Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1.
Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1.
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DOI：
10.1083/jcb.114.4.827
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发表时间：
1991-08
期刊：
影响因子：
--
通讯作者：
Gentry LE
中科院分区：
文献类型：
--
作者：
Sha X;Yang L;Gentry LE
在猴转化生长因子β1基因编码的转化生长因子β1原结构域，制备了一系列定点插入和缺失突变体。这些突变体在COS-1细胞中瞬时表达，并通过细胞和培养上清液的免疫印迹分析确定每个突变体被正确加工、正确折叠和分泌的能力。插入对应于氨基酸残基50、154和170的区域阻断了分泌；COS-1细胞的培养上清液没有显示免疫活性蛋白，而完整的细胞含有高水平的突变多肽。在原结构域中间第81、85和144位的插入影响成熟的转化生长因子β1的二硫键成熟。另一方面，在第110位插入似乎破坏成熟的转化生长因子β1多肽的稳定性，导致降解的生长因子。转化生长因子β1原结构域相对较小(10个氨基酸)到较大(125个氨基酸)的缺失突变，当表达为全长前转化生长因子β1时，似乎阻止分泌。相反，如果原结构域(命名为β1潜伏期相关肽[β1-LAP])是独立表达的，则区域40-110的缺失突变很容易由COS-1细胞分泌，而残基110-210的缺失要么破坏蛋白质结构的稳定，要么阻止其细胞内转运。使用放射性碘标记的转化生长因子β1的交联分析和生物测定表明，与成熟的转化生长因子β1结合需要50-85个残基β1-LAP。
A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110- 210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1- LAP are required for association with mature TGF beta 1.