PROTEOLYTIC PROCESSING OF THE ALPHA-SUBUNIT OF RAT ENDOPEPTIDASE-24.18 BY FURIN

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallope ptidase of the astacin family. It is found in brush-border membranes o f rodent kidney and human intestine. The membrane-bound enzyme is comp osed of alpha/beta dimers. Molecular cloning has shown that both subun its have a similar structural domain organization. Soluble alpha(2) di mers have also been observed in vivo and in transfected cells. The str uctures of all known alpha-subunits contain, upstream from the transme mbrane domain, the sequence RXKR, which corresponds to the RXK/RR cons ensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion proce ss of endopeptidase-24.18 alpha-subunit, we expressed in COS-1 cells r at alpha-subunits in which residues R(655) or S-656 (within the sequen ce R(652)pKRS656) were mutated to valine or leucine respectively. In c ontrast to the wild-type protein, the alpha R655V and alpha S656L muta nts were not secreted in the culture medium. Moreover, when cells expr essing 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 inc rease in the amounts of secreted alpha-subunit. This shift in molecula r mass was not observed with the mutant alpha-subunits. As observed fo r the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleav age occurred in the endoplasmic reticulum or in an early Golgi compart ment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We co nclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-s ubunit.