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\begin{document}

\title{Casein phosphopeptide in cow's milk is strongly allergenic}



\author[1]{Teruaki Matsui}%
\author[2]{Michihiro Naito}%
\author[1]{Katsumasa Kitamura}%
\author[1]{Atsushi Makino}%
\author[1]{Yoshihiro Takasato}%
\author[1]{Shiro Sugiura}%
\author[3]{Hidehiko Izumi}%
\author[1]{Komei Ito}%
\affil[1]{Aichi Children's Health and Medical Center}%
\affil[2]{Nagoya University of Arts and Sciences}%
\affil[3]{Nagoya University of Arts and Sciences School of Nutritional Science Graduate School of Nutritional Science}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\textbf{Conflict of interest disclosure}

The authors have no conflict of interest to declare.

\textbf{Financial support}

This study received no financial support.

\textbf{Keywords}

basophils, casein, phosphopeptide, food allergy, cow milk, milk
hypersensitivity

To the Editor:

Cow's milk (CM) is a common food allergen.\textsuperscript{1} It has a
high casein content, which comprises approximately 80\% of CM
proteins.\textsuperscript{2} A variety of processed foods and medicines
include processed products derived from CM. Thus, attention must be paid
to the symptoms induced by an allergic reaction to the accidental
ingestion of these products.\textsuperscript{3}

Casein phosphopeptide (CPP) is a processed product derived from caseins
by tryptic digestion.\textsuperscript{4} It binds with amorphous calcium
phosphate (ACP), increasing its ability to stabilize calcium phosphate
in solution.\textsuperscript{5} Thus, CPP-ACP acts as a biologic calcium
phosphate delivery vehicle and is used worldwide in various oral care
products, such as chewing gums, topical creams, and toothpaste, owing to
its anticariogenic properties.\textsuperscript{6}

Because the allergenicity of CPP has yet to be determined, it is unclear
whether CPP-ACP can induce allergic symptoms in patients with CM allergy
(CMA). Thus, the purpose of this study was to validate the allergenicity
of CPP-ACP. Our study was approved by the Research Ethics Board of Aichi
Children's Hospital (approval number: 2020114) and conformed to the
Declaration of Helsinki.

First, we searched the electronic medical record database of Aichi
Children's Health and Medical Center from February 2012 to March 2021 to
identify any CPP-ACP-related episodes. A total of eight patients were
identified (Table 1). Data on the patients' characteristics, clinical
information, episode details, and the associated results of the CM oral
food challenge (OFC) test and blood tests were extracted from the
medical records. The severity of the allergy-induced symptoms varied
between patients. Although the amount of product used was small, of the
six patients who reacted to the dental care product MI paste (GC Corp.,
Tokyo, Japan), which contains 10\% CPP-ACP, four developed anaphylaxis
and required an intramuscular adrenaline injection. Two patients reacted
to Recaldent gum (GC Corp.), which contains 0.18\% CPP-ACP, and one of
them experienced anaphylaxis. All patients had severe CMA and reacted to
\textless{}4 ml of CM during the OFC.

Next, we performed ImmunoCAP inhibition assays and basophil activation
tests using CPP or casein to examine the \emph{in vitro} allergenicity
of CPP. We recruited those patients with CMA who were diagnosed by a
positive CM-OFC test and were completely avoiding CM. Written informed
consent was obtained from the parents of the participants at enrollment.
Serum from patients 1a, 1b, and 1c and peripheral blood from patients
2a, 2b, and 2c were used for the inhibition assay and basophil
activation tests, respectively (Table S1).

The casein, CPP, and ovalbumin (FUJIFILM Wako Chemicals, Osaka, Japan)
used in this \emph{in vitro} study were characterized by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (Figure S1). All of the
casein fractions (\selectlanguage{greek}α\selectlanguage{english}s1-, \selectlanguage{greek}α\selectlanguage{english}s2-, \selectlanguage{greek}β-, \selectlanguage{english}and \selectlanguage{greek}κ-\selectlanguage{english}casein) were detected in the
casein, and CPP showed a smear with a molecular weight \textless{}20
kDa.

For the inhibition assay, each inhibitor was diluted with immunoglobulin
(Ig) E Sample diluent (Thermo Fisher Diagnostics) and incubated at 4\selectlanguage{ngerman}°C
overnight with the target serum at a 1:1 ratio. Then, casein-specific
IgE was measured by an ImmunoCAP test (Thermo Fisher Diagnostics, Tokyo,
Japan). CPP inhibited casein-specific IgE binding in a
concentration-dependent manner in all three patients (Figure 1).
Although the rate at which casein-specific IgE was inhibited was low at
lower concentrations, it was \textgreater{}80\% when applied at the
highest concentration (10 mg/ml). This inhibition was proved to be
antigen-specific because 10 mg/ml of ovalbumin did not interfere with
the detection of casein-specific IgE (Figure 1), and 10 mg/ml of casein
and CPP did not inhibit egg white-specific IgE using the identical sera
(Figure S2).

Next, the basophil activation test was performed using an Allergenicity
Kit (Beckman Coulter Inc., USA), according to the manufacturer's
instructions. Briefly, ethylenediamine tetraacetic acid
(EDTA)-containing whole blood was incubated with various concentrations
of casein, CPP, or ovalbumin for 15 min with a sufficient amount of
calcium solution to override the chelating capacity of EDTA.
Stimulations with 4 \selectlanguage{greek}µ\selectlanguage{english}g/ml anti-IgE and phosphate-buffered saline were
used for positive and negative controls, respectively. Phycoerythrin
cyanine 7-conjugated anti-CD-3, fluorescein isothiocyanate-conjugated
anti-CRTH2, and phycoerythrin-conjugated anti-CD203c antibodies were
added during the reaction. Using flow cytometry, the mononuclear cells
were gated by forward and side scatter, and basophils were identified as
CD3\textsuperscript{-}CD294 (CRTH2)\textsuperscript{+} cells. The
percentage of activated basophils was determined using a threshold
CD203c fluorescence intensity based on a comparison between the positive
and negative controls (Figure S3). CPP activated basophils in a
concentration-dependent manner comparable to that of casein in two of
the patients (Figure 2a and 2b) but was less effective than casein in
activating basophils at concentrations \textgreater{}10 \selectlanguage{greek}µ\selectlanguage{english}g/ml in the
remaining patient (Figure 2c).

These \emph{in vitro} data clearly indicate that CPP possesses
IgE-binding ability and induces basophil activation comparable to that
of casein, which supports the fact that CPP causes severe allergic
symptoms in patients with CMA. To date, many casein epitopes have been
reported through IgE epitope mapping using synthetic peptides to predict
CMA prognosis.\textsuperscript{7,8}However, only a few epitope sequences
were located in the CPP regions, and no common epitopes were detected in
the CPP regions of the different casein components.\textsuperscript{9}

We previously reported a strong correlation between the IgE levels
specific to \selectlanguage{greek}α\selectlanguage{english}s1- and \selectlanguage{greek}β-\selectlanguage{english}casein\textsuperscript{10} and confirmed strong
cross-reactivity between them using competitive enzyme-linked
immunosorbent assays.\textsuperscript{9} Although \selectlanguage{greek}α\selectlanguage{english}s1- and \selectlanguage{greek}β-\selectlanguage{english}casein
showed a fairly low level of homology of total amino acid sequences
(4\%--7\%), we found a partial similarity between the \selectlanguage{greek}α\selectlanguage{english}s1-casein
(E61--K70) and \selectlanguage{greek}β-\selectlanguage{english}casein (I12--E21) amino acid sequences, both of which
were included in the CPP region.\textsuperscript{9} Based on these
findings, we hypothesize the existence of common and dominant IgE
epitopes in these regions.

Tryptic hydrolysis has been used to identify CPP in \selectlanguage{greek}α\selectlanguage{english}s1-casein
(E61--K79), \selectlanguage{greek}α\selectlanguage{english}s2-casein (K1--Y20 and N46--K70), and \selectlanguage{greek}β-\selectlanguage{english}casein
(R1--R25).\textsuperscript{4,9} CPP contains highly phosphorylated
serine residues in and around the core structure of the SSSEE motif.
Therefore, the major phosphorylated epitopes in casein could have been
missed in studies using non-phosphorylated synthetic
peptides.\textsuperscript{9} Thus, phosphorylated serine residues in the
CPP motifs could be critical in forming the dominant IgE epitopes in
native casein components.

There are some limitations to our study. First, the inhibition assay
revealed that CPP had a lower inhibitory ability than casein, but showed
an almost identical basophil activating ability to that of casein. It
was difficult to interpret the difference between the methods, which
could be partially attributed to the small sample size. Nevertheless,
the clinical fact that an anaphylactic reaction was induced by a small
amount of CPP-ACP suggests strong allergenicity of CPP. Second, the CPP
reagent used in our experiments was a mixture of trypsin-digested casein
peptides, which might contain residual IgE epitopes, except for the CPP
region in casein.

In conclusion, despite the fact that major epitopes of CPP have yet to
be reported, CPP showed representative allergenic activity in casein. We
are currently performing experiments using dephosphorylated CPP peptides
and also planning to use synthetic phosphorylated CPP to confirm our
study findings.

\textbf{Acknowledgments}

We thank all the physicians, nutritionists, and nurses who performed the
OFCs and data collection, and Mr. Noboru Hayakawa (Clinical Laboratory
technician in Aichi Children's Health and Medical Center) and Ms. Kumiko
Matsumoto (Cell Biology Section, BML, Inc.) for their excellent
technical assistance.

\textbf{Impact statement}

CPP could cause severe allergic reactions in patients with CMA.
Moreover, it possesses IgE-binding ability and induces basophil
activation comparable to that of casein

\textbf{Data availability statement}

The datasets of this study are not publicly available to protect the
participants' personal data but can be availed from the corresponding
author on reasonable request.

\textbf{References}

1. Peters RL, Krawiec M, Koplin JJ, Santos AF. Update on food allergy.
Pediatr Allergy Immunol 2021; 32: 647-657.

2. Ito K, Futamura M, Moverare R, et al. The usefulness of
casein-specific IgE and IgG4 antibodies in cow's milk allergic children.
Clin Mol Allergy 2012; 10: 1.

3. Ebisawa M, Ito K, Fujisawa T, Committee for Japanese Pediatric
Guideline for Food Allergy TJSoPA, Clinical I, Japanese Society of A.
Japanese guidelines for food allergy 2020. Allergol Int 2020; 69:
370-386.

4. Cruz-Huerta E, Garcia-Nebot MJ, Miralles B, Recio I, Amigo L.
Caseinophosphopeptides released after tryptic hydrolysis versus
simulated gastrointestinal digestion of a casein-derived by-product.
Food Chem 2015; 168: 648-655.

5. Azarpazhooh A, Limeback H. Clinical efficacy of casein derivatives: a
systematic review of the literature. J Am Dent Assoc 2008; 139: 915-924.

6. Bijle MNA, Yiu CKY, Ekambaram M. Calcium-based caries preventive
agents: a meta-evaluation of systematic reviews and meta-analysis. J
Evid Based Dent Pract 2018; 18: 203-217.

7. Caubet JC, Lin J, Ahrens B, et al. Natural tolerance development in
cow's milk allergic children: IgE and IgG4 epitope binding. Allergy
2017; 72: 1677-1685.

8. Savilahti EM, Kuitunen M, Valori M, et al. Use of IgE and IgG4
epitope binding to predict the outcome of oral immunotherapy in cow's
milk allergy. Pediatr Allergy Immunol 2014; 25: 227-235.

9. Naito M, Matsui T, Yamada C, Tagami K, Ito K, Izumi H. Evaluation of
cross-reactivity between casein components using inhibition assay and in
silico analysis. Pediatr Allergy Immunol 2021; 32: 544-551.

10. Naito M, Matsui T, Tagami K, et al. Changes in casein
component-specific antibody levels during oral immunotherapy for milk
allergy. Pediatr Allergy Immunol 2020; 31: 421-424.



\textbf{Tables}

\textbf{TABLE 1} Allergic events caused by casein
phosphopeptide--amorphous calcium phosphate

\textbf{Figure legends}

\textbf{FIGURE 1} Inhibition assay of casein-specific IgE using
ImmunoCAP tests.

Different concentrations of casein (black circles), casein
phosphopeptide (white circles), or ovalbumin (gray circle) were mixed
with the target serum at a 1:1 ratio and then incubated at 4\selectlanguage{ngerman}°C
overnight. Casein-specific IgE was measured using ImmunoCAP tests.

\textbf{FIGURE 2} Basophil activation test stimulated by casein or
casein phosphopeptide.

The basophil activation test was performed using an Allergenicity Kit
(Beckman Coulter). Different concentrations of casein (black circles)
and casein phosphopeptide (white circles) were used as stimulators. The
percentage of activated basophils was determined using a threshold
CD203c fluorescence intensity based on a comparison between the positive
and negative controls.

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