Activation-induced aggregation and processing of the human Fas antigen - Detection with cytoplasmic domain-specific antibodies
Activation-induced aggregation and processing of the human Fas antigen - Detection with cytoplasmic domain-specific antibodies
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DOI：
10.1074/jbc.272.35.22307
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Yeh, ETH
中科院分区：
文献类型：
--
作者：
Kamitani, T;Nguyen, HP;Yeh, ETH
Fas（APO 1/CD 95）是一种1型跨膜蛋白，在受体介导的细胞凋亡中起重要作用。先前的研究已经表明，Pas在静息细胞中以单体形式存在，并且在交联时聚集以形成用于将另外的信号传导分子募集到细胞膜的复合物。为了研究受体活化后Pas抗原的分子命运，已经产生了对Fas的细胞死亡结构域特异性的单克隆抗体，该单克隆抗体（3D 5）可用于Western印迹分析，使用总细胞裂解物，以确定不同形式的Fas抗原，而无需免疫沉淀。受体交联后立即形成高分子量（>200 kDa）的抗SDS和β-巯基乙醇的Fas聚集体，约2 h后检测到97 kDa条带（p97），抗死亡结构域或C末端的抗体均能检测到p97，但抗胞外结构域的抗体不能沉淀p97。因此，p97最可能代表高分子量Fas聚集体的加工形式。尽管p97的产生遵循与CPP 32活化和聚（ADP-核糖）聚合酶切割类似的时间过程，但它不能被半胱氨酸蛋白酶、钙蛋白酶或蛋白酶体抑制剂抑制。
Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Pas exists in monomeric form in resting cells and aggregates upon cross-linking to form a complex that serves to recruit additional signaling molecules to the cell membrane, To study the molecular fate of the Pas antigen following receptor activation, a monoclonal antibody specific for the cell death domain of Fas has been generated, This monoclonal antibody (3D5) could be used in Western blot analysis using total cell lysates to identify different forms of Fas antigens without immunoprecipitation. High molecular mass (>200 kDa), SDS- and beta-mercaptoethanol-resistant Fas aggregates were formed immediately following receptor cross-linking, and a 97-kDa band (p97) was detected about 2 h later, p97 could be detected by antibodies against either the death domain or the C terminus, However, p97 could not be precipitated by antiextracellular domain antibodies. Thus, p97 most likely represents a processed form of the high molecular weight Fas aggregates, Although p97 generation followed a similar time course as CPP32 activation and poly(ADP-ribose) polymerase cleavage, it could not be inhibited by cysteine protease, calpain, or proteasome inhibitors.