Galanopoulou A S, Kent G, Rabbani S N, Seidah N G, Patel Y C
Fraser Laboratories, McGill University, Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada.
J Biol Chem. 1993 Mar 15;268(8):6041-9.
Mammalian prosomatostatin (PSS) is cleaved at a dibasic Arg-Lys site to produce somatostatin-14 (SS-14) and at monobasic Arg and Lys sites to yield SS-28 and PSS(1-10) (antrin), respectively. Furin, PC1, and PC2 are three recently discovered mammalian endoproteases localized either to the constitutive (furin) or regulated (PC1, PC2) secretory pathways. In this study we have compared the heterologous processing of PSS in transiently transfected endocrine (AtT-20 pituitary) and nonendocrine (COS-7 monkey kidney, PC12 pheochromocytoma) tumor cells. We have correlated the efficiency of processing of PSS to SS-14, SS-28, and PSS(1-10) with (i) secretion through the constitutive or regulated pathways; (ii) endogenous expression of mRNA for furin, PC1, and PC2; and (iii) exogenous expression of PC1 and PC2 in cells that do not contain these enzymes in order to delineate the putative role of these enzymes in mediating PSS cleavage at dibasic and monobasic sites and to localize the proteolytic events to specific compartments of the secretory pathways. COS-7 and PC12 cells expressed only furin, secreted constitutively, and processed PSS preferentially at monobasic sites to SS-28 (40-43%) and antrin (27-29%). Processing, however, was inefficient as suggested by large amounts of unprocessed PSS. In contrast, AtT-20 cells showed regulated secretion, expressed all three endoproteases (with high levels of PC1), and processed PSS efficiently to mainly SS-14. PC1, but not PC2, exogenously coexpressed with PSS in COS-7 cells produced significant conversion to SS-14 but not SS-28. This study shows that PSS is capable of monobasic cleavage in the constitutive secretory pathway. Such processing could be mediated by a furin-like enzyme but is relatively inefficient. PC1 can effect dibasic cleavage of PSS whereas PC2 is without influence on PSS processing at least within the constitutive secretory pathway. Although monobasic and dibasic processing of PSS in COS-7 cells correlates with furin-like and PC1 activity, respectively, the relative inefficiency of such processing suggests that compartmentalization of proteolytic events in secretory vesicles or other more specific endoproteases may be required.
哺乳动物前生长抑素（PSS）在一个双碱性精氨酸 - 赖氨酸位点被切割产生生长抑素 - 14（SS - 14），在单碱性精氨酸和赖氨酸位点被切割分别产生SS - 28和PSS(1 - 10)（胃泌素释放肽）。弗林蛋白酶、PC1和PC2是最近发现的三种哺乳动物内切蛋白酶，它们分别定位于组成型（弗林蛋白酶）或调节型（PC1、PC2）分泌途径。在本研究中，我们比较了在瞬时转染的内分泌（AtT - 20垂体）和非内分泌（COS - 7猴肾、PC12嗜铬细胞瘤）肿瘤细胞中PSS的异源加工过程。我们将PSS加工成SS - 14、SS - 28和PSS(1 - 10)的效率与以下因素相关联：（i）通过组成型或调节型途径的分泌；（ii）弗林蛋白酶、PC1和PC2的mRNA的内源性表达；（iii）在不含这些酶的细胞中外源表达PC1和PC2，以便阐明这些酶在介导PSS在双碱性和单碱性位点切割中的假定作用，并将蛋白水解事件定位到分泌途径的特定区室。COS - 7和PC12细胞仅表达弗林蛋白酶，组成型分泌，并且优先在单碱性位点将PSS加工成SS - 28（40 - 43%）和胃泌素释放肽（27 - 29%）。然而，大量未加工的PSS表明加工效率低下。相比之下，AtT - 20细胞表现出调节型分泌，表达所有三种内切蛋白酶（PC1水平较高），并有效地将PSS主要加工成SS - 14。在COS - 7细胞中与PSS共外源性表达的PC1而非PC2导致向SS - 14的显著转化，但未转化为SS - 28。本研究表明，PSS能够在组成型分泌途径中进行单碱性切割。这种加工可能由一种类似于弗林蛋白酶的酶介导，但效率相对较低。PC1可以影响PSS的双碱性切割，而PC2至少在组成型分泌途径内对PSS加工没有影响。尽管COS - 7细胞中PSS的单碱性和双碱性加工分别与类似弗林蛋白酶和PC1的活性相关，但这种加工的相对低效表明可能需要分泌小泡中蛋白水解事件的区室化或其他更特异性的内切蛋白酶。
