Roghani M, Becherer J D, Moss M L, Atherton R E, Erdjument-Bromage H, Arribas J, Blackburn R K, Weskamp G, Tempst P, Blobel C P
Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 1999 Feb 5;274(6):3531-40. doi: 10.1074/jbc.274.6.3531.
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-alpha convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-alpha-convertase.
金属蛋白酶解整合素是一类膜锚定糖蛋白，已知其在受精、成肌细胞融合、神经发生以及肿瘤坏死因子（TNF）-α的胞外域脱落过程中发挥作用。在此，我们报告了对广泛表达的金属蛋白酶解整合素MDC9的细胞内成熟过程和催化活性的分析。我们的结果表明，MDC9的前结构域在蛋白质出现在细胞表面之前，在分泌途径中被弗林蛋白酶样前体蛋白转化酶去除。MDC9的可溶性金属蛋白酶结构域能够切割胰岛素B链（一种通用的蛋白酶底物），这首次证明了MDC9具有催化活性。与TNF-α转化酶（TACE/ADAM17）相比，可溶性MDC9在切割候选底物肽方面似乎具有不同的特异性。MDC9的催化活性可被低纳摩尔浓度范围的异羟肟酸型金属蛋白酶抑制剂抑制，在一种情况下，对MDC9相对于TACE的选择性高达50倍。模拟MDC9或TACE预测的半胱氨酸开关区域的肽在低微摩尔浓度范围内抑制这两种酶，为通过半胱氨酸开关机制调节金属蛋白酶解整合素提供了实验证据。最后，当用佛波酯佛波醇12-肉豆蔻酸酯13-乙酸盐（一种已知的蛋白质胞外域脱落诱导剂）处理细胞时，MDC9会发生磷酸化。这项工作表明，在分泌途径中通过弗林蛋白酶样前体蛋白转化酶去除MDC9的抑制性前结构域是蛋白酶活性的先决条件。在前结构域去除后，诸如蛋白激酶C依赖性磷酸化等额外步骤可能参与调节MDC9的催化活性，MDC9可能靶向与相关TNF-α转化酶不同的底物。
