Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases
Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases
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DOI：
10.1042/bj3140951
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发表时间：
1996-03-15
影响因子：
4.1
通讯作者：
Murphy, RA
中科院分区：
文献类型：
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作者：
Seidah, NG;Benjannet, S;Murphy, RA
为了确定负责神经生长因子前体（proNGF）成熟的酶，在含有调节的和/或组成性分泌途径的细胞中使用牛痘病毒表达系统分析了其生物合成和通过前蛋白转化酶弗林蛋白酶、PC 1、PG 2、PAGE 4、PC 5和PC 5同种型PC 5/6-B的细胞内加工。结果表明，在两种细胞类型中，弗林蛋白酶以及在较小程度上的PACE 4和PC 5/6-B是最佳候选的proNGF转化酶。此外，16.5和13.5 kDa的两种加工的NGF形式在组成型分泌细胞系如LoVo和BSC 40细胞中是明显的，而在AtT 20细胞中仅观察到13.5 kDa的形式，其含有分泌颗粒。这两种形式显示出与成熟NGF相同的N-末端序列，并且也是在将NGF的C-末端Arg-Arg序列定点突变成Ala-Ala后产生的，这表明它们之间的差异不在C-末端。在BSC 40细胞中用弗林蛋白酶和嗜铬粒蛋白B或分泌粒蛋白II（但不含嗜铬粒蛋白A）表达proNGF消除了16.5 kDa形式。数据还显示，proNGF的前片段的N-糖基化和寡糖链的修剪对于该前体从内质网退出及其最终加工和分泌是必要的。硫酸盐标记实验表明，前体到达trans-Golgi网络后，proNGF被加工成成熟的NGF。该比较研究表明，鉴定的三种候选哺乳动物枯草杆菌蛋白酶/kexin样转化酶将proNGF加工成NGF，并且最终加工产物的性质取决于细胞内环境。
In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PG2, PAGE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Go-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment.