Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains.
Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.
Corbeil
D
D
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
Boileau
G
G
Lemay
G
G
Crine
P
P
D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D011333:Pro-Opiomelanocortin; D015260:Neprilysin