Intracellular activation of human adamalysin 19/disintegrin and metalloproteinase 19 by furin occurs via one of the two consecutive recognition sites
Intracellular activation of human adamalysin 19/disintegrin and metalloproteinase 19 by furin occurs via one of the two consecutive recognition sites
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DOI：
10.1074/jbc.m203532200
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发表时间：
2002-07-12
影响因子：
4.8
通讯作者：
Sang, QXA
中科院分区：
文献类型：
--
作者：
Kang, TB;Zhao, YG;Sang, QXA
Adamalysin 19（一种去整合素和金属蛋白酶19，ADAM 19或meltrin beta）是一种质膜金属蛋白酶。人ADAM 19酶原在其原结构域和催化结构域之间含有两个潜在的弗林蛋白酶识别位点（RX（K/R）R），（KRPRR）-K-196-R-200和（RRMKR）-R-199-R-203。蛋白质N-末端测序显示hADAM 19的细胞成熟形式起始于（EDLNSMK）-E-204，表明优选的弗林蛋白酶切割位点是（200）RMK（203）R向下箭头（204）EDLN。这些成熟的形式具有催化活性。α（1）-蛋白酶抑制剂Pittsburgh突变体和dee-Arg-Val-Lys-Arg-chloromethyl ketone（dee-Arg-Val-Lys-Arg-chloromethyl ketone）均能阻断hADAM 19的激活。hADAM 19的激活也被布雷菲德菌素A阻断，布雷菲德菌素A抑制蛋白质从内质网运输到高尔基体，或A23187，一种已知抑制弗林蛋白酶自激活的钙离子载体。当（KR）-K-202突变为AA时，酶原也被激活，表明（RPRR）-R-197是一个替代的激活位点。此外，在（RR）-R-199到AA突变体中仅检测到hADAM 19的前体形式，其消除了两个弗林蛋白酶识别位点。此外，在两个弗林蛋白酶缺陷的哺乳动物细胞系中，酶原没有转化为它们的活性形式;在这两个细胞系中hADAM 19和弗林蛋白酶的共表达恢复了酶原活化。最后，弗林蛋白酶和hADAM 19之间的共定位被确定在内质网-高尔基体复合体和/或trans-Golgi网络。这份报告是第一次彻底调查的细胞内激活adamalysin 19，证明弗林蛋白酶激活pro-hADAM 19的分泌途径，通过两个连续的弗林蛋白酶识别位点之一。
Adamalysin 19 (a disintegrin and metalloproteinase 19, ADAM19, or meltrin beta) is a plasma membrane metalloproteinase. Human ADAM19 zymogen contains two potential furin recognition sites (RX(K/R)R), (KRPRR)-K-196-R-200 and (RRMKR)-R-199-R-203, between its pro- and catalytic domains. Protein N-terminal sequencing revealed that the cellular mature forms of hADAM19 started at (EDLNSMK)-E-204, demonstrating that the preferred furin cleavage site was the (200)RMK(203)Rdown arrow(204)EDLN. Those mature forms were catalytically active. Both Pittsburgh mutant of alpha(1)-proteinase inhibitor and dee-Arg-Val-Lys-Arg-chloromethyl ketone, two specific furin inhibitors, blocked the activation of hADAM19. Activation of hADAM19 was also blocked by brefeldin A, which inhibits protein trafficking from the endoplasmic reticulum to the Golgi, or A23187, a calcium ionophore known to inhibit the autoactivation of furin. When (KR)-K-202 were mutated to AA, the proenzyme was also activated, suggesting that (RPRR)-R-197 is an alternative activation site. Furthermore, only pro-forms of hADAM19 were detected in the (RR)-R-199 to AA mutant, which abolished both furin recognition sites. Moreover, the zymogens were not converted into their active forms in two furin-deficient mammalian cell lines; co-expression of hADAM19 and furin in these two cell lines restored zymogen activation. Finally, co-localization between furin and hADAM19 was identified in the endoplasmic reticulum-Golgi complex and/or the trans-Golgi network. This report is the first thorough investigation of the intracellular activation of adamalysin 19, demonstrating that furin activated pro-hADAM19 in the secretory pathway via one of the two consecutive furin recognition sites.