Milhiet P E, Chevallier S, Corbeil D, Seidah N G, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
Biochem J. 1995 Jul 15;309 ( Pt 2)(Pt 2):683-8. doi: 10.1042/bj3090683.
Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.
内肽酶-24.18（EC 3.4.24.18；meprin）是一种虾红素家族的多亚基金属肽酶。它存在于啮齿动物肾脏和人类肠道的刷状缘膜中。膜结合酶由α/β二聚体组成。分子克隆表明，两个亚基都具有相似的结构域组织。在体内和转染细胞中也观察到了可溶性α2二聚体。所有已知α亚基的结构在跨膜结构域上游都含有序列RXKR，它对应于弗林蛋白酶特异性切割的RXK/RR共有序列。为了研究这个假定的切割位点在内肽酶-24.18α亚基分泌过程中的作用，我们在COS-1细胞中表达了大鼠α亚基，其中R655或S656残基（在序列R652PKRS656内）分别突变为缬氨酸或亮氨酸。与野生型蛋白不同，αR655V和αS656L突变体没有分泌到培养基中。此外，当表达α亚基的细胞用编码弗林蛋白酶的痘苗病毒感染时，免疫印迹显示内肽酶-24.18α亚基主要细胞相关形式的分子量从98 kDa变为85 kDa，并且分泌的α亚基量增加。突变的α亚基未观察到这种分子量的变化。正如在98 kDa物种中观察到的那样，85 kDa细胞相关蛋白对内切糖苷酶H处理敏感，这表明蛋白水解切割发生在内质网或早期高尔基体区室中。使用PACE4和PC5代替弗林蛋白酶进行的类似实验表明，这些酶不能产生85 kDa的物种。我们得出结论，弗林蛋白酶很可能是参与导致大鼠内肽酶-24.18α亚基分泌的蛋白水解作用的细胞酶。
