Molecular mechanism of cellular processing of tumor necrosis factor (TNF)

肿瘤坏死因子（TNF）细胞加工的分子机制

基本信息

批准号：
10660092
负责人：
UTSUMI Toshihiko
金额：
$ 2.37万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

To determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we have constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells.The in vitro translation/translocation assay using a canine pancreatic microsomal membrane system with a mutant, Δ-75--47,-32--1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence ; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside of the leader sequence of this mutant. When the mutant Δ-75--47, -32--1 was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment.In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.

为了确定原肿瘤坏死因子 (TNF) 的 76 个残基前导序列对于穿过内质网 (ER) 的膜易位和原 TNF 成熟的最低要求，我们构建了原 TNF 突变体，其中pro-TNF 的部分跨膜结构域直接连接到成熟结构域的 N 末端，并评估了它们跨 ER 膜的易位行为以及它们从转染细胞中的分泌使用具有突变体Δ-75--47,-32--1的犬胰腺微粒体膜系统进行的体外翻译/易位测定表明，TNF原跨膜结构域的N端一半由以下组成： 14个残基充当可切割信号序列；它产生了分子量与成熟TNF相似的切割形式。通过定点分析切割位点。诱变表明该位点位于该突变体的前导序列内部。当突变体Δ-75--47、-32--1在COS-1细胞中表达时，进一步观察到具有生物活性的可溶性TNF的有效分泌。从该突变体中删除疏水结构域抑制了转位，表明一定程度的疏水性对于 TNF 成熟结构域的膜转位是必不可少的。因此，跨膜结构域的 N 端一半。当与 TNF 的成熟结构域连接时，pro-TNF 的 14 个残基可以作为可裂解的信号序列发挥作用，并且可以通过该 14 个残基疏水片段实现 TNF 生物活性分泌形式的分泌。然而，在完整的 pro-TNF 中，这 14 -残基序列在细胞内加工过程中不能充当可裂解的信号序列，这表明 pro-TNF 的 76 个残基前导序列的其余部分抑制信号肽裂解并使得前导序列充当 II 型信号锚序列，产生跨膜形式的 TNF。