TNF-α的合成分泌机制不同于一般的分泌型蛋白

The differences between mechanisms of TNF α synthesis and secretion and typical secretory protein

目的拟借助微粒体膜和体外转录翻译系统来研究分泌型ＴＮＦα（Ｓ－ＴＮＦ－α）的产生机制及Ｓ－ＴＮＦ－α和跨膜型ＴＮＦα（ＴＭ－ＴＮＦ－α）的关系。方法首先将分子量为１７×１０３Ｓ－ＴＮＦ（不含编码ＴＮＦ信号肽的基因）、２６×１０３ＴＭ－ＴＮＦ（含信号肽基因）和１７×１０３Ｓ－ＴＮＦ突变体（Ｓ－ＴＮＦｍ，用ＩＬ－２信号肽密码子置换ＴＮＦ信号肽序列）的全长ｃＤＮＡ片段分别亚克隆于含Ｔ７启动子的ｐＧＥＭ－３Ｚｆ载体，然后利用体外转录翻译系统，在微粒体膜存在和不存在的情况下，体外翻译合成Ｓ－ＴＮＦ、ＴＭ－ＴＮＦ及其Ｓ－ＴＮＦｍ。结果经Ｗｅｓｔｅｒｎｂｌｏｔ分析结果表明：微粒体的存在并不改变２６×１０３ＴＭ－ＴＮＦ的分子量，但微粒体的存在能将Ｓ－ＴＮＦｍ上的ＩＬ－２信号肽切除，证实ＴＮＦ引导序列与一般分泌性蛋白的“信号肽”不同，在粗面内质网翻译过程中不被切除。进一步酶切分析表明ＴＭ－ＴＮＦ是通过某些金属蛋白酶酶解作用而被转换成Ｓ－ＴＮＦ的。结论实验结果提示１７×１０３Ｓ－ＴＮＦ产生机制可能是：ＴＮＦ产生细胞经ＬＰＳ等激活后，导致ＴＮＦ基因转录翻译增加，首先形成２６×１０３ＴＮＦ，并借助其信号肽疏水氨基酸部分将之“锚定”?

Objective Activated monocytes/macrophages may express two different molecular forms of TNF α, a secretory 17kD form S TNF α and a tansmembrane 26kD form TM TNF α. It is however still a matter in dispute whether 26kD TNF is present as an integrated protein on the cell membrane.The present study is to explore the mechanisms underlying the production of S TNF α and the interrelation between these two forms of TNF α. Methods Full length cDNA fragment of S TNF(without genes encoding TNF signal peptide), 26kD TM TNF(with gene encoding signal peptide) and S TNF mutant(S TNFm, in which TNF signal peptide sequence had been displaced by that of IL 2) were subcloned respectively into the vector pGEM 3Zf containing a T7 promoter, respectively. S TNF, TM TNF and S TNFm were then in vitro translated and synthesized in the presence or absence of microsomes. Results As shown in Western blot analysis, the presence of microsomes did not alter the molecular weight of TM TNF , but it did result in the cleavage of the IL 2 signal peptide from S TNFm,suggesting that the leader sequence of TNF might differ from the signal peptide of typical secretory protein in that it seemed not to have undergone cleavage during translation in the rough faced endoplasmic reticulum. Further enzymatic analysis revealed that TM TNF was converted into S TNF through the effect of certain metalloproteinase. Conclusions These results suggest that the mechanisms of TNF α production may be as follows: Activation of TNF α producing cells by LPS leads to augmented transcription/translation of TNF α gene, resulting firstly in the formation of 26kD TNF α , which is then anchored to the cell membrane as a protein with the aid of the hydrophobic amino acids of its signal peptide. This transmembrane TNF α serves to mediate biological activities through direct cell to cell contact. Under the action of certain proteinase, signal peptide of TNF may be cleaved from the TM TNF, resulting in the formation of 17kD secretory TNF α,which is released into the body fluids, exerting its effect locally or systemically.