The role of β2-glycoprotein I (β2GPI) carbohydrate chains in the reactivity of anti-β2GPI antibodies from patients with primary antiphospholipid syndrome and in the activation and differentiation of U937 cells

• Removal of the carbohydrate chains uncovers cryptic epitopes present in β2GPI.
• Anti-β2GPI-β2GPI glycoforms induced U937 cell differentiation and cytokine release.
• Sialic acid removal is enough for inducing the immune and biologic phenomena.

Several studies have shown that conformational changes of β2-glycoprotein I (β2GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-β2GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of β2GPI in this anti-β2GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-β2GPI antibodies from APS patients with native, partially glycosylated β2GPI (pdβ2GPI; without sialic acid) and completely deglycosylated β2GPI (cdβ2GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-β2GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three β2GPI forms were also studied. We found an increased reactivity of anti-β2GPI against pdβ2GPI and cdβ2GPI compared to native β2GPI. Both deglycosylated β2GPI isoforms showed higher inhibition of the anti-β2GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/β2GPI/glycoform complexes increased the synthesis of IL-6, IFNγ and TNFα and the expression of HLA-DR, CD14 and CD11c in U937 cells. CD and fluorescence studies of the glycoforms yielded considerable changes in the fluorescence signals. Our work suggests that the partial or complete removal of the carbohydrate chains uncover cryptic epitopes present in β2GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.