Human IgE Binding Capacity of Tryptic Peptides from Bovine α-Lactalbumin

Abstract

The specific IgE binding capacity of native bovine α-lactalbumin (α-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow’s milk protein allergy. The specific anti-bovine α-La IgE titers ranged from 0.6 to 125 IU/ml. Highly purified tryptic peptides from native and disulfide-bond-reduced α-La were obtained by reverse phase chromatography. By ELISA technique using immobilized native protein or peptides, 11 of the 19 sera reacted exclusively with intact protein while 8 of them also presented a specific IgE response to different tryptic peptides. Polyclonal IgE population specificity was not related to anti-bovine α-La IgE levels. Sequence (17G-K58) and larger peptides sharing this sequence were most strongly and frequently recognized. Competitive ELISA inhibition tests confirmed this IgE-specific response and gave also clear evidence for IgE binding to smaller peptides corresponding to sequences (6C-R10):S-S:(115L-L123) and (109A-L123). IgE binding to native α-La and large peptides confirmed the importance of conformational epitope(s). However, in some sera reduced and S-alkylated peptide (59I-K94) exhibited a similar or higher IgE binding capacity than the native corresponding fragment, suggesting the existence of sequential epitope(s) exposed through protein denaturation. Moreover, IgE binding sequences were also located within hydrophobic regions of α-La and/or within parts with high sequence homology to human α-La.

Profile Analysis and Immunoglobulin E Reactivity of Wheat Protein Hydrolysates

“…A number of studies have suggested that peptide fragmentation generated from proteolytic digestion or hydrolysis of food allergens can elicit IgE responses in already sensitized individuals [14,15,16]. Maynard et al [14] showed that 8 out of 19 exhibited a specific IgE response to different tryptic peptides among the sera from patients with cow’s milk allergy.…”

Protein digestibility and relevance to allergenicity.

“…A protein could appear stable or unstable in SGF (or SIF), depending on the relative amounts of enzyme and test protein used Fu et al 2002). Some studies of effects of proteolytic digestion on allergenicity have used comparatively low ratios (by weight) of enzyme:protein, ranging from 0.1 to 0.01 (Asselin et al 1989;Marquez and Lajolo 1981;Maynard et al 1997;Watanabe et al 1990). However, in vitro digestion assays for the purposes of safety assessment tend to employ higher ratios ranging from 25 to 5,000 (Fu 2002;Fuchs et al 1993;Noteborn et al 1995;Reed et al 1996).…”

Allergenicity of Milk Proteins

“…A study concerning the allergenic properties of α-La demonstrated that in 60% of the study patients, allergic sera were specific for intact α-La with only 40% binding to peptides obtained after tryptic hydrolysis. Residue 17-58 was the most frequently recognized in the sequence 59-93, 99-108, and 109-123 [79]. The linear epitopes were identified by using sera of patients suffering from persistent allergies and IgE to cow's milk levels > 100 kU(A)/L.…”