Galanopoulou A S, Seidah N G, Patel Y C
Fraser Laboratories, McGill University Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada.
Biochem J. 1995 Jul 1;309 ( Pt 1)(Pt 1):33-40. doi: 10.1042/bj3090033.
We have previously reported that rat prosomatostatin (rPSS) undergoes conversion at Arg decreases and Lys decreases monobasic sites to SS-28 and PSS-(1-10) respectively in COS-7 cells, and have proposed furin or a related enzyme of the constitutive secretory pathway as the endoproteinase responsible. Here we have tested directly the ability of furin to cleave rPSS at the two monobasic sites as well as at the RXRK dibasic site of SS-14 conversion (a furin motif, except for Lys substituting for Arg at P1). Recombinant vaccinia virus (VV) vectors were used to co-express rPSS with graded doses of furin in COS-7 cells and LoVo colon carcinoma cells deficient in furin. PSS and cleavage products in cell extracts and media were characterized by HPLC analysis and C-terminal [SS-14-like immunoreactivity (SS-14 LI)] and N-terminal [PSS-(1-10) LI] directed radioimmunoassays. There was a dose-dependent increase in SS-28 production from rPSS by furin in COS-7 cells from 29% (control) to 58% (high-dose furin) associated with a progressive decrease in unprocessed PSS from > 60% to approximately 20% of total SS-14 LI. Significant SS-14 production occurred only at high levels of furin infection. Control LoVo cells infected with VV:rPSS exhibited production of approximately 21% SS-28, approximately 15% PSS-(1-10) and 3.5% SS-14. Infection of LoVo cells with VV:hfurin (hfurin = human furin) enhanced SS-28 production to 30-34%. SS-14 synthesis also increased to 25-40%, probably by conversion from SS-28. Overexpression of furin in COS-7 or LoVo cells failed to increase PSS-(1-10) production. These results show that furin is a candidate SS-28 convertase. Arginine is the preferred residue at the P1 site of furin cleavage. Furin does not process rPSS to PSS-(1-10), suggesting the existence of another monobasic convertase with a preference for Lys rather than Arg at P1. Such an enzyme could also explain the presence of endogenous SS-28-, PSS-(1-10)- and SS-14-producing activities in LoVo cells.
我们之前报道过，大鼠前生长抑素（rPSS）在COS-7细胞中，分别在精氨酸减少和赖氨酸减少的单碱性位点处发生转化，生成SS-28和PSS-(1-10)，并提出弗林蛋白酶或组成型分泌途径的相关酶作为负责的内切蛋白酶。在此，我们直接测试了弗林蛋白酶在两个单碱性位点以及SS-14转化的RXRK双碱性位点（一个弗林蛋白酶基序，除了在P1位点赖氨酸替代精氨酸）切割rPSS的能力。重组痘苗病毒（VV）载体用于在COS-7细胞和缺乏弗林蛋白酶的LoVo结肠癌细胞中，将rPSS与不同剂量的弗林蛋白酶共表达。通过高效液相色谱分析以及C末端[SS-14样免疫反应性（SS-14 LI）]和N末端[PSS-(1-10) LI]定向放射免疫测定法，对细胞提取物和培养基中的PSS和切割产物进行表征。在COS-7细胞中，弗林蛋白酶使rPSS产生SS-28的量呈剂量依赖性增加，从29%（对照）增加到58%（高剂量弗林蛋白酶），同时未加工的PSS从总SS-14 LI的>60%逐渐减少到约20%。仅在高水平的弗林蛋白酶感染时才会产生显著的SS-14。用VV:rPSS感染的对照LoVo细胞产生约21%的SS-28、约15%的PSS-(1-10)和3.5%的SS-14。用VV:hfurin（hfurin = 人弗林蛋白酶）感染LoVo细胞可使SS-28的产生增加到30 - 34%。SS-14的合成也增加到25 - 40%，可能是由SS-28转化而来。在COS-7或LoVo细胞中过表达弗林蛋白酶未能增加PSS-(1-10)的产生。这些结果表明，弗林蛋白酶是SS-28转化酶的候选者。精氨酸是弗林蛋白酶切割P1位点的首选残基。弗林蛋白酶不会将rPSS加工成PSS-(1-10)，这表明存在另一种在P1位点偏好赖氨酸而非精氨酸的单碱性转化酶。这样一种酶也可以解释LoVo细胞中内源性SS-28、PSS-(1-10)和SS-14产生活性的存在。
