INSERTION OF HYDROPHILIC AMINO-ACID-RESIDUES IN THE SIGNAL PEPTIDE MEMBRANE ANCHOR DOMAIN OF NEPRILYSIN (NEUTRAL ENDOPEPTIDASE-24,11) RESULTS IN ITS CLEAVAGE - ROLE OF THE POSITION OF INSERTION

We have expressed in COS-1 cells mutants of neprilysin (neutral endope ptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was eith er substituted for V-42-T-M-I or inserted after T-38 in the signal pep tide/membrane anchor (SA) domain. These mutations were introduced in f ull-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP Delta cyto(H1) and NEP Delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP Delta cyto(H1), NEP(H2), and NEP Delta cyto(H 2) were secreted. Furthermore, carbonate treatment of isolated intrace llular membranes suggested that cleavage of the SA domain was performe d in the endoplasmic reticulum, presumably by signal peptidase. Sequen cing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala(46) but also at the carbox y side of AIa(41) in NEP(H2) and NEP Delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of t he cleavage site and, in the case of NEP(H1) and NEP(H2), the efficien cy of cleavage of the SA domain. (C) 1994 Academic Press, Inc.