Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of β-lactoglobulin

Author:

Endo Michio1,Yoshida Tadashi1,Ishii Keisatoi1,Iwamoto Taku2,Totsuka Mamoru2,Hattori Makoto1

Affiliation:

1. Department of Applied Biological Science, Tokyo University of Agriculture and Technology , Tokyo , Japan

2. Department of Applied Biological Chemistry, The University of Tokyo , Tokyo , Japan

Abstract

ABSTRACT To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it 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 D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

Funder

JSPS

Publisher

Oxford University Press (OUP)

Subject

Organic Chemistry,Molecular Biology,Applied Microbiology and Biotechnology,General Medicine,Biochemistry,Analytical Chemistry,Biotechnology

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