CONFORMATIONAL EPITOPES OF PEMPHIGUS ANTIGENS (DSG1 AND DSG3) ARE CALCIUM-DEPENDENT AND GLYCOSYLATION INDEPENDENT
CONFORMATIONAL EPITOPES OF PEMPHIGUS ANTIGENS (DSG1 AND DSG3) ARE CALCIUM-DEPENDENT AND GLYCOSYLATION INDEPENDENT
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DOI：
10.1111/1523-1747.ep12317587
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发表时间：
1995-08-01
影响因子：
6.5
通讯作者：
NISHIKAWA, T
中科院分区：
文献类型：
--
作者：
AMAGAI, M;ISHII, K;NISHIKAWA, T
天疱疮自身抗体的靶分子是跨膜桥粒组分，寻常型天疱疮（PV）中的桥粒芯糖蛋白3（Dsg 3）和落叶型天疱疮（PF）中的Dsg 1。在这项研究中，我们研究了钙和糖基化对天疱疮抗原的抗原性和构象表位的产生的影响。我们使用重组杆状病毒蛋白，PVIg和PFIg，这被认为是准确地反映其各自的蛋白Dsg 3和Dsg 1的胞外结构域的天然构象。这些杆状蛋白可以免疫吸附来自PV和PF患者相应血清的异质性自身抗体，完全阻断正常人皮肤的间接免疫荧光染色。使用乙二胺四乙酸（EDTA）或乙二醇-双（β-氨基乙基醚）-N，N，N '，N'-四乙酸（EGTA）从含有杆状蛋白的溶液中螯合钙，消除了PVIg和PFIg的免疫吸附;然而，对1 mM钙透析后，杆状蛋白的免疫吸附恢复。在衣霉素存在下产生的两种杆状蛋白的非糖基化形式保留了它们的免疫吸附能力。此外，免疫吸附的杆状蛋白被阻止不可逆的低pH值，高pH值，煮沸，但不是用非离子型去污剂Nonidet P-40的治疗。这些发现表明天疱疮抗原上构象表位的形成依赖于钙，但不依赖于糖基化，并提供直接证据表明钙在决定天疱疮抗原的抗原特性中起重要作用。
The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the antigenicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculoproteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens.