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.