Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11.
Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.
Lafrance
M H
MH
Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Vézina
C
C
Wang
Q
Q
Boileau
G
G
Crine
P
P
Lemay
G
G
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D004247:DNA; D015260:Neprilysin