Paquet L, Bergeron F, Boudreault A, Seidah N G, Chrétien M, Mbikay M, Lazure C
Laboratory of Structure and Metabolism of Neuropeptides, Clinical Research Institute of Montréal, University of Montréal, Québec, Canada.
J Biol Chem. 1994 Jul 29;269(30):19279-85.
The neuroendocrine granule-associated protein 7B2, unlike many other neuroendocrine precursor proteins stored in secretory granules, carries in its primary structure the Arg-Xaa-Arg/Lys-Arg processing site usually found in constitutively secreted precursor proteins and recognized by the ubiquitously expressed convertase, furin. pro7B2 (30 kDa), when expressed in endocrine (AtT-20, PC12, and GH4C1) or non-endocrine (Ltk-) cell lines using recombinant vaccinia viruses, was converted to a 23-kDa form. Mutation of the P4 Arg to Gly completely prevented this conversion. When excess pro7B2 was coexpressed with the pro-protein convertases PC1, PC2, or furin, only furin could induce complete processing. In addition, coexpression of pro7B2 in LoVo cells, which are devoid of endogenous furin activity, with each one of the three convertases, showed that only furin was able to induce processing of this precursor. pro7B2 processing in AtT-20 was completely abolished when protein transport into Golgi compartments was blocked by cell incubation at either 15 or 37 degrees C in the presence of monensin or brefeldin A. Furthermore, pulse-chase experiments in the presence of Na2[35S]SO4 showed that pro7B2 is Tyr-sulfated in the trans-Golgi network before it is processed. These results demonstrate that pro7B2 is first processed by a furin-like enzyme within the trans-Golgi network into a 23-kDa form that is then sequestered into secretory granules.
神经内分泌颗粒相关蛋白7B2与许多其他储存在分泌颗粒中的神经内分泌前体蛋白不同，其一级结构中带有通常在组成型分泌前体蛋白中发现的Arg-Xaa-Arg/Lys-Arg加工位点，该位点可被普遍表达的转化酶弗林蛋白酶识别。使用重组痘苗病毒在内分泌（AtT-20、PC12和GH4C1）或非内分泌（Ltk-）细胞系中表达时，pro7B2（30 kDa）会转化为23 kDa的形式。将P4位的精氨酸突变为甘氨酸可完全阻止这种转化。当过量的pro7B2与前体蛋白转化酶PC1、PC2或弗林蛋白酶共表达时，只有弗林蛋白酶能诱导完全加工。此外，在缺乏内源性弗林蛋白酶活性的LoVo细胞中，将pro7B2与这三种转化酶中的每一种共表达，结果表明只有弗林蛋白酶能够诱导该前体的加工。当在15或37℃下于莫能菌素或布雷菲德菌素A存在的情况下对细胞进行孵育，从而阻断蛋白质向高尔基体区室的转运时，AtT-20细胞中pro7B2的加工完全被消除。此外，在Na2[35S]SO4存在的情况下进行的脉冲追踪实验表明，pro7B2在被加工之前在反式高尔基体网络中发生了酪氨酸硫酸化。这些结果表明，pro7B2首先在反式高尔基体网络中被一种类弗林蛋白酶加工成23 kDa的形式，然后被隔离到分泌颗粒中。
