EXPRESSION OF AN ENZYMATICALLY ACTIVE GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED FORM OF NEUTRAL ENDOPEPTIDASE (EC-3.4.24.11) IN COS-1 CELLS

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membra ne protein found in a wide variety of cell types. We previously produc ed a secreted form of the enzyme by deletion of the cytoplasmic and tr ansmembrane domains and in-frame fusion of the cleavable signal peptid e of pro-opiomelanocortin [Lemay, Waksman, Rogues, Crine and Boileau ( 1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secrete d form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-a nchor attachment signal of decay-accelerating factor to produce a GPI- anchored form. Expression of this chimeric form in Cos-l cells resulte d in cell-surface activity. This activity could be released from the c ell surface by phosphatidylinositol-specific phospholipase C and radio labelling studies showed that the protein could incorporate [H-3]ethan olamine, indicating that the enzyme was GPI-anchored. The K-m value, u sing [D-Ala(2),Leu(5)]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 mu M) was comparable with that of wild-type NEP (70 +/- 4 mu M) , as were the sensitivities to the inhibitors phosphoramidon and thior phan. However, pulse-chase studies showed that the biosynthesis and ce ll-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a l ower rate of biosynthesis and/or cellular transport for GPI-anchored N EP compared with its transmembrane counterpart.