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

为了确定前肿瘤坏死因子（pro-tumor necrosis factor，TNF）的76个残基的前导序列对于跨内质网（endoplasmic reticulum，ER）膜转运和pro-TNF成熟的最低要求，我们构建了pro-TNF突变体，其中pro-TNF的跨膜结构域的一部分直接连接到成熟结构域的N-末端，体外翻译/易位试验使用具有突变体Δ-75--47，-32--1，揭示了由14个残基组成的pro-TNF的跨膜结构域的N-末端一半作为可切割的信号序列发挥作用;它产生了具有与成熟TNF相似的分子量的TNF的切割形式。通过定点突变分析切割位点表明，该位点位于该突变体的前导序列内。当突变体Δ-75--47，-32--1在COS-1细胞中表达时，观察到生物活性可溶性TNF的有效分泌。进一步删除该突变体的疏水结构域抑制了易位，表明一定程度的疏水性是不可缺少的成熟结构域的TNF的膜易位。因此，pro-TNF的跨膜结构域的N-末端的一半可以作为一个可切割的信号序列，当连接到TNF的成熟结构域时，可以用这个14个残基的疏水片段实现TNF的生物活性分泌形式的分泌。然而，在完整的pro-TNF中，这个14个残基的序列在细胞内加工期间不能作为一个可切割的信号序列，表明pro-TNF的76-残基前导序列的剩余部分抑制信号肽切割，并使前导序列能够作为产生跨膜形式TNF的II型信号锚定序列发挥作用。

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.