Reduced immunogenicity of beta-lactoglobulin by conjugation with acidic oligosaccharides.

ABSTRACT

Bovine beta-lactoglobulin (beta-LG) was conjugated with the acidic oligosaccharides, alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides (POs) by the Maillard reaction to reduce the immunogenicity of beta-LG. The molar ratios of beta-LG to ALGO and POs in the conjugates were 16 and 18. The carbohydrate-binding sites in the beta-LG-ALGO conjugate were partially identified to be (60)Lys, (77)Lys, (100)Lys, (138)Lys, and (141)Lys. The isoelectric point of each conjugate was lower than that of beta-LG. CD spectra indicated that the secondary structure of beta-LG was almost maintained after conjugation. The results of fluorescence studies indicated that the conformation around Trp had not changed in each conjugate and that the surface of each conjugate was covered with a saccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around (8)Lys-(19)Trp (beta-sheet, random coil, short helix) in the conjugates had changed, whereas the native structure was maintained around (15)Val-(29)Ile (beta-sheet) and (125)Thr-(135)Lys (alpha-helix). The beta-LG-ALGO and beta-LG-POs conjugates maintained 77 and 70% of the retinol binding activity of beta-LG. Conjugation with ALGO and POs substantially enhanced the thermal stability of beta-LG. The anti-beta-LG antibody response was markedly reduced after immunization with both conjugates in BALB/c, C57BL/6, and C3H/He mice. B cell epitopes of beta-LG and the conjugate recognized in these mice were determined with 15-mer multipin peptides, and the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, whereas the antibody response to each epitope was dramatically reduced. In particular, effective reduction of the antibody response was observed in the vicinity of the carbohydrate-binding sites. Conjugation of beta-LG with these acidic oligosaccharides was effective in reducing the immunogenicity of beta-LG. The conjugates obtained in this study are edible, so they would be very useful for food application.