Identification and mutation of primary and secondary proteolytic cleavage sites in murine stem cell factor cDNA yields biologically active, cell-associated protein.
Identification and mutation of primary and secondary proteolytic cleavage sites in murine stem cell factor cDNA yields biologically active, cell-associated protein.
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DOI：
10.1016/s0021-9258(17)42248-4
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发表时间：
1994-01
期刊：
影响因子：
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通讯作者：
M. Majumdar;Lijun Feng;E. Medlock;Deniz Toksoz;David A. Williams
中科院分区：
文献类型：
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作者：
M. Majumdar;Lijun Feng;E. Medlock;Deniz Toksoz;David A. Williams
Steel小鼠黑素细胞、生殖细胞和造血细胞的表型异常证明了干细胞因子（SCF）在发育中的关键作用。造血微环境中SCF作为膜相关因子或可溶性因子的产生对造血干细胞和祖细胞产生多效性影响，并对红细胞的产生产生显著影响。尽管这两种形式的SCF的产生受到高度调控，但膜相关和可溶性SCF的生理作用尚不清楚。我们已经证明，来源于胎儿造血微环境的小鼠基质细胞产生可溶性小鼠SCF依赖于两个不同的蛋白水解裂解位点。外显子6的主要位点在这些细胞中被优先利用。位于外显子7的次要位点仅在没有主站点时使用。这一次要位点的蛋白水解过程似乎具有物种特异性，因为人类蛋白序列在这一位点存在差异，而在小鼠基质细胞中，编码这一位点的人类cDNA表达的蛋白在很大程度上仍与膜相关。对编码两个蛋白水解裂解位点的小鼠SCF cDNA进行定点诱变，可在小鼠基质细胞上产生膜相关的和具有生物活性的SCF。这些结果表明，对次级蛋白水解裂解位点加工的调控可能在膜相关SCF蛋白的功能中起关键作用。
Phenotypic abnormalities of melanocytes, germ cells, and hematopoietic cells of Steel mice demonstrate the critical role of stem cell factor (SCF) in development. Production of SCF in the hematopoietic microenvironment as either a membrane-associated or soluble factor leads to pleiotropic effects on hematopoietic stem and progenitor cells and significant effects on the production of erythroid cells. Although the production of these two forms of SCF is highly regulated, the physiologic role(s) of membrane-associated and soluble SCF remain unclear. We have demonstrated that the generation of soluble murine SCF by murine stromal cells derived from the fetal hematopoietic microenvironment is dependent on two distinct proteolytic cleavage sites. The primary site in exon 6 is preferentially utilized in these cells. The secondary site located in exon 7 is utilized only in the absence of the primary site. Proteolytic processing at this secondary site appears to be species-specific, since the human protein sequence diverges at this site, and protein expressed from the human cDNA encoding this site in murine stromal cells remains largely membrane-associated. Site-directed mutagenesis of the murine SCF cDNA encoding both proteolytic cleavage sites leads to the generation of membrane-associated and biologically active SCF on murine stromal cells. These results suggest that the regulation of processing of the secondary proteolytic cleavage site could play a critical role in the function of membrane-associated SCF protein.