Lusson J, Benjannet S, Hamelin J, Savaria D, Chrétien M, Seidah N G
J.A. DeSève Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7.
Biochem J. 1997 Sep 15;326 ( Pt 3)(Pt 3):737-44. doi: 10.1042/bj3260737.
In order to define the functional importance of the conserved RRGDL motif in the P-domain of the mammalian proprotein convertases(PCs) we generated and cellularly expressed three mutant PC1 vaccinia-virus (VV) recombinants: ARGDL-PC1, RAGDL-PC1 and RRGEL-PC1. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demonstrated that, in part, this effect was due to both an increased degradation of the mutant PC1s within the endoplasmic reticulum and to a diminished level of zymogen processing in the same compartment. In addition, within cells containing secretory granules such as PC12 and GH4C1 cells, such mutations prevented the C-terminal auto-processing of PC1 into the fully mature 66 kDa form stored in the secretory granules of regulated cells. Since the 66 kDa PC1 is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the generation of maximal intracellular PC1 activity. In regulated cells, co-expression of POMC with PC1 or its mutants together with the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PC1 mutants, whereas it only partially inhibited the ability of wild-type PC1 to process POMC. This suggests that RRGDL mutations prevent PC1 from entering secretory granules and hence the formation of the 66 kDa PC1, and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocytochemical data demonstrating that RRGDL mutants exhibit an intracellular localization pattern different from that of the granule-associated wild-type PC1,but similar to that of the Golgi-localized convertase PC5-B.
为了确定哺乳动物前体蛋白转化酶（PCs）P结构域中保守的RRGDL基序的功能重要性，我们构建并在细胞中表达了三种突变型PC1痘苗病毒（VV）重组体：ARGDL-PC1、RAGDL-PC1和RRGEL-PC1。在功能上，这些突变体导致促阿片黑素细胞皮质激素（POMC）加工成β-促脂素垂体激素（β-LPH）的水平降低，尤其是在组成性分泌的BSC40细胞中。脉冲追踪分析表明，这种效应部分是由于内质网中突变型PC1的降解增加以及同一区室中酶原加工水平降低所致。此外，在含有分泌颗粒的细胞如PC12和GH4C1细胞中，此类突变阻止了PC1的C末端自加工成储存于调节性细胞分泌颗粒中的完全成熟的66 kDa形式。由于66 kDa PC1是该酶最具活性的形式，因此有人提出RRGDL序列对于产生最大细胞内PC1活性至关重要。在调节性细胞中，POMC与PC1或其突变体以及主要在组成性分泌途径中起作用的通用PC抑制剂α1-抗胰蛋白酶波特兰（α1-PDX）共表达，结果表明后者完全抑制了PC1突变体形成β-LPH的能力，而它仅部分抑制野生型PC1加工POMC的能力。这表明RRGDL突变阻止PC1进入分泌颗粒，从而阻止66 kDa PC1的形成，并导致PC1突变体错误分选至组成性分泌途径。免疫细胞化学数据进一步支持了这一结论，该数据表明RRGDL突变体表现出与颗粒相关的野生型PC1不同的细胞内定位模式，但与高尔基体定位的转化酶PC5-B相似。
