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* Allergy Immunology Division, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226;
Zablocki Veterans Affairs Medical Center, Milwaukee, WI 53295; and
Northwestern University Medical School, Chicago, IL 60611
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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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Our previous study on structure function relationship of Asp f 2 demonstrated the presence of continuous as well as discontinuous IgE binding epitopes in Asp f 2 (10). The immunologic evaluation of Asp f 2 using overlapping synthetic peptides revealed the presence of nine IgE binding epitopes varying in length from 3–7 aa. The study also revealed that either -N- or -C-terminal region of the protein is essential for allergen IgE interaction since the recombinant construct without -N- and -C-terminal regions failed to bind the IgE Ab. Incorporation of either of the terminal regions of Asp f 2 to the same construct resulted in regaining the IgE Ab binding capacity. As the identification of the structural entities on the allergen molecule that interact with IgE Ab is an essential prerequisite for the development of effective therapeutic strategies, we have investigated further the role of cysteine residues of Asp f 2 in allergen IgE interaction.
The primary structure analysis of Asp f 2 revealed the presence of eight cysteine residues throughout the molecule. To study the involvement of cysteine residues in the secondary structure as well as in IgE binding of Asp f 2, we devised a number of deletion and point mutants by manipulating various cysteine residues especially near the C-terminal region of the protein. Several mutants were constructed by adding C-terminal sequences including three C-terminal cysteines C204, C257, and C267 one at a time to the non-IgE binding recombinant construct Asp f 2 68–203(68–203) to evaluate the contribution of these residues in the conformational epitope of Asp f 2. The distinct differences in IgE binding of Asp f 2 68–266(68–266) and Asp f 2 68–267(68–267) identified C267 as an essential amino acid for Asp f 2 Ab interaction. The immunologic evaluation of point mutants of Asp f 2 68–268(68–268) at C204A and C257A also indicated the involvement of C204 in IgE binding with sera from Asperigillus-sensitized patients. Moreover, immunologic evaluation of reduced and alkylated Asp f 2 and its mutants suggest that disulfide bonds between certain cysteine residues may be critical for allergen IgE interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three groups of subjects were included in this study. Serum samples (n = 10) from patients with allergic bronchopulmonary aspergillosis (ABPA) were collected at the Allergy-Immunology Clinics of Medical College of Wisconsin affiliated Hospitals (Milwaukee, WI) and Allergy-Immunology Clinic of Northwestern University Medical School (Chicago, IL). These patients fulfilled the criteria for ABPA as described by Rosenberg et al. (11, 12). The serum samples from 10 patients with asthma and skin prick test positivity to a mixture of Aspergillus extracts but without clinical features of ABPA, as well as 10 normal subjects, were also used in this study. The institutional review committee approved the human study.
Expression of Asp f 2 and its deletion mutants in pET expression system
To express the recombinant constructs in prokaryotic system
along with C-terminal His tags, the PCR-amplified products carrying
various gene fragments were cloned and expressed in pET 23
(b+) vector as previously described
(9). The primers used for the PCR amplification of
deletion mutants and the complete Asp f 2 were shown in Table I
. The recombinant constructs were
affinity purified using Ni2+-nitrilotriacetic
acid agarose column. The purified proteins were analyzed on SDS-PAGE
and stained with Coomassie brilliant blue R250 as previously described
(6).
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The gene encoding the extreme N-terminal sequences (67 aa) and
C-terminal sequences (81 aa) of Asp f 2 were expressed as recombinant
fusion proteins in pMALc2 vector. The primers were designed with
ECOR1/HindI11 restriction sites at 5' and 3' ends of Asp f 2
1–67(1–67) and ECOR1/PST 1 restriction sites at 5' and 3' ends of Asp f 2
188–268(188–268) as shown in Table I
. Escharichia coli (TB1) was
transformed with pMALc2 vectors with or without inserts. The
recombinant Asp f 2 constructs expressed along with MBPs were affinity
purified using amylose resin. The protein bound to amylose resin was
eluted using 10 mM maltose and maltose-binding protein (MBP) without
the fusion component was used as a control in ELISA
(13).
Construction of Asp f 2 point mutants
Two point mutants of Asp f 2 68–268(68–268) with C204A and C257A were prepared using the QuikChange site directed mutagenesis kit from Stratagene (Stratagene, La Jolla, CA). The plasmids isolated from the colonies were subjected to automatic DNA sequencing. All the colonies exhibited incorporation of alanine in place of cysteine at their respective positions. The clones with mutated gene fragments were overexpressed in pET system using the same protocol as described above (9).
Ag-specific ELISA
The binding of specific IgE and IgG Ab from the sera of Asperigillus-sensitized patients and normal controls to various deletion and point mutants of Asp f 2 was analyzed by an indirect ELISA (7).
Inhibition ELISA
To study the structural similarities between various recombinant constructs and Asp f 2, an ELISA inhibition study was conducted using pooled serum samples from patients with ABPA. The serum samples were diluted 1/10 and incubated with decreasing concentrations of 50 µg to 10 ng of different deletion and point mutants of Asp f 2. The preincubated samples were centrifuged and added to the washed and blocked Asp f 2-coated plates. Further steps in the assay were conducted with reagents as described before (10). The dose-dependent decrease in Ab titer of preincubated serum was determined from the ELISA absorbance values in comparison with uninhibited serum.
SDS-PAGE and Western blots
Purified recombinant constructs (5 µg/lane) were resolved on 15% SDS-PAGE gel (7, 14). The resolved proteins were stained with Coomassie brilliant blue R250. The SDS-PAGE-separated proteins were then transferred on to nitrocellulose membrane (Schleicher and Schuell, Keene, NH) by using transblot apparatus (Bio-Rad, Hercules, CA) as previously described (14). After blocking, the membranes were incubated separately with pooled sera from patients with ABPA, patients with allergic asthma, and normal subjects at a dilution of 1/10 in blocking buffer. After overnight incubation at 4°C, the strips were treated with biotinylated goat anti-human IgE (1/1000) and incubated for 1 h. The addition of streptavidin peroxidase and color development was conducted as previously described (14).
Determination of Kd of IgE allergen interaction by ELISA
A quantitative estimation of Kd
was conducted as described previously (15). Briefly, ELISA
plates were coated with different concentrations (1 nM to 5 µM) of
Asp f 2 and its various mutants in PBS (pH 7.4) overnight at 4°C.
After blocking with 1% BSA in PBS, pooled serum sample from patients
with ABPA (1/25) was added to the plates and incubated for 3 h at
room temperature. The following procedures of addition of biotinylated
anti-IgE Ab and detection of Ag-specific Abs in the plate using
streptavidin peroxidase were the same as mentioned above. The resulting
absorbance values at 490 nm were used to determine the Ab-bound
fraction 
. Using Klotz equation, a plot of 1/ and
1/(Ag)t was obtained where was the fraction
of Ab that was bound and (Ag)t was the total Ag
concentration (15). Kd
values were determined from the slope of the plot.
Immunologic evaluation of reduced and alkylated Asp f 2 and its mutants
To study the involvement of free cysteines and the disulfide bond between two cysteines in IgE Ab binding, wild protein and the mutants were first denatured with 8 M urea to expose all the free sulfhydryl (SH) groups and disulfide bonds buried within the protein for further reactions. A set of denatured samples were then subjected to reduction using 20 mM DTT as described previously (16). The denatured samples without DTT treatment were designated as (-DTT) samples and after DTT treatment as (+DTT) samples. For modifying the thiol groups that were available in (-DTT) and (+DTT) samples, these were treated separately with 250 mM iodoacetamide and iodoacetic acid as detailed elsewhere (16). The modified protein samples were then resolved in a nondenaturing gel and visualized by the differences in the mobility of iodoacetic acid and iodoacetamide-treated samples. The (-DTT) and (+DTT) samples were then evaluated further for their IgE Ab binding using ELISA (9).
Statistical analysis
The mean OD values of Ag Ab reactions by ELISA using serum samples from two different subject groups were compared with those of the control group by using Student’s t test (two-tailed independent samples) with Statworks program (Cricket Software, Philadelphia, PA). Values for ABPA, asthma, and control groups were compared and p < 0.05 was taken as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and B
describes the different constructs that were used in this study.
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To determine whether various fragments were capable of binding to
IgE, a solid-phase ELISA was carried using 10 individual sera from
patients with ABPA. As shown in Fig. 2A, fragment Asp f 2 68–203(68–203)
without C14, C204,
CC257, and C267 failed to
show IgE Ab binding. However, Asp f 2 1–203(1–203) with an additional
C14 at the N-terminal region and Asp f 2
68–268(68–268) with the remaining C-terminal cysteine residues,
C204, C257, and
C267 exhibited IgE Ab binding comparable to the
full-length allergen Asp f 2 1–268(1–268). The mean ELISA IgE absorbance
(OD490) for Asp f 2 1–203(1–203) and Asp f 2
68–268(68–268) was 0.688 ± 0.776 and 0.610 ± 0.826,
respectively, compared with 1.42 ± 1.43 for Asp f 2 1–268(1–268).
However, sera from patients with asthma and normal controls failed to
show significant IgE binding with Asp f 2 and its mutants by ELISA
(data not shown).
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To determine whether C14 alone or the three
C-terminal cysteines C204,
C257, and C267 together
could provide the essential conformation of IgE Ab binding, we designed
two small fragments, Asp f 2 1–67(1–67) with C14 and
Asp f 2 188–268(188–268) with all the three C-terminal cysteines. The IgE Ab
binding of these constructs with sera from 10 ABPA patients are shown
in Fig. 2B. Of the two fragments, only Asp f 2 188–268(188–268)
exhibited ELISA results comparable to the mature allergen, whereas Asp
f 2 1–67(1–67) failed to show IgE Ab binding with ABPA sera. The mean
ELISA absorbance for Asp f 2 188–268(188–268) was 0.778 ± 0.963
compared with 0.934 ± 0.615 for the full-length allergen. This
finding clearly indicates that certain conformation or the tertiary
structure of the allergen is essential for the allergen IgE Ab
interaction. In contrast, IgG Ab binding of the various deletion and
point mutants of Asp f 2 were comparable with the native allergen,
indicating that cysteine residues were not involved in epitope IgG
interaction.
C-terminal C267 is essential for the allergenicity of Asp f 2
To define the specificity of epitope Ab interaction near the
C-terminal region of Asp f 2, we devised three more constructs by
incorporating the remaining three cysteines at the C-terminal region,
C204, C257, and
C267 one at a time to Asp f 2 68–203(68–203) as
described above. The IgE Ab binding of various deletion mutants Asp f 2
68–255(68–255), Asp f 2 68–266(68–266), and Asp f 2 68–267(68–267) with serum samples
from 10 patients with ABPA are shown in Fig. 2C.
Deletion mutant Asp f 2 68–255(68–255) with five cysteines at the
C-terminal region failed to show IgE binding. A marked difference was
noticed in IgE binding of the deletion mutants Asp f 2 68–266(68–266) and
Asp f 2 68–267(68–267); addition of only one amino acid,
C267, resulted in a 4-fold increase in IgE
binding, indicating the involvement of C267 in
Asp f 2 Ab interaction. The statistical significance of IgE Ab binding
of Asp f 2 and its recombinant constructs with sera from ABPA patients
were compared with the Ab binding with normal subjects Fig. 2D. The values of p < 0.008 for Asp f 2 and
among other IgE binding C-terminal fragments, Asp f 2 68–268(68–268) and Asp
f 2 188–268(188–268), exhibited values of p < 0.048 and
p < 0.022, respectively.
Dissociation constant Kd IgE Ab interaction with Asp f 2 as determined by ELISA was 0.044 + 0.003 µM. The Kd values 0.04 + 0.004 and 0.027 + 0.0007 µM for Asp f 2 68–268(68–268) and C257A mutant of Asp f 2 68–268(68–268), respectively, were also comparable with the complete allergen (data not shown).
The IgE binding of these mutants under denaturing condition as
determined by immunoblots using pooled sera from patients with ABPA was
shown in Fig. 3. The mutants and the
complete allergen exhibited some reactivity under denatured condition
in Western blots. As seen in ELISA, the fragments Asp f 2 68–203(68–203) and
Asp f 2 68–255(68–255) failed to show IgE binding in blots, whereas there
was gradual increase in IgE binding from fragment Asp f 2 68–266(68–266) to
Asp f 2 68–268(68–268).
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Further analysis of the conformational similarity among various
recombinant constructs was conducted by competitive ELISA inhibition
assay (Fig. 4). Pooled serum from
patients preincubated with various concentrations of deletion mutants
were evaluated for IgE binding to recombinant Asp f 2. No inhibition of
IgE Ab binding to Asp f 2 was observed with serum samples incubated
with Asp f 2 68–203(68–203) and Asp f 2 68–255(68–255). In contrast, Asp f 2
68–266(68–266) exhibited 30% inhibition in IgE binding compared with 56%
inhibition by Asp f 2 68–267(68–267). Interestingly, the addition of
single cysteine residue at C267 imparted a
conformation more comparable to the complete Asp f 2 molecule. Hence,
indirect ELISA and competitive inhibition ELISA emphasizes the
importance of C267 in proper folding and
conformation of the allergen Asp f 2.
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To define the involvement of C204 and
C257 individually in IgE Ab binding, we
constructed two point mutants of Asp f 2 68–268(68–268) by replacing
C204 and C257 with alanine.
The mutant Asp f 2 68–268(68–268) with C204A exhibited the same molecular
size as Asp f 2 68–268(68–268) in the range of 29 kDa, whereas mutant Asp f
2 68–268(68–268) with A257C demonstrated a molecular size of 35 kDa (Fig. 5). On reducing gel, C257A mutant
exhibited a size almost 5–6 kDa higher than that of Asp f 2 68–268(68–268).
When both the mutants were evaluated for IgE binding with sera from 10
individual ABPA patients, the mutant A204C exhibited complete loss in
IgE-binding activity. The mutant C257A exhibited an ELISA absorbance of
0.648 ± 1.08 compared with 0.681 ± 1.036 for Asp f 2
68–268(68–268) as shown in Fig. 6A.
This result indicates that C204 is essential for
the allergen IgE interaction, whereas removal of
C257 failed to show any reduction in IgE Ab
binding. Akin to the results obtained in indirect ELISA, competitive
inhibition ELISA exhibited 74% inhibition in IgE binding by C257A of
the solid phase coated with Asp f 2 68–268(68–268) as shown in Fig. 6B, whereas A204C failed to show significant inhibition in
IgE binding.
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To address the issues of whether the free -SH groups of these cysteine residues or a particular conformational constraint due to disulfide bond formation between the cysteine residues were essential for Ab allergen interaction, we conducted a series of biochemical and immunologic analyses of Asp f 2 and its mutants.
Asp f 2 (-DTT samples) when subjected to -SH modification with
iodoactamide and iodoacetic acid failed to show any difference in the
mobility in gel (Fig. 7). However, Asp f
2 (+DTT samples) on similar alkylation exhibited distinct differences
in mobility in the gel confirming the presence of free -SH groups in
the reduced protein. In indirect ELISA, Asp f 2 (-DTT samples)
exhibited strong IgE binding with sera from patients with ABPA, and the
mean ELISA absorbance value (OD490, 2.20) was
comparable with absorbance for native Asp f 2
(OD490, 2.427) as shown in Fig. 8. In contrast, alkylated Asp f 2 (+DTT
samples) failed to show significant IgE Ab binding with almost 20-fold
reduction in absorbance compared with the native Asp f 2 and Asp f 2
(-DTT samples). Among the Asp f 2 mutants included in this study, Asp
f 2 68–267(68–267) and the point mutant C257A of Asp f 2 68–268(68–268) also
exhibited similar reactivity after denaturation and reduction with DTT
as the native Asp f 2 (Fig. 8). A significant loss in IgE Ab binding of
the Asp f 2 and its mutants after reduction and alkylation reaffirmed
the critical role of cysteine disulfide bonds in allergenicity of Asp
f 2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three-dimensional structure of Asp f 2 has not been known yet; however, the molecular cloning and sequencing has revealed the presence of eight cysteine residues throughout the molecule (9, 10). Asp f 2, when denatured with 8 M urea to expose the free thiol groups (if any) present in the native protein and further treated with thiol group modifying agents such as iodoacetic acid and iodoacetamide, failed to show any modification, thereby suggesting that all eight cysteines of Asp f 2 were present in disulfide bonds and no free thiol group was available or exposed to surface even after denaturation. At the same time, denatured proteins on alklylation with iodoacetic acid and iodoacetamide exhibited similar IgE binding as the native protein in ELISA. These two findings together suggest that free thiol groups may not be involved in the conformational epitopes of Asp f 2. In contrast, contribution of intrachain disulfide bonds between cysteine residues of Asp f 2 in IgE Ab binding was evident by significant loss in IgE binding of denatured as well as reduced allergen protein.
Akin to this finding, several earlier studies have reported that cysteine residues with sulfydryl groups in their side chains are involved in interchain or intrachain disulfide bond formation, and hence often play a crucial role in protein folding and conformation (17, 18). However, the IgE Ab binding of denatured and reduced Asp f 2 and its mutants in Western blots could be due to the residual Ab binding conformation remaining in the protein because of the incomplete denaturation and reduction, or due to the interaction of IgE Abs with epitopes involving amino acids other than cysteines.
The involvement of intrachain disulfide bonds in IgE binding has recently been reported for major allergens from different sources (19). Disulfide binds between cysteines in close proximity are predicted to be on the protein surface and hence available for Ab binding, whereas disulfide bonds between nonlocal cysteine residues appear to be involved in stabilizing the protein molecules (19, 20, 21). A recent study by Smith et al. (20) demonstrated that a small loop between C73-C78 is mainly involved in conformational epitope of Der p 2, since disruption of this disulfide bond resulted in complete loss of IgE Ab binding of the allergen. In a similar study on another mite allergen Lep d 2, the disulfide bond between C72-C77 appeared to be mainly involved in IgE allergen interaction (23). In our study, the IgE binding of the fragment Asp f 2 188–268(188–268) with C204, C257, and C267 indicate that intrachain disulfide loops between cysteine residues residing in close proximity might be involved in conformational epitopes of Asp f 2. Similar conformational constraints in IgE Ab binding was observed near the N-terminal region of Asp f 2. Deletion mutants Asp f 2 1–67(1–67) and Asp f 2 68–203(68–203) failed to show IgE Ab binding, while the mutant Asp f 2 1–203(1–203) encompassing five N-terminal cysteine residues exhibited comparable IgE binding with the complete Asp f 2 allergen. In contrast, deletion of cysteine residues residing far apart within the molecule such as in Asp f 3, another Af allergen, failed to contribute significantly in IgE binding. This indicates that small loops between cysteine residues within the allergen are probably involved in epitope paratope interactions (our unpublished observations).
To precisely delineate the role of these three individual cysteine residues in the allergen IgE interaction, our main approach was to construct various deletion mutants of Asp f 2 by incorporating one cysteine at a time in the fragment of Asp f 2 68–203(68–203). A high IgE binding of Asp f 2 68–267(68–267) compared with Asp f 2 68–266(68–266) indicated the direct involvement of C267 in the conformational constraints of Asp f 2. However, at this point, the involvement of C204 and C257 in conjunction with C267 in IgE Ab binding was not clearly defined. Hence, in the next set of experiments, two point mutants were constructed to examine whether C267 alone is sufficient to impart the proper conformation for IgE binding or C204 and C257 are also involved in the context of C267 for allergen IgE interaction. Of the two point mutants Asp f 2 68–268(68–268) with alanine substitution, C204A and C257A, respectively, mutant C204A failed to show IgE binding with patient’s sera, indicating that C204 along with C267 are essential for IgE Ab binding of Asp f 2. The involvement of disulfide bonds in IgE Ab binding of Asp f 2 as evidenced from the reactivity of DTT-treated protein also raises the possibility that C204 and C267 are engaged in disulfide bond in native allergen.
Interestingly, the point mutant C257A with comparable IgE binding as the wild construct exhibited an increase in molecular mass of 5–6 kDa in SDS-PAGE. As we used reducing gel, the size difference could be either due to the aggregation of the protein in gel or due to the formation of incorrect disulfide bond in the mutated protein (22).
The role of cysteine residues in IgE Ab binding was also reported for a
number of pollen allergens (23). Immunological evaluation
of various deletion mutants of a major pollen allergen Par j 1
demonstrated that cysteine residues at positions 14 and 29 were
essential for IgE binding of the protein. In fact, high-resolution
crystal structure studies have shown that the bond between cysteine 14
and 29 is one of the four essential covalent bonds conserved among
several homologous lipid transfer proteins from plant
(24). In contrast, studies conducted on various allergens
from different sources indicated that the involvement of cysteine
disulfide bonds in Ab binding are restricted only to certain allergens
based on their primary and secondary structures. As in the case of
major ragweed allergens Amb t 5 and Amb a 5, the contribution of
individual amino acids in Ab binding is evident by the antigenic
noncross-reactivity of these structurally similar allergens. These two
allergens with 45% sequence identity, conserved cysteine residues, and
similar 
-sheet and 
-helical structures carry distinct Ab-binding
epitopes, represented mainly by nonconserved amino acids of the
allergen (25, 26). Similarly, for Timothy grass pollen
allergen Phl p 5 b, the allergenic variant generated by
incorporating a cysteine residue near the N-terminal region of the
protein in addition to the wild-type cysteine residue present near the
C-terminal region failed to affect the IgE binding of the allergen,
indicating that the disulfide bond formation does not contribute to the
IgE-binding property of Phl p 5b (27).
In summary, this study demonstrates the contribution of cysteine residues as well as the conformational constraints due to disulfide bonds between cysteine residues to the allergenicity of Asp f 2. In addition, almost 100-fold reduction in IgE binding of recombinant fragment Asp f 2 68–203(68–203) and point mutant C204A suggests that targeted substitution or deletion (cysteine or other surface-exposed amino acids) could result in nonanaphylactic allergenic variants which may in turn be useful for safer immunotherapy. In our recent study using T cell clones generated from ABPA patients, immunodominant T cell epitopes were identified near the N-terminal region of the allergen which were distinct from IgE-binding epitopes (28). Therefore, the hypoallergenic variants of Asp f 2 generated and characterized in this study may be explored further in place of T cell peptides as multiple T cell epitopes with different MHC restrictions to be recognized by a number of patients as mode of therapeutic strategy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Banani Banerjee, Zablocki Veterans Affairs Medical Center, Research Service 151-I, 5000 West National Avenue, Milwaukee, WI 53295. E-mail address: banerjee{at}mcw.edu
3 Abbreviations used in this paper: Af, Aspergillus fumigatus; ABPA, allergic bronchopulmonary aspergillosis; MBP, maltose-binding protein; SH, sulfhydryl.
Received for publication May 29, 2002. Accepted for publication September 12, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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