Sugar-induced structural modifications of β₂GPI. (A) Bioinformatic analysis of potential glycation sites within the primary structure of β₂GPI. In the primary structure of human β₂GPI, the underlined sequence represents the signal peptide, the lysine residues (K) in bold and underlined indicate the potential glycation sites, and the cysteine residues (C) in bold and underlined indicate the residues involved in secondary structure Sushi domain formation. (B) SDS-PAGE analysis of human β₂GPI (left) and human serum albumin (right) incubated for 10 days at 37°C in the presence of 250mM glucose (G-β₂GPI) or mannitol (M-β₂GPI). The results of 1 representative experiment of 3 are shown. (C) Nontryptophan AGE fluorescence of β₂GPI. The emission spectra of G- and M-β₂GPI are reported. The protein was incubated with sugars (250mM) for 10 days at 37°C in the dark (see “Methods”), whereas sugars alone were incubated in separate tubes under the same experimental conditions. At the end of the incubation, the spectra were collected at an excitation of 370 nm, and the reported traces show the fluorescence emission after subtraction of the fluorescence due to the sugars alone. The results of 1 representative experiment of 3 are shown. (D) Dot-blot analysis of β₂GPI preparations. G-β₂GPI, M-β₂GPI, or native β₂GPI (0.5 μg) were spotted onto Immobilon-P strips. Each strip was exposed overnight to serum obtained from patients with APS or from control healthy subjects (diluted 1:100) at room temperature. Bound Abs were visualized with HRP-conjugated anti–human IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.43 software. The results of 1 representative experiment of 10 are shown.