Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells.
Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.
Lemay
G
G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Waksman
G
G
Roques
B P
BP
Crine
P
P
Boileau
G
G
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011333:Pro-Opiomelanocortin; D004247:DNA; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+); D015260:Neprilysin