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

To reduce the immunogenicity of ß-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.

Introducing site-specific glycosylation using protein engineering techniques reduces the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-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.

Comparison of carbonaceous aerosols in Tokyo before and after implementation of diesel exhaust restrictions.

We compared the status of carbonaceous aerosols in Tokyo before and after the implementation of a diesel vehicle regulation intended to reduce the quantity of particulate carbon from diesel engines in one of the largest scale ever attempts at vehicle exhaust control. Radiocarbon (14C) in elemental carbon (EC) and total carbon (TC) were analyzed to identify fossil fuel carbonaceous particles emitted from diesel-powered vehicles. One-sided paired-month t-tests showed no distinct difference in the absolute concentrations of particles in terms of total mass (19.5 to 18.0 microg m(-3); p = 0.321), EC (3.6 to 3.3 microg m(-3); p = 0.272), and TC (6.3 to 6.2 microg m(-3); p = 0.418) for the finest particles (d(a) < 1.1 microm) after the implementation of the regulation. The ratios of the concentrations of the chemical constituents were, however, altered after the regulation. EC/TC was significantly decreased from 56.7% to 50.2% (p = 0.039). Although it was not statistically significant, the percentage of fossil carbon in EC also decreased (67.8% to 63.8%; p = 0.104). Since EC is predominantly of combustion origin, the observed decrease was likely due to the decrease in fossil EC emissions from diesel-powered vehicles. The decrease in EC/TC after the implementation of the regulation was also likely to have resulted from attachment to diesel vehicle exhaust systems of particulate filters as required as part of the regulation by the Tokyo Metropolitan Government. The EC/TC of fossil carbon of the finest particles decreased from 66.2% to 55.2% (p = 0.066), but EC/TC of biomass carbon did not decrease but rose slightly from 43.6% to 44.5% (p > 0.5). Thus, the relative ratios of components of carbonaceous aerosol particles, such as 14C, could provide a better understanding of the atmospheric pollution status, despite short-term fluctuations, than do measurements of absolute concentrations.