Metalloprotease-disintegrin MDC9: Intracellular maturation and catalytic activity

Metalloprotease disintegrins are a family of membrane-anchored glycoprotein s that are known to function in fertilization, myoblast fusion, neurogenesi s, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we re port 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 convert ase 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 cata lytically active. Soluble MDC9 appears to have distinct specificities for c leaving candidate substrate peptides compared with the TNF-cu convertase (T ACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one cas e with up to 50-fold selectivity for MDC9 versus TACE, Peptides mimicking t he predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation o f metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MD C9 is shown to become phosphorylated when cells are treated with the phorbo l ester phorbol la-myristate 13-acetate, a known inducer of protein ectodom ain shedding. This work implies that removal of the inhibitory pro-domain o f MDC9 by a fur-in-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 dif ferent substrates than the related TNF-alpha-convertase.