THE PRO DOMAIN OF PRE-PRO-TRANSFORMING GROWTH FACTOR-BETA-1 WHEN INDEPENDENTLY EXPRESSED IS A FUNCTIONAL BINDING-PROTEIN FOR THE MATURE GROWTH-FACTOR
THE PRO DOMAIN OF PRE-PRO-TRANSFORMING GROWTH FACTOR-BETA-1 WHEN INDEPENDENTLY EXPRESSED IS A FUNCTIONAL BINDING-PROTEIN FOR THE MATURE GROWTH-FACTOR
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DOI：
10.1021/bi00481a014
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发表时间：
1990-07-24
期刊：
影响因子：
2.9
通讯作者：
NASH, BW
中科院分区：
文献类型：
--
作者：
GENTRY, LE;NASH, BW
转化生长因子β1(TGF-β1)通过蛋白水解衍生自称为前原-TGF-β1的390个氨基酸前体分子的羧基末端。先前的研究表明，前TGF-β.1原的前片段可能在无活性的潜在复合物的形成中发挥重要作用。这些潜在形式被认为对于TGF-β1活性的调节很重要。为了更详细地了解这种潜在复合物，我们在组织培养细胞中独立于成熟生长因子表达了前原TGF-.beta.1的前结构域。通过将Arg-278密码子从CGA改变为终止密码子TGA，将终止密码子基因工程化到前TGF-β.1原的cDNA中。所得蛋白质在成熟生长因子的氨基末端丙氨酸残基之前被截短。瞬时表达研究和免疫印迹表明该前体片段很容易由 COS-1 细胞制造和分泌；当通过 SDS 聚丙烯酰胺凝胶电泳分析时，表达的 Pro 片段的主要形式表现为二硫键连接的二聚体 (Mr 80 000)。使用水貂肺指示细胞的生物测定表明，pro 结构域与外源添加的成熟 TGF-β1 形成无活性复合物。用热或酸处理该复合物导致活性TGF-β1的释放，表明其体外结构类似于天然的潜在TGF-β1复合物。还发现前体TGF-β1与两个密切相关的家族成员TGF-β1.2和TGF-β2形成潜在结构。使用放射性碘标记的TGF-β1的交联研究表明原结构域的二聚体与成熟TGF-β1之间存在一对一的关联。
Transforming growth factor .beta.1 (TGF-.beta.1) is proteolytically derived from the carboxyl terminus of a 390 amino acid precursor molecule termed pre-pro-TGF-.beta.1. Previous studies have suggested that the pro piece of pre-pro-TGF-.beta.1 may play an important role in the formation of an inactive, latent complex. These latent forms are thought to be important in the regulation of TGF-.beta.1 activity. To understand this latent complex in more detail, we have expressed the pro domain of pre-pro-TGF-.beta.1 in tissue culture cells independent of the mature growth factor. A stop codon was genetically engineerred into the cDNA of pre-pro-TGF-.beta.1 by changing the Arg-278 codon from CGA to the STOP codon TGA. The resulting protein is truncated just prior to the amino-terminal Ala residue of the mature growth factor. Transient expression studies and immunoblotting indicate that this pro piece is readily made and secreted by the COS-1 cells; the major form of the expressed pro piece, when analyzed by SDS-polyacrylamide gel electrophoresis, behaves as a disulfide-linked dimer (Mr 80 000). Bioassays, using mink lung indicator cells, reveal that the pro domain forms an inactive complex with exogenously added mature TGF-.beta.1. Treatment of this complex with heat or acid results in the release of active TGF-.beta.1, indicating an in vitro structure similar to natural, latent TGF-.beta.1 complexes. The pro piece TGF-.beta.1 was also found to form latent structures with two closely related family members, TGF-.beta.1.2 and TGF-.beta.2. Cross-linking studies using radioiodinated TGF-.beta.1 suggest a one to one association between the dimer of the pro domain and mature TGF-.beta.1.