Itai T, Tanaka M, Nagata S
Department of Genetics, Osaka University Medical School, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka, Japan.
Eur J Biochem. 2001 Apr;268(7):2074-82. doi: 10.1046/j.1432-1327.2001.02085.x.
Tumour necrosis factor (TNF)-alpha-converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processing of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-alpha, although the culture medium of sTACE-transfected cells could cleave a peptide containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-alpha indicated that both mTACE and sTACE could interact with the proform of TNF-alpha. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-alpha, showed a dominant negative effect on not only TNF-alpha secretion but also FasL secretion. These results suggest that binding of the membrane-anchored but not the soluble form of TACE to TNF-alpha results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.
肿瘤坏死因子（TNF）-α转换酶（TACE）是一种膜蛋白，属于ADAM（一种去整合素和金属蛋白酶）家族，可切割多种膜蛋白，包括TNF-α的前体形式。在本研究中，我们构建了膜结合全长TACE（mTACE）及其截短的可溶性形式（sTACE）的表达载体。当将人TNF-α表达载体导入人293细胞时，共表达mTACE可增强TNF-α向其成熟形式的加工，并且这种加工受到金属蛋白酶抑制剂的抑制。另一方面，尽管sTACE转染细胞的培养基可切割含有TNF-α切割位点的肽，但共表达sTACE对TNF-α的加工没有影响。Fas配体（FasL）转染的293细胞释放出大量可溶性FasL，共表达mTACE和sTACE均未增强这种脱落。用TACE和TNF-α共转染的细胞进行免疫沉淀和蛋白质印迹分析表明，mTACE和sTACE均可与TNF-α的前体形式相互作用。在相同的分析中，mTACE和sTACE均未与FasL相互作用。缺乏催化结构域的TACE突变体也可与TNF-α相互作用，不仅对TNF-α分泌，而且对FasL分泌均显示出显性负效应。这些结果表明，膜锚定形式而非可溶性形式的TACE与TNF-α的结合导致有效的胞外域脱落，并且FasL分泌酶是一种与TACE相似但不完全相同的金属蛋白酶。
