Expression of an enzymically 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 membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.