The Journal of Allergy and Clinical Immunology
Volume 112, Issue 2 , Pages 433-437, August 2003

Mutational analysis of major, sequential IgE-binding epitopes in αs1-casein, a major cow's milk allergen☆☆☆

Division of Pediatric Allergy and Immunology and the Jaffe Institute for Food Allergy, Mount Sinai School of Medicine. New York, NY

Received 28 January 2003; received in revised form 27 March 2003; accepted 10 April 2003.

Article Outline

Abstract 

Background: Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising. Objective: We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of αs1-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity. Methods: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera. Results: With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition. Conclusion: This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity. (J Allergy Clin Immunol 2003;112:433-7.)

Keywords:  B-cell epitope, αs1-casein, children, cow's milk allergy, immunoglobulin E, linear, mutational analysis, SPOTs membrane

Abbreviations:  AA , Amino acid, CMA , Cow's milk allergy, OD , Optical density

 

Cow's milk is usually 1 of the first foods incorporated into the human diet, either replacing or supplementing milk from breastfeeding. Cow's milk allergy (CMA) affects ~2.5% of children <3 years of age1, 2 and involves the skin, the respiratory tract, or the gastrointestinal tract. Immunoglobulin E (IgE)-mediated reactions account for ~60% of milk-induced allergic disorders.1, 3 Although most infants with IgE-mediated CMA “outgrow” their sensitivity by the third year of life, 15% retain their sensitivity into the second decade.4, 5 At present there is no treatment for milk allergy other than avoidance, and this is very difficult because of the ubiquitous nature of milk in processed foods.

Caseins are major milk allergens and account for 80% of the total protein in cow's milk.6 They comprise 4 fractions with little primary structural homology: αs1-, αs2-, β-, and κ-casein, constituting 32%, 10%, 28%, and 10% of the total protein content, respectively.7 αs1-Casein is a single-chain phosphoprotein with 199 amino acids (AAs), characterized by a high content of proline residues distributed throughout the molecule8 and a lack of disulfide bonds, resulting in a reduced tertiary structure.9 These factors increase the likelihood that the major allergenic epitopes of this protein are linear rather than conformational.

Previous studies that evaluated the allergenic epitopes of peanut proteins showed that substitution of critical AAs led to a loss or reduction of IgE binding.10, 11, 12 By modifying the peanut allergen, cDNAs encoding proteins with reduced IgE-binding capacity were developed. These modified peanut allergens demonstrated a greatly reduced IgE-binding capacity; however, they retained the ability to stimulate T-cell proliferation.13, 14 In a murine model of peanut anaphylaxis, treatment with modified peanut proteins was shown to “desensitize” mice to peanut and protected the peanut-allergic mice against peanut-induced anaphylaxis.13, 15

In our previous studies, we characterized the sequential IgE-binding epitopes for all casein and whey proteins.16, 17, 18, 19 In the present study, we identified the AAs on αs1-casein crucial for IgE binding to the allergenic epitopes and provide the necessary information to produce hypoallergenic variants of this major milk allergen.

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Methods 

Patient population 

Sera from 15 patients with documented IgE-mediated cow's milk hypersensitivity (mean age, 8.5 years; range, 2-18 years) were used to identify the AAs essential for IgE binding. Milk-specific IgE antibodies in these sera ranged from 25 to >100 kU/L (median, >100 kU/L), as measured by the CAP system FEIA (Pharmacia Diagnostics, Uppsala, Sweden). In 11 children, CMA was confirmed by open (n = 3) or double-blind, placebo-controlled food challenges (n = 8). The remaining 4 children had a history of a severe reaction to an isolated ingestion of milk protein with either anaphylaxis or breathing difficulties and were therefore not challenged. Informed consent was obtained, and the study was approved by the Institutional Review Board.

Peptide synthesis 

In our previous studies, we identified 6 major, sequential IgE-binding regions in αs1-casein at AA 17-36, AA 83-102, AA 109-120, AA 139-153, AA 159-174, and AA 173-194. To determine the AAs essential for IgE binding within these IgE-binding regions, peptides 10-14 AAs in length, representing the native epitopes, and “engineered” peptides with single-AA changes at each position were synthesized on a cellulose membrane (SPOTs membrane; Genosys Biotechnologies, Woodlands, Tex) as described previously.16 In brief, with use of the 9-fluorenyl-methyloxycarbonyl method, cycles of coupling, blocking, and deprotection were repeated until the peptides of the desired composition and length were synthesized. Peptide synthesis reactions were monitored by bromophenol blue color reactions during each cycle. Alanine was substituted for each AA of the peptide, and if alanine was present in the native epitope, the substitution was done with glycine. Because of their length, the IgE-bonding regions at AA 17-36 and AA 173-194 were both divided into 2 separate peptides AA 17-30 and AA 23-36 as well as AA 173-186 and AA 179-192, respectively, resulting in 8 native peptides and their corresponding mutants.

To determine the effect of substitution on epitope-specific IgE binding, the native and mutated peptides were probed with pooled or individual sera from patients with CMA, as described below. If no significant loss of IgE binding was attained with individual patient sera, substitutions of 2-4 native AAs were done at the same time in positions that previously had given the greatest reduction of IgE binding when probed with the pooled sera. Membranes containing synthesized peptides were either probed immediately with serum IgE or stored at −20°C until needed.

IgE-binding assays 

Native and mutated peptides were blocked overnight at room temperature with human serum albumin in phosphate-buffered saline containing Tween-20. Pooled sera from 15 patients or 8 randomly selected, individual patient's serum was diluted 1:50 and incubated with the membrane-bound peptides overnight at 4°C. After being washed with phosphate-buffered saline, the membranes were incubated for 3 hours with goat anti-human IgE diluted 1:10,000 (Kirkegaard & Perry Laboratories, Gaithersburg, Md), followed by peroxidase-streptavidin conjugate (Kirkegaard & Perry Laboratories). The membranes were then developed with a chemiluminescent-avidin-horseradish peroxidase detection system (Amersham, Arlington Heights, Ill), and the optical density (OD) of each individual peptide spot was measured by means of reflection densitometry. The OD of each peptide spot was recorded as the difference between the OD of the peptide spot and that of the background. Critical AAs were defined as the those that resulted in loss of IgE binding when substituted by another residue.

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Results 

Pooled sera from 15 patients and individual sera from 8 randomly selected patients whose serum was included in the pool were used to identify AAs essential for epitope-specific recognition by IgE antibodies. In the first phase of experiments, each residue within an epitope was substituted with alanine (or, when alanine was present, with glycine) 1 at a time. Fig 1 shows an example of the immunolabeling with pooled sera for peptide AA 109-120.

  • View full-size image.
  • Fig. 1. 

    Selected part of the SPOT membrane, showing 1 of the synthesized, wild-type (WT ) peptides (AA 109-120) and the corresponding mutated peptides with single-AA substitution. Shown is the IgE labeling with pooled sera from 15 CMA patients. Single-AA changes at AA 113, AA 114, and AA 117 resulted in loss of IgE binding to this epitope.

In comparison with the native peptide, substituting alanine at AA 113, AA114, and AA117 resulted in loss of IgE binding.

In 2 of 8 peptides, substitution of a single AA resulted in the total loss of IgE binding by the pooled sera (Fig 2).

  • View full-size image.
  • Fig. 2. 

    Identification of the critical AA within the major αs1-casein epitopes. Shown is IgE binding of the pooled sera from 15 CMA patients to each individual wild-type and mutated peptide determined by densi-tometry. The arrows mark the AAs that resulted in total abrogation of IgE binding, and the stars indicate the AAs that were chosen for double substitution.

The remaining peptides showed diminished but not totally abrogated binding after single-AA changes (Fig 2). For those peptides, 2 residues were changed at the same time by using combinations of residues that, when mutated individually, had resulted in decreased IgE binding with the pooled sera. With this strategy, IgE binding by the pooled sera was abrogated to the remaining peptides, except peptide AA 89-102 (Fig 3, A ).
  • View full-size image.
  • Fig. 3. 

    Native and mutated peptides with single- (eg, P114A) or double- (eg, F23A/F24A) AA substitutions were synthesized on cellulose membranes. IgE binding to these membrane-bound peptides was measured by optical densitometry. In A, the results are shown for pooled sera from 15 CMA patients. The black bars represent binding to the native peptides and the gray bars , binding to the mutated ones. In B, IgE binding from 8 individual patient sera, randomly selected from the pool, is shown. Each color represents the OD measurement of an individual patient, resulting in a cumulative OD for all patients.

In an effort to determine whether individual patient sera would give results similar to those of the pooled sera, 8 patients were randomly selected from the same pool of 15, and their sera were tested individually in the same manner. The results are shown in Fig 3, B . Most patients demonstrated the same pattern that was found with the pooled sera. However, in 4 of 8 peptides, modification of the critical AAs identified with the pooled sera did not result in abrogation of binding with at least 1 individual patient's serum (Fig 3). For these patients, AAs other than those implicated by the patient pool were identified, indicating a more heterogeneous pattern in IgE recognition than expected. An example is shown in Fig 4.

  • View full-size image.
  • Fig. 4. 

    Peptides 10-14 AAs in length, representing the major IgE-binding epitopes of αs1-casein, and “engineered” peptides with single-AA changes at each position were synthesized on a cellulose membrane. IgE binding to the native and mutated peptides was determined with pooled and individual patient sera. The results for 2 IgE-binding epitopes (AA 23-36 and AA 159-172) are presented in this figure. Each column represents a mutated peptide in which an AA change has been made at the indicated position. IgE binding to each modified peptide was measured by OD and is shown for the pooled patient sera in row 1 and for the individual patients in the following rows. The results are expressed as the percentage of IgE-antibody binding to the mutated peptides relative to the wild-type peptides, ranging from no reduction in binding, represented in black squares , to total abrogation of binding, represented in white squares . Although no single-AA substitution resulted in abrogation of IgE-binding in every patient, substitutions highlighted in red showed at least a clear reduction in the majority of patients. Therefore, these AAs were substituted simultaneously, and the results are shown in the last column.

For peptide AA 89-102, no further reduction of IgE binding could be found with multiple AA substitutions, even when 13 different combinations of AA substitution of 2 or 3 AAs were used at the same time. Only when 4 AAs were substituted simultaneously was IgE binding abrogated by the pooled sera as well as in 5 of 6 individual patients (Fig 5).

  • View full-size image.
  • Fig. 5. 

    Selected part of the SPOT membrane, showing the synthesized, wild-type (WT ) peptides for AA 89-102 and the corresponding mutated peptides with multisided AA substitution. Shown is IgE labeling with pooled sera from 15 CMA patients.

Among a total of 94 AAs present in the 6 major IgE-binding regions, 46 were hydrophobic residues (49%), 26 were polar (28%), and 29 were charged (31%). At least 16 AAs were identified as being critical (Fig 6).

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  • Fig. 6. 

    Synthesized peptides (underlined) representing the major IgE-binding epitopes on αs1-casein are shown. The numbers on the top indicate the position of the AA relative to the first AA encoded by this sequence. All hydrophobic AA residues are marked in red . The squares indicate the AAs critical for IgE binding.

Of these, hydrophobic AAs appeared to be the most common (75%). In addition, the AAs critical for IgE binding were more often located in the center of the epitope.

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Discussion 

Within the last few years, the primary AA sequences of a number of food allergens and their IgE-binding regions have been identified. To develop safe immunotherapeutic agents for food allergy, modified, recombinant proteins need to be engineered that will not bind serum and mast cell IgE. Therefore, critical AAs within the IgE-binding regions need to be identified to provide the necessary information to alter the cDNAs that encode proteins capable of activating milk-specific T cells, but not mast cells, basophils, or IgE-bearing, antigen-presenting cells.

On the basis of the IgE-binding epitopes previously identified by our group,16 we sought to determine the critical AAs within the major B-cell epitopes of αs1-casein. Some studies have indicated that IgE directed against the casein fractions in cow's milk appears to be dominant in older children and adults with CMA when compared with younger children.20, 21 This suggests that caseins play an important role in persistent cow's milk allergy and indicates that they are important components for future immunotherapeutic agents. αs1-Casein is a protein with minimal tertiary structure due to its lack of disulfide bonds. Consequently, the binding activity of IgE antibodies to denatured α-casein is almost identical to that of native α-casein, indicating that IgE antibodies are mainly directed against linear epitopes of αs1-casein.22 Therefore, modification of the protein for use in immunotherapy could be based on mutation of the sequential epitopes of caseins.

In the search for critical AAs for IgE binding, alanine and glycine were used for AA substitution because they are relatively small, neutral AAs that do not significantly alter the charge or solubility of peptides. This approach has been used successfully for other allergens.10, 11, 12, 23

Unlike other allergenic food proteins studied, only 2 of 8 major allergenic epitopes studied had abrogation of IgE binding after a single-AA substitution. For the other 6 epitopes, at least 2 AAs had to be substituted in parallel to abrogate or markedly reduce IgE binding. Similar results were obtained for the major shrimp allergen, Pen a1, for which 2 or more substitutions were generally necessary to abolish IgE binding to allergenic epitopes.24 This finding stands in clear contrast to results previously obtained in the identification of critical AAs within the IgE-binding regions of the 3 major peanut allergens, Ara h 1, Ara h 2, and Ara h 3.10, 11, 12 In each of the IgE-binding epitopes within the peanut allergens, a single substitution led to abrogation or marked reduction of IgE binding.

Comparing the results of the pooled sera and individual patient serum, the same pattern of reduced IgE binding was observed among the same critical AAs in the majority of patients. These results imply that a single- or double-AA substitution within IgE-binding epitopes should be adequate for decreasing the affinity of allergen-specific IgE antibodies. However, for several epitopes, IgE antibodies from at least 1 patient appeared to bind equally well to the mutated peptides, suggesting a more heterogeneous pattern of epitope recognition than previously reported. This indicates that “engineered” recombinant vaccines might still provoke adverse reactions in some patients. To our knowledge, this is the first report that used individual patient sera to determine critical AAs within IgE-binding regions. The next steps would be to produce recombinant, native and mutated proteins and test for differences in allergenicity with the use of immunoblots, histamine-release assays, and even animal models.

Our results showed that the critical AAs in αs1-casein are most commonly hydrophobic. Similar hydrophobic residues appear to be critical for IgE binding in the peanut allergen Ara h 1,25 whereas no obvious type of AA was apparent in Ara h 2 and Ara h 3.11, 12 In addition, AAs in the center of the epitope appear to be more important for IgE binding in αs1-casein than those found at the periphery of the epitope. This was also observed for the shrimp allergen Pen a 1, the peanut allergen Ara h 3, and the walnut allergen Jug r 1.12, 23, 24

In this study, we have identified the critical AAs within the IgE-binding epitopes recognized by CMA patients. Determination of AAs critical for IgE binding provides crucial information for altering αs1-casein cDNA to encode a protein with reduced IgE-binding capacity. Such mutated proteins or peptides could be used in future immunotherapeutic approaches for this disease.

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☆ Supported by grants AI 44236 and AI 24439 from the National Institute of Allergy and Infectious Diseases, grant M01 RR00071 from the Division of Research Resources, National Institutes of Health, and the Bunning Family Fund.

☆☆ Reprint requests: Kirsten Beyer, MD, Department of Pediatrics, Charite-CVK, Augustenburgerplatz 1, 13353 Berlin, Germany.

PII: S0091-6749(03)01548-3

doi:10.1067/mai.2003.1617

The Journal of Allergy and Clinical Immunology
Volume 112, Issue 2 , Pages 433-437, August 2003

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