INTRODUCTION
Casein (CN), a major allergen for patients with persistent cow’s milk (CM) allergy, comprises approximately 80% of CM proteins.
1 Bovine CN is constituted of four components, αs1-, αs2-, β-, and κ-CN, and exists as a micelle with a 30
–300 nm diameter.
2 CN is an unfolded phosphoprotein and interacts with calcium phosphate in milk.
3 αs-CN is defined as an insoluble fraction in 0.4 M calcium chloride, while κ-CN is the soluble fraction. β-CN is defined as soluble in 3.3 M urea and insoluble in 1.7 M urea. Bovine αs1-, αs2-, and β-CN are comprised of 199, 207, and 209 residues, respectively, and are calcium-sensitive.
3 Bovine κ-CN is a glycoprotein comprised of 169 residues and is not calcium-sensitive. The homology of amino acid sequences among CN components is extremely low (Table S1).
We previously reported that specific IgE levels to purified αs1-CN and β-CN, but not κ-CN, showed simultaneous decreases during CM oral immunotherapy.4 Therefore, we hypothesized that αs1- and β-CN have cross-reactive epitopes, or alternatively, these two CNs might share remarkably similar antigenic properties. In this study, we investigated cross-reactivity among CN components.
Furthermore, Candreva et al. analyzed the in silico similarity between proteins at the peptide level.5 Cross-reactivity between milk and soy proteins has been suggested by other immunological studies.6 Thus, we also employed an in silico analysis at the peptide level using the peptide similarity tool on the Structural Database of Allergenic Proteins (SDAP).
METHODS
2.1 Materials
Each CN component (αs1-, β-, and κ-CN) was prepared following the
previously described methods.4 Protein-Free Blocking
Buffer (PFBB) was purchased from Thermo Fisher Scientific (Roskilde,
Denmark). Alkaline phosphatase (AP)-conjugated goat anti-human IgE
was purchased from Bethyl
Laboratories (Montgomery, AL, USA). Anti-α-casein mouse monoclonal
antibody was purchased from Cosmo Bio (Tokyo, Japan; out of production).
Peroxidase-conjugated anti-mouse
IgG was purchased from Jackson ImmunoResearch (PA, USA).