title: Introducing site-specific glycosylation using protein engineering techniques reduces the immunogenicity of β-lactoglobulin.
abstract: To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared wild-type bovine BLG variant A (wt) and three site-specifically glycosylated BLGs (D28N, D137N/A139S, and P153A), and expressed them in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the glycosylated BLGs were conjugated with a ~4 kDa high-mannose chain. Each glycosylated BLG retained ∼80% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface structure was slightly changed by using protein engineering techniques, but that the site-specifically glycosylated BLGs were covered by high-mannose chains without substantial disruption of wt conformation. Antibody responses to the glycosylated BLGs tended to be weaker in BALB/c, C57BL/6, and C3H/He mice. We conclude that site-specific glycosylation is an effective method to reduce the immunogenicity of BLG, and that masking of epitopes by high-mannose chains is effective to reduce immunogenicity.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tatsumi
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Sasahara
authors.forename: Noriki
authors.initials: N
authors.lastname: Kohyama
authors.forename: Satomi
authors.initials: S
authors.lastname: Ayano
authors.forename: Michio
authors.initials: M
authors.lastname: Endo
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yamada
authors.forename: Mamoru
authors.initials: M
authors.lastname: Totsuka
authors.forename: Makoto
authors.initials: M
authors.lastname: Hattori
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005260:Female; D006031:Glycosylation; D007782:Lactoglobulins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D007782:Lactoglobulins