EXPRESSION OF MEPRIN SUBUNIT PRECURSORS - MEMBRANE ANCHORING THROUGH THE BETA-SUBUNIT AND MECHANISM OF ZYMOGEN ACTIVATION

The biosynthesis of the membrane-bound metalloendopeptidase meprin was studied after the introduction of cDNAs encoding the rat meprin alpha and beta subunits into human 293 cells. When expressed individually t he meprin a subunit was found to be primarily secreted into the cultur e medium, while the beta subunit was determined to be an integral memb rane protein, Coexpression of the alpha and beta subunits results in t he localization of both subunits to the plasma membrane. In this case the alpha subunit is specifically released from the cell surface by di thiothreitol, indicating the a subunit is associated with the membrane via disulfide bond(s) to the beta subunit. Meprin expressed in 293 ce lls is similar to the rat kidney enzyme in that it forms disulfide-lin ked dimers and has a similar pattern of glycosylation. However, the ex pressed protein displayed no detectable peptidase activity against fou r meprin substrates. Processing of the a subunit was followed after th e introduction of sequences coding for the human c-myc peptide epitope EQKLISEEDL into its cDNA in the region of its prosequence and at the COOH terminus. Detection of the resulting proteins using a monoclonal an tibody specific for the c-myc epitope indicates the a subunit is pr ocessed at its COOH terminus but retains the prosequence which is abse nt from the enzyme purified from rat kidney. Limited trypsin digestion of meprin precursors expressed in 293 cells results in both the activ ation of the enzyme and the removal of the prosequence. This result su pports the hypothesis of Bode et al. (Bode W, Gomis-Ruth, F.X., Huber, R., Zwilling, R., and Stocker, W (1992) Nature 358, 164-167) that mep rin and other astacin family proteases require removal of NH2-terminal prosequences at the junction of the astacin protease domain for zymog en activation. Trypsin-activated meprin was assayed with the protein s ubstrate azocasein and three synthetic peptide substrates. The membran e-bound beta subunit was found to be more active than the secreted a s ubunit against azocasein but much less active toward the synthetic pep tide substrates.