EXPRESSION OF RAT ENDOPEPTIDASE-24.18 IN COS-1 CELLS - MEMBRANE TOPOLOGY AND ACTIVITY

Endopeptidase-24.18 (E-34.18; EC3.4.24.18) is a metallopeptidase of th e astacin family and is highly expressed in kidney brush-border membra nes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/be ta dimers [(alpha/beta)(2)]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, t he cloned cDNAs for the rat alpha and beta subunits were transiently e xpressed either alone or together in COS-1 cells. Immunoblotting of ce ll extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-b ound. In alpha/beta-transfected cells, the alpha subunit remained memb rane-bound, but could be released from the cell surface after papain t reatment or after incubation with 10 mM dithiothreitol. Furthermore, m utants of the alpha subunit in which the putative C-terminal anchor do main was deleted could still form cell-associated alpha/beta dimers. T hese results are consistent with a topological model of E-24.18 in whi ch the beta subunit is anchored in the plasma membrane and the alpha s ubunit is retained at the cell surface through disulphide bridge(s) wi th the beta subunit. Both the alpha and beta recombinant subunits expr essed in COS-1 cells showed little azocasein-degrading activity. Howev er, activity of either individual subunits of alpha/beta dimers was in creased after mild trypsin digestion, suggesting that in COS-1 cells t he enzymes are synthesized as zymogens. Finally, inactivation of the a lpha subunit by site-directed mutagenesis of Glu-157, which is believe d to play a role in catalysis, showed that both subunits participate i n the enzymic activity of the heterodimer.