Kusakabe Makoto, Cheong Pak-Leng, Nikfar Reza, McLennan Ian S, Koishi Kyoko
Department of Anatomy and Structural Biology, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
J Cell Biochem. 2008 Jan 1;103(1):311-20. doi: 10.1002/jcb.21407.
The TGF-beta family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-beta 1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-beta 2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-beta 2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-beta2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-beta 2 or a chimeric TGF-beta consisting of the TGF-beta1 LAP region, the TGF-beta 2 cleavage site and the TGF-beta 2 mature peptide. The constructs included a c-myc epitope tag in the N-terminal region of the mature peptide. The TGF-betas produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-beta 2, but efficiently processed the chimeric TGF-beta. This indicates that the insensitivity of both forms of TGF-beta 2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-beta1 and -beta 2 may be part of the mechanism that generates isoform-specific functions of the TGF-betas.
转化生长因子-β（TGF-β）家族成员最初是以潜在的前原多肽形式产生的。活性成熟肽通过细胞蛋白酶从潜在形式中切割出来。例如，TGF-β1主要由一种类枯草杆菌蛋白酶原蛋白转化酶弗林蛋白酶进行加工。TGF-β2有一个弗林蛋白酶的共有切割位点，因此被认为是由弗林蛋白酶切割的。然而，TGF-β2通常以潜在形式分泌，这似乎与其对弗林蛋白酶的假定敏感性不一致。我们在此报告，TGF-β2的常规（短）形式及其带有一个额外外显子的剪接变体（长形式）对弗林蛋白酶均不敏感。用含有TGF-β2短形式或长形式或由TGF-β1的潜伏相关肽（LAP）区域、TGF-β2切割位点和TGF-β2成熟肽组成的嵌合TGF-β的表达载体转染NIH 3T3和CHO细胞。构建体在成熟肽的N端区域包含一个c-myc表位标签。用蛋白质免疫印迹法和免疫细胞化学法分析转染细胞产生的TGF-β。从这些细胞中收获细胞内蛋白质并与弗林蛋白酶一起孵育。弗林蛋白酶只能低效地切割TGF-β2的长形式和短形式，但能有效地加工嵌合TGF-β。这表明TGF-β2的两种形式对弗林蛋白酶不敏感是其LAP区域三级结构的结果，而非其切割位点的原因。TGF-β1和-β2的这种差异加工可能是产生TGF-β亚型特异性功能机制的一部分。
