Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.
Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2-benzyl-1-oxopropyl]- glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor-NEP complexes. In the present paper, we demonstrate that, for short incubation times, the fluorescent probe preferentially labeled brush border membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in videomicrofluorimetry suggesting that part of the inhibitor was bound or localized close to the basolateral plasma membrane. Confocal experiments confirmed the transcytosis of FTI/NEP complex, from the apical to the basolateral domain. Using [3H]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral membrane-bound molecules was estimated to be about 12 and 23%, respectively. The coincubation of the cells with FTI and 2B12, a monoclonal antibody raised against the rabbit enzyme, greatly modified the fluorescence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (> 1 h), in addition to the basolateral labeling, some intracellular fluorescent vesicles were observed essentially localized in the vicinity of the nucleus. The colocalization of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC-Con A) strongly suggests an endosomal/lysosomal internalization pathway when FTI was incubated in the presence of 2B12 mAb.
Milhiet
P E
PE
Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, URA D1500 CNRS, Faculté de Pharmacie, Paris, France.
Giocondi
M C
MC
Crine
P
P
Le Grimellec
C
C
Roques
B P
BP
Garbay-Jaureguiberry
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004285:Dogs; D005452:Fluoresceins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D015260:Neprilysin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D010670:Phenylthiourea / Q000031:analogs & derivatives
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; C077979:N-(fluoresceinyl)-N'-(1-(6-(3-mercapto-2-benzyl-1-oxopropyl)amino)-1-hexyl)thiocarbamide; D010670:Phenylthiourea; D015260:Neprilysin