IgE and IgG Binding Epitopes on a-Lactalbumin and ß-Lactoglobulin in Cow’s Milk Allergy (1)
It has been revealed the importance of the caseins as major milk allergens, but showed also significant reactivity to the whey proteins a-lactalbumin (ALA) and ß-lactoglobulin (BLG). ALA is a monomeric globular protein of 123-amino acid residues with a molecular weight of 14.4 kD and four disulfide bridges. Its hydrophilic profile suggests that the major antigenic site is located on peptide corresponding to amino acids (AA) 5–18, which corresponds to the homologous sequence 124–134 on the BLG molecule. BLG occurs naturally in the form of a 36-kD dimer with each subunit corresponding to a 162-residue polypeptide. It possesses two disulfide bridges and one free cysteine. The hydrophilic profile of the protein suggests the antigenicity of sequence corresponding to AA 124–134.
IgE antibodies from 8 of 11 patients showed binding to at least one linear epitope.
The complete AA sequence of the protein with IgE binding epitopes depicted is shown in figure 2. None of the linear epitopes were recognized by the younger patients or by the control subjects.
Of the 5 controls, 3 recognized the first epitope, and 2 showed binding to the third IgG binding epitope.
In comparison to ALA, all patients recognized at least one epitope on BLG. The region corresponding to AA 119–128, which showed weak binding with sera from the older patients, and was therefore not defined as a major epitope, proved to show intensive binding among the younger patients.
The complete AA sequence of the protein with IgE binding epitopes is depicted.
Of the 5 control subjects, 4 recognized the third epitope, and 1 showed binding to the fifth and 1 to the sixth epitope. 2 control subjects recognized the region corresponding to AA 35–46, and 1 bound to AA 115–126, which were also IgG binding sites for 2 of the patients, but not considered as major IgG binding epitopes.
The fragment corresponding to AA 5–18 was shown by RAST inhibition to exhibit IgE binding capacity. Trypsin-digested ALA, a sequence corresponding to AA 17–58 and larger peptides sharing this sequence were most strongly and frequently recognized, and binding to smaller peptides corresponding to sequences AA 6–10:S-S:115–123 and AA 109–123 was also reported. There is AA 97–108 as a major linear epitope, which corresponded to a major epitope (AA 95–113). The tryptic fragments AA 1– 8, 25–40, 41–60, 102–124 and 149–162 as major IgE binding epitopes in a population of milk-allergic patients whose ages. Three IgG binding sequences on ALA which were AA 7–18, 53–62, 89–108, in partial agree- ment with earlier findings using rabbit antisera. IgG antibodies from milk-allergic patients show a marked preference for conformational epitopes whereas normal individuals recognize both conformational and sequential ones. The IgG binding sequences gener- ally colocalized with the IgE binding epitopes, except for three short regions (AA 1–6, AA 19–26 and AA 47–52) on ALA, and one extended region (AA 31–50) on BLG, which appeared to be exclusive allergenic epitopes. BLG is absent in human milk, but has extensive homology to several human and animal proteins of the lipocalin family, some of which are found in endogenous body fluids in humans.