Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.
Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.
Département de Chimie Organique, U266 INSERM, URA D1500 CNRS, Faculté de Pharmacie, Paris, France.
P E
PE
Milhiet
F
F
Dennin
M C
MC
Giocondi
C
C
Le Grimellec
C
C
Garbay-Jaureguiberry
C
C
Boucheix
B P
BP
Roques
D000626:Aminopeptidases / Q000032:analysis; D000818:Animals; D018826:CD13 Antigens; D002478:Cells, Cultured; D004285:Dogs; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D015260:Neprilysin / Q000032:analysis* / Q000037:antagonists & inhibitors; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D010670:Phenylthiourea / Q000031:analogs & derivatives; D010784:Photomicrography; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D005456:Fluorescent Dyes; C077979:N-(fluoresceinyl)-N'-(1-(6-(3-mercapto-2-benzyl-1-oxopropyl)amino)-1-hexyl)thiocarbamide; D010670:Phenylthiourea; D000626:Aminopeptidases; D018826:CD13 Antigens; D007703:Peptidyl-Dipeptidase A; D015260:Neprilysin