Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada
1 To whom correspondence should be addressed. e-mail: ymine{at}uoguelph.ca
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Abstract |
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Keywords: food allergy/IgE epitope mapping/ovalbumin/peptide synthesis/SPOTs assay
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Introduction |
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It has been reported that treatment of ovalbumin with carboxymethylation, 6 M urea or heating at 95°C had no significant effect on its binding capacity to human IgE derived from egg-allergic patients sera (Mine and Zhang, 2002). This indicates that anti-ovalbumin IgE antibodies from egg-allergic patients recognize mainly sequential epitopes. The IgE class of antibodies for ovalbumin also have been shown to exist in sera from patients with allergy (May et al., 1977
). Epitope mapping of specific regions in ovalbumin have been done by several groups using enzyme-digested fragments and synthetic peptides (Elsayed and Stavseng, 1994
; Masuda et al., 2000
). The studies on IgE antibody responses to ovalbumin have been shown earlier (Elsayed et al., 1991
; Kahlert et al., 1992
). Protein structure also plays a critical role in determining the immunodominant IgE-binding epitopes and there seems to be a link between the allergen structure and the IgE-binding epitopes (Shimojo et al., 1994
; Sen et al., 2002
).
Although a few IgE epitopes have been identified earlier, there are no obvious sequence motifs shared by these epitopes. In the present study, we have determined the entire mapping of the IgE-binding epitopes in the primary sequence of ovalbumin, and the critical amino acids within each of the IgE-binding epitopes of ovalbumin that are important to IgE binding were identified. Interestingly, the substitution of a single amino acid within each of the peptides led to either reduction or loss of IgE binding. In some cases, substitution led to an increase in IgE-binding activity. This information is important to design diagnostic and therapeutic approaches to reduce the risk of the food hypersensitivity caused by ovalbumin.
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Materials and methods |
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The individual peptides were synthesized on a derivatized cellulose membrane using Fmoc amino acid active esters (SPOTs synthesis) according to the manufacturers instructions (Sigma Genosys Biotechnologies, Woodlands, TX). The synthesis of peptides using the SPOTs technique was designed based on the primary amino acid sequence of ovalbumin (Nisbet et al., 1981). Synthesis of each peptide began by esterifying a Fmoc amino acid to the cellulose membrane derivatized with a dimer of a ß-alanine-NH2 group and was monitored at certain stages using colour development of bromophenol blue. In short, the primary sequence was synthesized so that it contained synthetic peptides that were 12 amino acids in length and offset by two amino acids. Coupling reactions were followed by acetylation with acetic anhydride (4%, v/v) in N,N-dimethyformamide to render peptides unreactive during the subsequent steps. After acetylation, Fmoc protective groups were removed by the addition of piperidine to render nascent peptides reactive. The remaining amino acids were added by this same process of coupling, blocking and deprotection until the expected desired peptide was generated. After addition of the last amino acid in the peptide, the amino acid side chains were deprotected using a solution of dichloromethanetrifluoroacetic acidtriisobutylsilane (1:1:0.05, v/v/v) and washed with methanol. Membranes containing the synthetic peptides were either probed immediately or stored at 20°C until needed (Frank and Overwin, 1996
).
IgE-binding assay
The cellulose membranes containing the synthesized peptides were washed with Tris-buffered saline (TBS) and blocked overnight with the blocking buffer (Sigma-Genosys Biotechnologies) with 5% (w/v) sucrose. After blocking, the membranes were washed with TBST (50 mM Tris, pH 8.0, 136 mM NaCl, 2.7 mM KCl and 0.05% Tween-20) for 10 min and incubated with three pooled sera, each consisting of six egg-allergic patients sera, for 4 h at 1:20 dilution at room temperature. The membranes were washed with TBST and incubated with monoclonal anti-human IgE alkaline phosphatase conjugate (Sigma, St. Louis, MO; 1:2000 dilution) at room temperature for 2 h. After incubation, the membranes were washed with TBST and the bound antibodies were detected using the chemiluminescent substrate CDP-Star (Boehringer Ingelheim, Mannheim, Germany) and enhancer Nitro-Block II (Tropix, Bedford, MA) both diluted to 1:100 with 0.1 M TrisHCl, 0.1 M NaCl, pH 9.5. The membrane was visualized under a Molecular Light Imager (EG & G Berthold, Bad Wildbad, Germany). The quantified signal of each spot was obtained using Win Light software (EG & G Berthold) and the value was expressed as relative percentage of signal intensity.
Analysis of the critical amino acids
The amino acids critical to each ovalbumin IgE-binding site were identified by substitution of a single amino acid at each position in the epitope by synthesizing multiple peptides. Peptides were synthesized on a SPOTs membrane with each amino acid sequentially replaced with either alanine (A), glycine (G) or glutamine (Q) and probed as described in the previous section.
Regeneration of SPOTs membrane
The membrane was regenerated according to the manufacturers instructions (Sigma-Genosys Biotechnologies) by washing with regeneration buffer A [8 M urea, 1% (w/v) sodium dodecyl sulphate, 0.1% (v/v) ß-mercaptoethanol], followed by washing with regeneration buffer B [50% (v/v) ethanol, 10% (v/v) acetic acid]. The membrane was then washed with methanol and dried and stored at 30°C. Regenerated membrane was used for evaluating human IgE-binding activity with different pooled sera from egg-allergic patients.
Human serum
Human serum was collected from 18 patients exhibiting allergies to egg white. The RAST scores were determined according to the procedures of the manufacturers kit (Phadebas RAST kit; Pharmacia Diagnostics, Tokyo, Japan). Table I gives a brief summary of each patients clinical history. The serum samples were kindly provided by Dr A.Urisu (Department of Pediatrics, Fujita Health University, Japan) and Dr Morikawa (Department of Pediatrics, Gunma University, Japan) and stored at 80°C until use. The serum samples were pooled into three groups consisting of six patients sera each for the experiments. A pool of three non-allergic individuals sera was also used as a control. Sera from food hypersensitivity with positive double-blind, placebo-controlled food challenges (DBPCFC) to egg white were used in this study.
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Data were analysed by using analysis of variance (ANOVA-SPSS version 7.5 for Windows; SPSS, Chicago, IL) and the level of significance was defined at P < 0.05.
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Results |
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One hundred and eighty-seven overlapping peptides were synthesized on cellulose membranes to determine which regions of the primary sequence of ovalbumin protein were recognized by allergic patients sera. Each synthesized peptide consisted of 12 amino acids and was offset by two amino acids from the previous peptide. This approach led to the epitope mapping of the IgE-binding region of the entire ovalbumin sequence. Prior to testing the membrane with egg-allergic patients sera, it was incubated with non-allergic control sera to evaluate non-specific antibody binding (data not shown). Figure 1a shows a typical SPOTs membrane probed with the pooled serum of group 1. Spot numbers 20, 119122 and 126 showed strong signals, while numbers 4648 and 9596 showed lower intensity. Two groups of pooled sera were used in this manner and the average spot intensities are summarized in Figure 1b. Five prominent epitope regions were identified. The IgE-binding regions were represented by amino acid residues 3849, 95102, 191200, 243248 and 251260. Epitopes 15 were designated as L38T49, D95A102, E191V200, V243E248 and G251N260, respectively. Figure 2 corresponds to the entire primary sequence of the ovalbumin allergen where the epitopes can be seen to be distributed along the entire length of the sequence that is recognized by the sera obtained from patients with egg hypersensitivity.
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The amino acids that were critical for IgE binding to ovalbumin were determined by synthesizing multiple peptides with a single amino acid mutation at each IgE-binding site. Each amino acid in each of the five epitopes was sequentially replaced by alanine (A), glycine (G) or glutamine (Q) depending on the amino acid composition of the epitope and re-proved with human sera from egg-allergic patients. Figure 3 represents an immunoblot strip containing the wild-type and mutant peptides for each IgE epitope after being probed with pooled sera group 1. Two other serum pools exhibited a similar pattern of spots and the average signals of three groups are shown in Figure 3b. Binding to epitopes, L38T49, D95A102, E191V200 and V243E248 were strictly regulated by human IgE antibodies, since a single mutation caused complete loss of IgE binding and no visible spots were detected except with the signal of A102G. The epitope G251N260 showed a different pattern from the others. In some cases (G251A, L252A and E253A), the amino acid substitutions were found to significantly increase binding, while in other cases (L255A, E256A, S257A and I258A), they were found to significantly reduce or completely eliminate IgE binding to the epitope. Amino acids within each of the IgE epitopes of ovalbumin that are critical to IgE antibody binding were identified in this manner and are summarized in Table II. The amino acids of all five epitopes were then classified as either hydrophobic, polar or charged residues and enumerated (Figure 4), and the properties of those amino acids considered to be critical were examined.
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Discussion |
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Five distinct IgE-recognition sites were identified in the primary sequence of the ovalbumin protein by the pooled sera obtained from 18 patients with egg hypersensitivity and these immunodominant epitopes seem to be distributed throughout the ovalbumin primary sequence. There have been several reports on IgE-binding sites of ovalbumin using synthetic peptides or chemical cleavage by means of western blots and 2D-PAGE quantitative precipitation inhibition assay, including amino acids 110 (Elsayed et al., 1988), 323339 (Johnsen and Elsayed, 1990
), 3446, 4755 (Elsayed and Stavseng, 1994
), 41172 and 301385 (Kahlert et al., 1992
). Furthermore, detailed IgE epitope mapping in the C-terminal region comprising residues 347385 of ovalbumin was carried out using immunoblot and histamine release assays (Honma et al., 1996
). The amino acid residues 347366, 347385, 357366, 357376 and 367385 were identified as allergenic epitopes of ovalbumin (Honma et al., 1996
). The present sequential IgE epitope mapping results seem to be different from earlier results. We could not identify any IgE-binding sites in the N- or C-terminal regions where previous workers have reported them in the sequence of 119 and 347385. The differences in these results may be attributed to a difference in the patient populations employed in each study. Elsayed and Stavseng (Elsayed and Stavseng, 1994
) selected three of 16 patients sera due to their total IgE and RAST class, and pooled sera were used. Furthermore, earlier studies (Elsayed et al., 1988
; Kahlert et al., 1992
) did not provide sufficient clinical information about the patients serum used. Three patients who showed RAST scores for egg white class 3 were used for C-terminal IgE-binding site mapping (Honma et al., 1996
). Therefore, the general applicability of the previous work may limit comparison to the present results which were obtained from three pooled groups of six patients sera each, all having egg allergy.
It is well known that 4060% of egg-hypersensitive patients outgrow the allergy (becoming clinically tolerant) with age (Bock and Atkins, 1990
). The outgrowing of hypersensitivity is observed even in individuals maintaining high IgE antibodies and a positive skin response to egg white. More recently, it has been shown that six epitopes in cows milk proteins (
s1-casein,
s2-casein and
-casein) were not recognized by IgE antibodies from any patients with transient milk allergy, but showed binding by the majority of the patients with persistent allergy (Vila Sexto et al., 2001
; Järvinen et al., 2002
). This might reflect different epitope recognition of antigens between patients with persistent egg allergy and those who have outgrown their egg allergy. Although no studies have been done comparing the allergens or IgE-binding epitopes for individuals with persistent egg allergy versus those who have outgrown their egg allergy, serum from both patients with respect to levels of egg-specific IgE and the epitopes on egg allergens may be different. This could offer a possible explanation about some of the differences in epitopes that were mapped in this study and previous studies where this matter was not paid any attention. IgE-binding epitopes in ovalbumin could be different between patients with active egg allergy and those who have outgrown egg allergy. This is of great interest in predicting the persistence of egg allergy. Recently, a newer radio-allergo-sorbent test (CAP-RAST) system has been employed and its values correlated with the probability of positive DBPCFC (Sampson and Ho, 1997
; Sampson, 2001
). In this study, the Phadebas RAST test, as well as earlier work, was employed for screening human sera from egg allergy, and oral challenge testing was conducted. However, studies comparing Phadebas RAST IgE concentrations with CAP-RAST IgE were not done. Further investigation using a large number of patients sera with sufficient characterization of the egg-allergic individuals could provide more reliable information to address these conflicts.
The substitution of a single amino acid was accomplished by synthesizing multiple peptides with a single amino acid mutation in each to determine the critical amino acids involved in IgE binding. We observed that the substitution of a single amino acid in some cases led to the loss of IgE-binding activity, in some cases decreased the activity and in a few cases increased the IgE-binding activity. The IgE epitopes L38T49, D95A102, E191V200 and V243E248 showed strictly regulated epitope structure because single amino acid substitution throughout the sequence, except A102, caused a complete loss of IgE-binding activity. In contrast, only three amino acids in the G251N260 epitope (10 amino acids in length) were identified as critical, indicating that this IgE epitope antibody binding is less stringent than others. Some substitutions, however, were found to enhance human IgE binding. The substitution of G251, L252 and E253 with alanine (A) in epitope G251N260 produced noticeably darker spots (Figure 3) than with the native signals, indicating an increase in human IgE binding. This apparent increase in IgE binding may be due to an increase in the similarity of peptide secondary structure, which mimics its original structure in the entire ovalbumin molecule. This suggests that the synthesized epitope (peptide) structure is not always completely identical to that in the native protein structure. This should be studied in detail in the future to address the structural homology between synthesized peptides and native protein, and how each may relate to allergic reactions.
The amino acids of the epitopes were classified as hydrophobic, polar or charged (Figure 4). There were a total of 46 amino acids in all of the sequential IgE epitopes. Hydrophobic residues were found to be the most frequently occurring (25/46), followed by polar residues (11/46) and then charged residues (10/46). Of these polar residues, aspartic acid (D), glutamic acid (E), lysine (K) and arginine (R) appeared to be more important. Most of the critical residues belonged to the hydrophobic group, followed by polar and charged residues. In fact, relative epitope hydrophobicities, as determined by the method of Kyte and Doolittle (Kyte and Doolittle, 1982), identified all five IgE epitopes as relatively hydrophobic regions of the protein (Figure 5). Therefore, the ovalbumin IgE epitopes identified in this study tend to consist mainly of hydrophobic residues, but charged residues (D, E, K and R) seem to have an important role.
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Acknowledgements |
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Received January 9, 2003; revised April 23, 2003; accepted August 28, 2003.