Hernández-Ramírez Diego F, Olivares-Martínez Elizabeth, Núñez-Álvarez Carlos A, Chavelas Eneas A, García-Hernández Enrique, Gómez-Hernández Gregoria, Llorente Luis, Cabral Antonio R
Departament of Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zúbiran, Mexico.
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, México 04510, D.F., Mexico.
Biochem Biophys Res Commun. 2014 Oct 10;453(1):94-100. doi: 10.1016/j.bbrc.2014.09.064. Epub 2014 Sep 23.
Several studies have shown that conformational changes of β(2)-glycoprotein I (β(2)GPI) when bound to negatively charged components expose cryptic epitopes and subsequent binding of anti-β(2)GPI from patients with antiphospholipid syndrome (APS). However, the role of the carbohydrate chains of β(2)GPI in this anti-β(2)GPI reactivity is poorly understood. We therefore studied the reactivity and inhibition of anti-β(2)GPI antibodies from APS patients with native, partially glycosylated β(2)GPI (pdβ(2)GPI; without sialic acid) and completely deglycosylated β(2)GPI (cdβ(2)GPI). To determine the potential biologic importance of these glycoforms and their interaction with anti-β(2)GPI in vitro, stimulation assays were performed with the U937 cell line. Circular dichroism (CD) and fluorescence analysis of the three β(2)GPI forms were also studied. We found an increased reactivity of anti-β(2)GPI against pdβ(2)GPI and cdβ(2)GPI compared to native β(2)GPI. Both deglycosylated β(2)GPI isoforms showed higher inhibition of the anti-β(2)GPI reactivity than the native protein in soluble-phase. Likewise, the antibody/β(2)GPI/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 β(2)GPI. The differentiation and increased synthesis of pro-inflammatory cytokines by U937 cells in vitro may have pathogenetic implications.
多项研究表明，β2糖蛋白I（β2GPI）与带负电荷成分结合时的构象变化会暴露出隐蔽表位，随后抗磷脂综合征（APS）患者的抗β2GPI会与之结合。然而，β2GPI的糖链在这种抗β2GPI反应性中的作用却知之甚少。因此，我们研究了APS患者的抗β2GPI抗体与天然、部分糖基化的β2GPI（pdβ2GPI；不含唾液酸）和完全去糖基化的β2GPI（cdβ2GPI）的反应性及抑制情况。为了确定这些糖型的潜在生物学重要性及其在体外与抗β2GPI的相互作用，我们用U937细胞系进行了刺激试验。还研究了三种β2GPI形式的圆二色性（CD）和荧光分析。我们发现，与天然β2GPI相比，抗β2GPI对pdβ2GPI和cdβ2GPI的反应性增加。两种去糖基化的β2GPI异构体在可溶性相中对抗β2GPI反应性的抑制作用均高于天然蛋白。同样，抗体/β2GPI/糖型复合物增加了U937细胞中IL-6、IFNγ和TNFα的合成以及HLA-DR、CD14和CD11c的表达。糖型的CD和荧光研究在荧光信号上产生了相当大的变化。我们的研究表明，部分或完全去除糖链会暴露出β2GPI中存在的隐蔽表位。U937细胞在体外促炎细胞因子的分化和合成增加可能具有致病意义。
