3.4 In silico analysis of the known IgE epitopes of CN components
To identify the putative cross-reactive epitope peptides in αs1-CN, we also applied in silico analysis on the previously reported IgE epitopes of αs1-CN10-13 against the amino acid sequences of αs2-, β-, and κ-CN. Unexpectedly, we could only find six peptide sets that showed PD values lower than 10 (Table 3). Furthermore, the lowest PD value of 7.16 was detected in αs2-CN.
We arranged the amino acid sequences of casein components and filled the reported IgE epitopes in order to visualize the location (Figure 3).10-15 The four “SSSEE” motifs, known as the core motif of casein phosphopeptide (CPP), are shown in bold characters, but the reported IgE epitopes were scarcely located on the “SSSEE” motif except one located at the N-terminus of αs2-CN.
DISCUSSION
The purpose of this study was to investigate the cross-reactivity among CN components. The data clearly indicated that IgE (also IgG4 and IgA) specific to αs1- and β-CN were strongly associated, and the presence of strong cross-reactivity was confirmed using competitive ELISA. We also found lower cross-reactivity between αs1-CN and κ-CN. Unfortunately, we could not examine αs2-CN due to purification difficulties.
Bernard et al. reported that the sum of the IgE levels to the different CN components was higher than the IgE levels to whole CN, suggesting the presence of cross-reactive epitopes between the CN components.16 Generally, cross-reactivity is expected between proteins with high sequence homology.17However, an article which compiled cross-reactivities between non-homologous allergens was recently reported.18 With regard to CN, the homology of full-length amino acid sequences between CN components is extremely low (4–7%). To assess this theoretical gap in knowledge, we employed in silico analysis to find the common peptide sequences in each CN component and found highly homologous amino acid sequences in αs1-, αs2-, and β-CN. The sequences commonly included the unique ”SSSEE” motif, known as the basic structure of CPP.
During CN digestion, κ-CN, which covers the surface of CN micelles, is broken down and subsequently the other CNs too.19 CN is well known as a protein with high digestibility.20However, the fact that CN is a major allergen suggests that the major IgE epitope should be placed in the digestion-resistant structure.21 CPP, which results from the digestion of CN, has been identified as a trypsin-resistant peptide due to the highly phosphorylated serine residues in and around the core structure of “SSSEE”. CPP plays a key role in the effective absorption of calcium due to the formation of micellar calcium phosphate at the phosphorylated serine residues. Based on this nutritional function, CPP is widely used medically to prevent osteoporosis and in dental care to ameliorate the calcium deposition on teeth. Remarkably, dental treatments using small amounts of CPP paste have the potential to be allergenic and cause anaphylaxis. This clinical fact suggests that CPP presents strong allergenic activity. CPP derived from αs- and β-CN. CPP identified in αs1-CN (D43–K79), αs2-CN (K1–K32 and N46–K70) and β-CN (R1–K28) (Figure 3).22,23
In this study, we did not find strong cross-reactivity and detect a highly homologous amino acid sequence between αs1-CN and κ-CN. The CPP motif does not exist in κ-CN, and the C-terminal half of κ-CN consists of a unique water-soluble glycoprotein designated as a glycomacropeptide.24 Epitope-mapping of κ-CN reveals many IgE binding sites, which might have individual allergenic properties with low cross-activity between αs- and β-CNs. Epitope-mapping of whole CN components has been extensively performed worldwide, using overlapping peptides and peptide microarray techniques.10-13 Nonetheless, the reported IgE-epitopes are different with each other, and no consensus of the dominant IgE epitope has been established. Furthermore, prior to this study, cross-reactive epitopes among CN components have not been reported.
Recently, IgE- and IgG4-epitope mapping was carried out to develop a prediction tool for the prognosis of CM allergy.25- 29 However, in these reports, there were few epitope sequences located in the CPP regions and no common epitopes were found in the CPP regions on the different CN components. We supposed the reason that IgE binding ability to synthetic peptide is low compared with natural CN is because serine residues were not phosphorylated in the synthetic peptides.
Generally, IgE-binding epitopes have hydrophilic properties and are present on the surface of a granular protein molecule. Phosphorylated serine residues comprise the water-soluble part, and low phosphorylated residues comprise the water-insoluble part of the CN molecule. Otani et al. reported that the IgG binding ability to dephosphorylated αs1-CN was lower than that to native αs1-CN.30Furthermore, whileO -phospho-L-serine inhibited IgG binding, L-serine scarcely inhibited binding, thereby indicating the importance of the phosphorylated serine residue in the epitope. Therefore, phosphorylated serine residues in the CPP motifs might have the critical role in the formation of the dominant and cross-reactive IgE epitopes in the native CN components.
A major limitation of this study is that we suggest the presence of cross-reactive epitopes between αs1-CN and β-CN based on bioinformatics finding of the presence of highly homologous amino acid sequences in the CPP regions; however, we need to combine these findings with direct experimental data. Therefore, future experiments are expected to define the IgE-binding capacity of highly purified CPPs from natural CN and evaluate the effect of dephosphorylation. If this agenda were achieved, this would suggest the following: (i) CPP can induce milk allergy, (ii) possibility of cross-reaction caused by the similarity of CPP motifs on the different CN components, (iii) change in IgE binding ability by dephosphorylating the side chain of an amino acid, and (iv) partial negation of epitope analysis using the conventional overlapping peptides.
In conclusion, despite the homology among CN components being extremely low, αs1-CN shows cross-reactivity with β-CN. The sequence of ”SSSEE”, which is a basic structure of CPP, may have a potential of being the cross-reactive epitope.