting point for a
more focal analysis of the epitopes on Der p 2.
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EXPERIMENTAL PROCEDURES |
Production of rDer p 2--
Recombinant Der p 2 (rDer p 2) was
expressed and purified from Escherichia coli cultures
as previously described (9). Briefly, the protein was recovered from
the insoluble fraction of the cell sonicate and resolubilized with 6 M guanidine (one-fifth the original cell culture volume),
and after dialysis against buffer (10 mM Trizma (Tris
base), 1 mM EDTA, pH 8.5), the protein was purified by mAb
affinity chromatography. 15N-Labeled protein was obtained
by growing the bacteria on a minimal medium with
15N-ammonium sulfate as the sole nitrogen source.
Monoclonal Antibodies--
The murine mAb used in this study
were produced at the University of Virginia Lymphocyte Culture Center
and have been described in detail elsewhere (17). The mAb
DpX was
produced by Dr. Rob Aalberse and colleagues (18). Radioimmunoassay and
ELISA have shown that this panel of 8 mAb defines three antigenic
regions on Der p 2 (17), and the mAb 7A1,
DpX, and 6D6 were selected as representative of these regions. For this study, mAb were purified from ascites fluid by precipitation with
(NH4)2SO4 followed by affinity
chromatography using an rDer p 2 antigen affinity column. Protein
concentrations were calculated using extinction coefficients of
E0.1% = 1.42 and E0.1% = 0.72 for
murine IgG and rDer p 2, respectively. The resins used for the mAb
purification and the amide proton exchange rate protection
studies (described below) were constructed using Affi-Gel-10 (Bio-Rad
Laboratories, Hercules, CA) according to manufacturer protocols.
Briefly, 25 mg of protein was coupled per 1 ml of gel bed in a buffer
of 0.1 M MOPS, 80 mM NaCl, pH 7.5. Typically
greater than 95% of the protein coupled to the matrix. The capacities
of the columns were as follows: rDer p 2 column, 27 mg of mAb; mAb 6D6
column, 5 mg of rDer p 2; mAb 7A1 column, 10 mg of rDer p 2; and mAb
DpX column, 13 mg of rDer p 2.
Monoclonal Ab Inhibition of IgE Binding to rDer p
2--
Increasing concentrations of mAb were used to inhibit IgE
binding to rDer p 2 in a modified enzyme immunoassay. The antigen was
bound directly to the microtiter plate or presented by mAb
DpX. Sera
were added along with increasing concentrations of mAb so that the
final concentration of serum was 1:4 or 1:8, and the concentration of
mAb ranged from 0.01 to 100 µg/ml. IgE binding was detected using
biotinylated goat anti-human IgE (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) and streptavidin horseradish peroxidase
(Sigma). The percent inhibition was calculated from the
A405 of IgE Ab binding to rDer p 2 in the
absence of inhibitor mAb. The positive control experiment used the
anti-Der p 2 chimeric mAb 2B12-IgE developed by Schurmann et
al. (19). Sera from 4 mite-allergic subjects (radioallergosorbent
assay positive to Dermatophagoides pteronyssinus
extract), pooled sera from 7 additional subjects, and 1 radioallergosorbent assay negative control subject were tested.
Measurement of Amide Exchange Rates--
The amide exchange
kinetics of the mAb·rDer p 2 complex was measured using the
method described by Williams et al. (13). Briefly
15N-rDer p 2 was loaded on the mAb column in
phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, 100 mM NaCl) and washed with three column-volumes of
phosphate-buffered saline. This was followed with three more
column-volumes of NMR sample buffer (10 mM sodium
phosphate, 200 mM K2SO4, 10 mM NaCl, 1 M EDTA, pH 6.6) made with 99%
D2O. Time 0 was considered the time at which the second
volume of buffer was added. The columns were left at 4 °C for
48 h and then rinsed with 3 volumes of D2O before
elution with 0.2 M acetic acid. The eluted protein was
titrated to pH 3.2 using NaOH to quench the amide exchange. The 48-h
control sample was made by concentrating and diluting the protein 3 times with the deuterated exchange buffer. The exchange was considered initiated after the first dilution, because the sample was greater than
94% D2O at this point. After a 48-h incubation at 4 °C
the sample was titrated to pH 3.2 with HCl to quench the reaction. The
protein was then placed into column elution buffer by 3 rounds of
concentration and dilution. The control sample for the 0-h time point
and the sample used for the assignment of the spectra were in
H2O containing 0.2 M acetic acid.
Collection of NMR Spectra--
All NMR experiments were carried
out on a Varian Unity Plus spectrometer operating at a proton frequency
of 500 MHz. Amide peak intensities were obtained from two-dimensional
HSQC spectra acquired using 256 t1 points and 1024 t2 points and with 64 transients (20). The
three-dimensional HSQC-NOESY spectra (21) were obtained as
previously described (9). All spectra were transformed and analyzed
using Felix 95.0 (Biosym/Molecular Simulations, San Diego, CA) with
standard protocols.
Prediction of Epitopes and Construction of Alanine Point
Mutants--
Residues potentially within a given epitope were
predicted by taking the midpoint between the two protected residues
that were furthest apart and determining which residues had atoms that were less than 12.5 Å from this point. A 12.5-Å radius circle would
have an area of 490 Å2 whereas a sphere of this radius
would have a surface area of 1,960 Å2 (22). Considering
that the surface of protein antigen epitopes are convoluted, the area
predicted should exceed the 600-900-Å2 surface area of
known protein epitopes (22) and thus over-predict the size of the epitope.
The accessible surface area was determined using a 9.0-Å probe (23).
Residues with accessible side chains (see Table II) were changed to
alanine using the QuickChange kit from Stratagene (La Jolla, CA).
Plasmid DNA was isolated using Wizard Plus minipreps (Promega, Madison,
WI) and sequenced using automated methods. Because a polymerase chain
reaction-based method was used to create the mutations, the entire
coding sequence of each mutant was sequenced to ensure that no
unintended changes were introduced.
Competitive Inhibition ELISA--
The ability of rDer p 2 and
the various alanine mutants to inhibit mAb binding to solid phase rDer
p 2 was measured using a goat anti-mouse IgG (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) conjugated to horseradish peroxidase
(9, 24). The results were calculated as the percentage inhibition using
the value of maximum inhibition by rDer p 2 as 100% inhibition. The
curves were fit to a sigmoidal function using Sigma Plot (SPSS Science, Chicago, IL) to determine the concentration of 50% inhibition (IC50). Relative binding constants were calculated,
according to Equation 1, as follows.
|
(Eq. 1)
|
The change in free energy of the binding reaction as a result of
the mutation was calculated from Equation 2 (13),
|
(Eq. 2)
|
where the standard error in the determination of the 
G is
~0.1 kcal/mol. Consequently, a 
G greater than 0.5 kcal/mol was
considered significant.
 |
RESULTS |
Effect of mAb on IgE Ab Binding--
The mAb used in this study
have been characterized by a variety of techniques including inhibition
radioimmunoassay and ELISA and have been shown to define three
antigenic regions on Der p 2 (17). To demonstrate that these antigenic
regions also represent IgE-binding regions, we determined the ability
of each mAb to inhibit the binding of IgE from allergic sera. Fig.
1, panel A shows the control
experiment using the murine-human chimeric IgE mAb that contains the
2B12 variable region. The data in panel A clearly show that
inhibition of binding of the 2B12-IgE hybrid occurs only when the
inhibiting mAb bind to the same antigenic region as does the 2B12 mAb
itself. For example, only mAbs 1D8, 6D6, 4G7, and 2B12, which were
previously shown by classical competitive inhibition studies to bind to
the same antigenic regions on Der p 2 (17), are capable of inhibiting
the binding of the 2B12-IgE chimeric mAb in a
dose-dependent manner. In contrast, mAb 7A1 and
DpX,
each of which binds to one of two other nonoverlapping regions, do not
inhibit. These results are in complete agreement with a more detailed
study on the competitive inhibition of these and other mAb for binding
to Der p 2 (17). Panels B and C show the ability
of mAb to competitively inhibit binding of IgE in sera from two house
dust mite-allergic subjects, and panel D shows results using
pooled sera from 7 additional mite-allergic subjects. Each serum gave
unique curves, and the maximum inhibition at 100 µg/ml ranged from 10 to 50% for any given mAb. Thus, the ability of the mAb to inhibit IgE
binding to rDer p 2 suggests that these mAb are appropriate markers for
IgE epitopes.

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Fig. 1.
mAb inhibition of IgE binding to Der p
2. Increasing concentrations of mAb were used to inhibit IgE Ab
binding to rDer p 2 presented by mAb DpX in an ELISA. Panel
A shows the inhibition of the mouse-human chimeric IgE mAb
(containing the mAb 2B12 combining site). Inhibitor mAb were 2B12
(open circles), 1D8 (filled circles), 7A1
(filled squares), 4G7 (filled triangles), 6D6
(filled diamonds), DpX (open squares), and an
isotype matched control mAb 10A6 (dashed line). Panels
B and C show the inhibition of serum IgE binding from
two mite-allergic subjects, B. R., and M. W. The inhibitor mAb
were 2B12 (open circles), 1D8 (filled circles),
7A1 (filled squares), 4G7 (filled triangles), 6D6
(filled diamonds). Panel D shows inhibition of
pooled sera IgE Ab by 1D8 (filled circles), 7A1
(filled squares), 4G7 (filled triangles), and
DpX (open squares). Values plotted in panels
B, C, and D have been corrected for
background binding, determined using the mAb 10A6, which was
~5%.
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Amide Exchange Experiments--
Fig.
2 shows sections of HSQC spectra of rDer
p 2 for experiments conducted with mAb 7A1. Peaks for residues 73, 94, 97, and 101 are shown. The panels show spectra acquired in
H2O (left), after 48 h in D2O
(center), and after 48 h in D2O of a
complex of Der p 2 and mAb 7A1 (right). These spectra
clearly show that residues 94 and 101 were strongly protected from
amide proton exchange while complexed with mAb. In contrast residue 97 was weakly protected, and residue 73 was not protected at all. A
similar analysis of spectra obtained for rDer p 2 complexed with mAb
DpX and mAb 6D6 (data not shown) showed that residues 72, 73, and 75 were protected by mAb
DpX, and residues 111 and 116 were protected by mAb 6D6. The protected residues are highlighted on the
structure of rDer p 2 in Fig. 3. Also
displayed in Fig. 3 are the accessible surfaces of those residues that
were predicted to be involved in the full structural epitope. The amino
acid residues predicted to be in the epitope for each mAb are listed in
Table I.

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Fig. 2.
Representative exchange data for mAb
7A1. Portions of the HSQC spectra of Der p 2 containing
resonances from residues 73, 101 (panel A), 97 (panel
B), and 94 (panel C) are shown. The left
column of spectra were obtained for a sample in H2O.
The middle column of spectra were obtained from a sample of
Der p 2 in D2O. The right column
(Protected) of spectra were obtained from a sample that was
bound to mAb 7A1 for 48 h in the presence of
D2O.
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Fig. 3.
Epitopes of mAb 7A1, mAb
DpX, and mAb 6D6. Residues that were
found to have altered exchange rates are mapped on the structure of Der
p 2. For each epitope those residues that were found to be protected
from amide-proton exchange by mAb are colored red. The
molecular surface for the residues predicted to be within each epitope
is displayed with cyan-colored dots. The residues that form
the DpX epitope reside primarily on the upper -sheet whereas
those that form the 6D6 epitope reside primarily on the bottom
-sheet.
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Alanine Scanning Mutagenesis of the mAb 7A1 Epitope--
A number
of mutants were constructed to test the contribution to binding of
different residues in the predicted epitope for mAb 7A1 (Table
II). We selected mAb 7A1, because
previous studies had shown that Lys-100 was important for binding (8).
Only those amino acids with side chain exposure to solvent were
analyzed. For example, although Trp-92 and Val-94 are both within the
predicted epitope, they do not show any side chain exposure to solvent. Also systematically excluded from mutagenesis were glycine and proline
residues. The binding of mAb 7A1 to these mutants was measured using
competitive inhibition ELISA, and the calculated IC50
values for these mutants are shown in Table II. A total of 8 residues
(amino acids 30, 31, 33, 57, 93, 96, 97, and 102) showed a 
G
value greater than 0.5 kcal/mol, strongly suggesting that each of these
residues contribute significantly to the binding affinity (Fig.
4).

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Fig. 4.
Functionally important residues in the 7A1
epitope. A, space-filling model showing the residues
(white) protected by the 7A1 mAb during the NMR amide-proton
protection assay. B, the residues important for mAb 7A1
binding are mapped onto a surface representation of Der p 2. The view
is looking down the end of the -barrel where mAb 7A1 is proposed to
interact. The color scheme is as follows: red, residues that
when mutated to alanine significantly affect the binding of Der p 2 to
mAb 7A1. White, Lys-97, which was the only residue protected
in the amide-protection assay, which, when mutated, significantly
affected binding. Green, residues that when mutated to
alanine did not affect antibody binding. Blue, residues that
were not mutated.
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The reduction in binding affinity, which occurred as a result of the
mutation, was likely to be caused by changes in the direct interaction
of the mAb with the altered residue. Alternatively, the mutation may
have affected the secondary or tertiary structure of Der p 2. As a
control, the alanine mutants were used to inhibit mAb
DpX binding to
rDer p 2. The only mutant that did not interact with mAb
DpX in a
manner similar to the native protein was N93A (data not shown). Thus
the effect of this mutation cannot be definitively attributed to direct
interactions of residue Asn-93 with mAb 7A1.
Although the control experiments with mAb
DpX could identify gross
changes in the structure, it was possible that more subtle changes
occurred in the mutant proteins. Consequently, a detailed analysis of
the K33A mutant was performed to assess the magnitude of the changes in
the structure of Der p 2 due to this mutation. The HSQC spectrum of
K33A was very similar to rDer p 2, and a portion of this spectrum is
shown in Fig. 5A. The
boxes overlaid on this section of the spectrum indicate the
position of peaks in the HSQC spectrum of rDer p 2. One peak
disappeared (at 120 ppm nitrogen and 7.32 ppm proton), this peak
corresponded to Lys-33. Two new peaks from Ala-33 appeared that were
not in the spectrum of rDer p 2. The presence of two peaks indicated
that this residue exchanged slowly between two different conformations.
However, the structure of these two conformations must be similar based on the similar pattern of NOEs for Ala-33 shown in Fig. 5C.
In an analysis of the full HSQC spectra, the only other peaks that shifted in the spectrum of K33A were Gly-32 and Val-94.

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Fig. 5.
NMR spectra of the K33A Der p 2 mutant.
A region of the HSQC spectrum of K33A is shown in panel
A. The boxes indicate the previous positions of
resonances for rDer p 2. The amide peak of residue 33 has moved from
the right side of the spectrum (empty box) to the
left of the spectrum (unboxed peaks). The two new
peaks are from Ala-33. Panels B and C show a
slice of the HSQC-NOESY spectrum for the amide of residue Lys-33 in
rDer p 2 (panel B) whereas panel C shows the
spectrum for the amide of residue Ala-33 in K33A. Annotated on these
spectra are the NOE assignments. The Ala-33 peak in panel C
contains the same sequential and long range NOEs as Lys-33 but lacks
the intraresidue peaks of the Lys-33 side chain. This spectrum also
contains a new peak that is likely the methyl group of Ala-33.
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The HSQC spectrum shown in Fig. 5A suggests that the
replacement of Lys-33 by Ala was not totally benign. To show that the changes in chemical shifts were not indicative of a significant structural rearrangement of the K33A protein, a three-dimensional 15N1H HSQC-NOESY was acquired to detect changes
in interproton distances. A section of this spectrum, corresponding to
the amide proton of Ala-33, is shown in Fig. 5C. For
comparison the region of the same spectrum for residue Lys-33 in rDer p
2 is shown in Fig. 5B. Indicated on these spectra are the
NOE assignments from the previous structure determination. The same
sequential and long range NOEs were found in the spectra of rDer p 2 and the spectrum of the K33A mutant. The NOEs from residue Ala-33 that
are similar to those from Lys-33 are the sequential NOEs to the alpha
protons of residues 32 and 34 and the long range peaks to the methyl
protons of Val-94. Examining the NOEs from the new amide peak indicated that the differences in the spectra were the lack of intraresidue peaks
due to the Lys-33 side chain protons and a new peak that was likely the
methyl of Ala-33. These changes would be expected because of
replacement of Lys by Ala. An examination of the NOEs from Val-94 and
Gly-32 showed similar results; the pattern of NOEs did not change as a
result of the K33A mutation (data not shown). These results indicate
that there are no large structural changes induced by the K33A mutant;
thus, this residue is likely to be in direct contact with mAb 7A1.
 |
DISCUSSION |
To date, no data exist describing IgE Ab binding sites for any
allergen at the molecular level. The lack of allergen-specific monoclonal IgE Ab precludes epitope mapping using crystallographic analysis of allergen-antibody complexes. The NMR methods used here and
in other studies (11-13) have clearly shown that it is possible to
obtain an approximate mapping of epitopes using this technique. The
fidelity of the epitope map depends on several factors, the most
significant being the ability to assign resonances and follow the
exchange kinetics of these resonances. In the case of Der p 2 89 residues were assigned at pH 3.2. Of these, 31 residues exhibited
exchange rates that were either too rapid or too slow to evaluate for
protection. Although the NMR experiments were performed under
conditions that minimized amide exchange (pH 3.2) the time required to
prepare the sample and acquire the NMR spectra was significantly longer
than the half-life of the amide proton. This time allowed potentially
protected residues with very fast exchange rates in the free protein to
exchange with the deuterated buffer. Additional residues were removed
from the analysis if their amide hydrogens exchanged too slowly for a
significant difference in protection to be seen with the incubation
times used here.
Even in the context of these experimental constraints, the residues
that were protected are in close proximity to other residues that have
been previously implicated to influence the binding of that particular
mAb. For mAb 7A1, this study showed that residues 101, 97, and 94 were
protected whereas previous studies had shown that mutations of Lys-100
reduce the affinity of Der p 2 for mAb 7A1 (8). For the second mAb,
DpX, the protected residues were 72, 73, and 75. Residue Cys-73
forms a disulfide bond with Cys-78, and mutants that lacked this
disulfide, as well as chemically reduced Der p 2, showed reduced
affinity for
DpX and IgE Ab (6, 7). The final mAb examined was 6D6,
which belongs to a group of mAb that recognizes different naturally
occurring Der p 2 isoforms at position 114, either asparagine or
aspartic acid (9, 10). The protected residues for mAb 6D6 were 111 and
116. Interestingly, mutation of Lys-100 to alanine had no effect
whereas previous studies showed that mutation to threonine did affect
7A1 binding (8), although, in the latter study, mutation to arginine
had no effect. Other studies also suggest that different mutations of
the same residue can have varying effects on antibody binding (24,
25).
Because the protection data for all three mAbs correlate well with
previous mutagenesis data it is likely that the protected residues are
within the structural epitope. Finding only 2-3 residues with
different exchange rates in Der p 2 is consistent with studies in other
systems. For example, in a study of exchange rate differences for three
mAb binding to lysozyme, 2-5 residues within the structural epitope
for a given mAb were protected (12). Other studies that used this
method to localize mAb epitopes generally compared exchange rates for
free antigen versus antigen protected by mAb (11, 13). Based
on those results, we chose to only measure the differences in amide
exchange after 48 h to rapidly assess where the mAb might bind.
This time was chosen, because it resulted in a large difference between
the strongly protected residues that are contained in the epitope
versus residues with different exchange rates due to long
range conformational changes (12, 13). A more extensive examination of
exchange rates with more time points may reveal other protected
residues. Although the results shown in Fig.
1, panel A show no competitive
inhibition (i.e. no outright blockage) of 2B12
binding by the 7A1 and
DpX mAb, it is still possible that the
affinity of one mAb could be affected by the presence of another mAb,
although there is no evidence for such in this study or others
previously reported (17) in which reciprocal inhibition studies were conducted.
It is possible that binding of mAb to Der p 2 causes a conformational
change in a region recognized by another mAb. Indeed, using three mAb
specific for the antigen hen egg white lysozyme, we have shown that
upon binding, conformational effects are observed at sites distal to
the epitope (12, 13). However, these conformational effects are not
sufficient to prevent binding of another mAb at the distal site. In
that case and in the present study, if there are long range changes in
amide protection, i.e. protected residues remote
from the bound antibody that are within the epitope of another
antibody, the affinity must be affected on the basis of thermodynamic
arguments. However, it is impossible to predict the size or direction
of the change from the current data, but they cannot be larger than the
typical remote protection factors of about 1-2 kcal/mol (12, 13).
Although we have not conducted experiments with Der p 2 similar to the
lysozyme experiments, one would expect to see similar results,
i.e. long range conformational effects.
Nevertheless, also similar to the studies in the lysozyme-anti-lysozyme system, it is clear from the results shown in Fig. 1 (panel
A) and from studies previously reported on mAb binding to Der p 2 (17) that the failure of the 7A1 and
DpX mAb to inhibit 2B12 binding
cannot be accounted for by conformational effects. This strongly
supports the conclusion that inhibition by the other mAbs is due to
binding at the same site as the 2B12 mAb. The same arguments apply
directly to the data on mAb inhibition of binding of IgE from patient
serum, whether it be from one patient or a pooled serum from several
patients. Binding of a mAb inhibits the binding of any IgE that binds
to the same, or overlapping, epitope(s). It is important to recognize
that binding of a single mAb can significantly reduce total patient IgE
binding even when the patient IgE can be expected to have maximum
heterogeneity as in the case of the pooled sera.
To further characterize the epitopes localized by the exchange data, we
utilized the known buried surface area of other protein-antibody complexes to predict which residues may be contained within epitopes on
Der p 2. A potential difficulty associated with this method of
identifying the structural epitope is illustrated for mAb
DpX. Previous mutagenesis studies implicated residues 44-46 in the binding
of
DpX to Der p 2 (8). These residues are 15-19 Å from the
protected residues and are thus well outside the predicted epitope. It
is possible that the residues protected by mAb
DpX (amino acids 72, 73, and 75) are on the opposite side of the epitope from residues
44-46, and the true center of the epitope lies between these two
clusters. A more likely possibility is that alteration of residues
44-46 resulted in significant changes in the conformation of Der p 2. In the original study (8) these residues were changed from NQN to HPP.
The 44-46 HPP mutant reacted poorly with both
DpX and 7A1, as well
as with the other two mAbs studied. The epitopes for the
DpX and the
7A1 antibodies are shown here to be quite distant. This implies that
the triple mutation likely affects the structure of Der p 2 and that
residues 44-46 do not directly contact
DpX. An evaluation of the
structure of the HPP mutant by another technique, such as NMR, would
help resolve this issue.
Although all three of the mAb studied here will bind simultaneously to
Der p 2, the predicted epitope for mAb
DpX overlaps considerably
with the predicted epitope for mAb 6D6. Because both mAb can bind Der p
2 at the same time (17), the residues that are protected must not be
central to the epitopes. This gives the impression that the mAb
DpX
epitope is rotated away from the protected residues of mAb 6D6 or
vice versa. In the case of lysozyme, two mAb were shown to
interact with the same residues but with different atoms, and the two
mAb could simultaneously bind the antigen (26). These results provide
the precedent that epitopes of mAb 6D6 and mAb
DpX could be closely
associated and still simultaneously bind Der p 2.
To test the predicted epitope of mAb 7A1, single alanine mutants were
generated, and the contribution of their side chain to the free energy
of mAb binding was determined. Alanine scanning mutagenesis has been
used to examine the energetic contribution of side chains to antibody
interactions for human growth hormone, Staphylococcal nuclease, and
lysozyme (14-16). These studies found that the 
G values ranged
between 1 and 4.5 kcal/mol, but the majority of residues showed 
G
between 1.0 and 2.0 kcal/mol. Residues with 
G values in the
neighborhood of 1.0 kcal/mol or greater were considered to be
functionally important. In each case, only 2 to 5 residues met this
criterion for any given antibody. The energetically significant
residues were found to be discontinuous with respect to the primary
sequence. In our study a total of 7 residues showed changes in 
G
greater than 0.5 kcal/mol. Of these, 5 would be considered to be
functionally important for mAb binding based on the criteria stated
above. In studies of this type it is essential to show that no large
changes have occurred in the structure because of the alanine mutation.
In this study one mutant, K33A, was chosen for a more detailed
structural analysis, because it showed a large difference in 
G,
and the chemical shift of its amide is resolved in NMR spectra.
Although there are a few minor perturbations in the spectra, the NOE
analysis indicated that no significant changes occurred because of the substitution of the alanine at position 33 in the uncomplexed Der p 2. In addition, the mutagenesis data reported here are confirmed by a
recent report on the effect of mutagenesis of the Der f 2 allergen on
binding of two mAb that see the same antigenic regions as do the 7A1
and
DpX (27).
There are two possible explanations for the observation that changing
Asn-93 to an alanine significantly affects binding of both the 7A1 and
DpX mAb. First, Asn-93 may indeed have been recognized by both mAb
although competitive inhibition showed that cross-inhibition did not
occur. The crystal structures of two lysozyme-anti-lysozyme mAb
complexes showed that the two mAb could bind to the same residue,
albeit at different atoms, and yet not competitively inhibit (26).
Second, we have shown that binding of mAb at one region can cause
conformational effects elsewhere in the antigen molecule (12, 13). The
side chain of Asn-93 forms two hydrogen bonds with the main chain
nitrogen and oxygen of Val-94. These same two Val-94 atoms also
form hydrogen bonds with main chain atoms of Lys-33 for which
independent mutational evidence suggests it is a binding residue. Thus
mutation of Asn-93 to alanine could indirectly affect 7A1 binding
through Lys-33.
Considering the number of residues found to be protected from exchange,
the number of residues found to be energetically important, and the
distribution of these residues, Der p 2 and the mAbs that interact with
it fit the general paradigm of protein-antibody interactions. The
energetics of the interaction of Der p 2 with mAb 7A1 is similar to
that found with other antigen-antibody complexes; only a small number
of discontinuous residues contribute significantly, as individual amino
acids, to the binding affinity. These results will provide insights
into the molecular nature of the epitopes of this clinically important
antigen and will serve as a basis for the generation of modified forms
of Der p 2 for immunotherapy.
Of particular importance to the use of sequence variants in
immunotherapy are our results comparing the reactivity of patient IgE
with natural and rDer p 2 (6, 9). These studies showed that the IgE of
some patients reacted with natural and not with rDer p 2. Natural Der p
2 is a mixture of isoforms that differ at positions 40, 47, 127, and
114 (28). The rDer p 2 used in these studies corresponds to the rDer p
2.0101 isoform that carries aspartic acid at position 114. Subsequent
experiments have clearly shown that native reactivity could be restored
by changing position 114 of the rDer p 2 to asparagine, the amino acid
found in the other Der p 2 isoforms. Taken together, these results
strongly suggest that the patient sera that did not react with rDer p
2.0101 were specific for a single site that included position 114. In addition, the results presented here suggest that the patient IgE Ab
recognized a limited number of epitopes on Der p 2, in that 50% of IgE
reactivity could be inhibited by a single mAb (Fig. 1). Overall, these
results suggest that Der p 2, as seen by at least a subset of patients,
could be considered monovalent (a single epitope) or paucivalent (a few
epitopes), rather than polyvalent. Thus, selected amino acid changes
within each known epitope may produce a null allergen permitting its
use in immunotherapy without fear of hyperreactivity because of
preexisting IgE Ab. All changes required to produce this null allergen
must be made on the same molecule. If a separate variant allergen was
produced for each epitope and a mixture of these variants used for
immunotherapy, each variant in the mixture would possess all the other
native epitopes, and although the overall concentration of each epitope would be reduced, the mixture would react with preexisting IgE with the
risk of inducing hypersensitivity reactions during immunotherapy.