ANALYTICAL

BIOCHEMISTRY

205,

57-64 (1%X?)

Detection of Neutral Endopeptidase-24.11 by Flow Cytometry and Photomicroscopy a New Fluorescent Inhibitor’

/CD1 0 Using

Pierre-Emmanuel M ilhiet, Francine Dennin, Marie-Ckile Giocondi,* Christian Le Grimellec,” Christiane Garbay-Jaureguiberry, Claude Boucheix,? and Bernard P. Roques2 Dkpartement de Chimie Organique, U266 INSERM, URA 01500 CNRS, Faculte’ de Pharmacie, 4 Avenue de lObservatoire, 75270 Paris Cedex 06, France; *U251 INSERM, HGpital Bichat, 16 rue H. Huchard, 75018 Paris, France; and -fU268 INSERM, HGpital P. Brousse, Avenue P. Vaillant-Couturier, 94800 Villejuif, France

Received December 26, 1991

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CDlO). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyll-N’-[ 1-(6-(3-mercapto-2benzyl - 1 - oxopropyl)amino - 1 - hexyllthiocarbamide (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 IC,, value of 10 n M and a good selectivity compared to that of aminopeptidase N (> 100 pM) and angiotensin converting enzyme (32 PM). 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 episcopit fluorescence microscopy, a specific labeling was obtained with 100 n M FTI which was completely displaced by 10 p M 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 n M 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. 10 1992 Academic Press, Inc.

i The authors are grateful to Dr. A. Gouyette for mass spectra recording, N. Morellet for helpful discussions, M. Prenant for cell culture, and C. Dupuis for typing the manuscript. Special thanks are given to A. Beaumont for her help during the course of this work and for English revision. This work was supported by the Minis&e de la Recherche, Rhone Poulenc Rorer, and Merck, Sharp and Dohme. 0003.2697/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

Neutral endopeptidase (NEP3; E.C. 3.4.24.11) is a 94kDa mammalian ectoenzyme, first isolated from the brush border membranes of the kidney (1). NEP, which is a zinc-metallopeptidase, has been identified as the common acute lymphoblastic leukemia antigen (CALLA, CDlO) (2,3). The CD10 antigen is a differentiation marker of early stages of the lymphoid B cell lineage and it is found at the surface of B lympoid lineage-derived acute lymphoblastic leukemia (4). The enzyme has a wide tissue distribution (5), including the central nervous system (6), neutrophils (7), and lymphoid tissues (8). NEP inactivates a variety of biologically active peptides (9) by cleaving the amide bond, generally on the amino side of hydrophobic residues. The enzyme is in particular involved in the degradation of enkephalins and atria1 natriuretic peptide (reviewed in (10)). The sequence of NEP, including residues thought to be involved in the active site, is highly conserved in different species such as rabbit (ll), rat (12), and human (13), although both tissue and species differences in glycosylation have been observed. A tritiated NEP inhibitor, [3H]HACBOGly (N-[3(&S)-[(hydroxyamino)carbonyl]-2-benzyl-l-oxopropyllglycine), which directly labels the active site, has been consequently used as a potent tool to study the enzyme distribution in different ’ To whom correspondence should be addressed. ’ Abbreviations used: NEP, neutral endopeptidase-24.11; CALLA, common acute lymphoblastic leukemia antigen; MDCK, Madin Darby canine kidney; APN, aminopeptidase N; ACE, angiotensin converting enzyme; FITC, fluorescein isothiocyanate; FTI, Auorescent thioinhibitor; mAb, monoclonal antibody; MFI, mean fluorescence intensity; HACBOGly, N-[3(R, S)-[(hydroxyamino)carbonyl]2-benzyl-l-oxopropyllglycine; TFA, trifluoroacetic acid; GAM, goat anti-mouse antibody. 57

Inc. reserved.

58

MILHIET

tissues and species (6,14,15). The major advantage of inhibitors over monoclonal antibodies (mAb) is that they recognize NEP independently of the species and the degree of glycosylation of the enzyme. A recent study has shown, for example, that out of 20 human mAbs tested, only 4 cross-reacted with a rat cell line (16). In addition, NEP levels have been shown to remain almost constant after inhibitor treatment (P. E. Milhiet, submitted for publication) while mAb binding generally leads to an internalization of NEP. For instance, a 90% internalization of the enzyme has been observed after 24 h with the mAb 55 on the leukemic cell line Laz 221 (17). This modulation has decreased the therapeutic interest of mAb since the target disappears from the cell surface with treatment. In order to overcome this problem and to investigate the modulation and cellular traffic of NEP without the tissue and species restrictions observed with mAbs, a fluorescent derivative of the zinc-directed NEP inhibitor thiorphan (18) has been synthesized. Fluorescent probes have already shown to be potentially useful for microscopic visualization (19) to study ligand-receptor interaction, as demonstrated in the case of enkephalins (20,21). In this study, we describe the synthesis and evaluation of the first fluorescein-NEP inhibitor conjugate (FTI), obtained by coupling the chemical precursor of thiorphan, 3-acetylthio-2-benzylpropanoic acid, to fluorescein isothiocyanate (FITC) by a diaminohexane linker. As the inhibitor has a Ki of 10 nM for the enzyme and retains the fluorescent properties of FITC, it appears to be a very potent probe for detecting membranebound NEP for biological studies or diagnostic applications. It should be particularly useful for detecting the membrane-bound enzyme by flow cytometry, which until now has relied on using mAbs. MATERIALS

AND

METHODS

Chemicals were obtained from Aldrich (France), except for fluorescein isothiocyanate, which was purchased from Jansen (France). Thiorphan (18), captopril (22), and HACBOGly (23) were synthesized in the laboratory following the previously described methods. Bestatin was purchased from Bachem (Switzerland) and D- [3H]Ala2-leucine-enkephalin was from the CEA (France). Cellular culture products were from GIBCO (France). Analytical

Methods

Melting points were determined on an electrothermal digital apparatus. The purity of the synthesized compounds was checked either by thin-layer chromatography on Silica gel plates (Merck, 60F254) in the following solvent systems (v/v): A, CH,Cl,/MeOH (9/l); B, n-butanol/NH,OH (17/5); C, ethyl acetate/pyridine/

ET AL.

CH,COOH/H,O (1000/20/6/11) or by HPLC using a reverse-phase Nucleosil C, column (Socihti! Franqaise Chromato Colonne, France) with CH,CN/0.05% TFA (pH 4) as the mobile phase. The eluted peaks were monitored at 210 nm (flow rate, 1 ml/min). The structure of the compounds and all intermediates was confirmed by ‘H NMR spectroscopy (Bruker WH 270). The final product was analyzed by mass spectroscopy on a double-focusing magnetic instrument (VG 7’250, VG Instruments) equiped with a fast atom bombardment gun (Ion Tech.). Fluorescence spectra were performed on a 4800 SLM Aminco spectrofluorimeter. Synthesis N-(tert-Butoxycarbonyl)-6-amirwhexane nitrile (2). A solution of 6-aminohexane nitrile (3.5 g, 30 mmol) in 90 ml of dioxaneiwater (l/l) and 30 ml of 1 M NaOH was reacted with di-tert-butyl dicarbonate (8.2 g, 36 mmol) at room temperature for 30 min. The mixture was filtered, the filtrate concentrated to remove dioxane, and the solution acidified (pH 2) with KHSO,. The aqueous phase was extracted with EtOAc and subsequently washed with H,O. The EtOAc layer was dried over Na,SO,, filtered, and concentrated to yield compound 2 (6.3 g, 99%): mp 52°C; R, (A) 0.84. N’-(tert-Butoxycarbonyl)-1,6-diaminohexane (3). A solution of 2 (1.7 g, 8 mmol) in MeOH (15 ml) and 6 M HCl (1.3 ml) was added to Pd/C (660 mg) in MeOH saturated under H,. The mixture was shaken under H, for 12 h. The catalyst was removed by filtration on a Celite pad and the filtrate evaporated to dryness. The residue was dissolved in water. The aqueous phase was washed with EtOAc, alkalinized at pH 10, and then extracted twice with ethyl acetate. The combined organic layers were dried and concentrated in uacuo to give 773 mg (42%) of 3: mp 107°C; R, (B) 0.5. N1-(3-Acetylthio-2-benzyl-l-oxopropyl)-NG-(tert-butoxycarbonyl)-1,6-diaminohexane (5). A mixture of 3 (630 mg, 3 mmol) and 3-acetylthio-2-benzylpropanoic acid 4, synthesized as previously described (18) (690 mg, 3 mmol) in the presence of 1-hydroxybenzotriazole (431 mg, 3.3 mmol) in dry tetrahydrofuran was cooled to 0°C. Dicyclohexylcarbodiimide (658 mg, 3.3 mmol) was added under stirring. The reaction mixture was stirred at 0°C for 90 min and then at room temperature overnight. The solution was filtered and evaporated to dryness and the residue dissolved in EtOAc. The organic phase was successively washed with water (40 ml), 10% citric acid (30 ml), water (10 ml), aqueous NaHCO, (30 ml), water (30 ml), and saturated aqueous NaCl(10 ml), dried over anhydrous Na,SO,, and evaporated to dryness. The crude product, purified by flash chromatography over silica gel (CH,Cl,/MeOH, 20/l as the solvent), provided 746 mg (59%) of compound 5: mp 113.4”C; R, (A) 0.7.

FLUORESCENCE

DETECTION

N1[3-Acetylthio-2-benzyl-l-oxopropyl]-1,6-diaminohexane (6). The protected amine 5 (386 mg, 0.88 mmol) was treated with trifluoroacetic acid (1.3 ml) in dry dichloromethane (5 ml) at 0°C. After 30 min at room temperature, the solvent was evaporated and the solid phase extensively washed with ethyl ether and dried under vacuum to yield 323 mg of 6 (82%): mp 110°C; R, (B) 0.2. N-[5-Fluoresceinyl]-N’[6-(3-acetylthio-2-benzyl-l-o~opropyl)amino-l-hexyl] thiocarbamide (7). One hundred fifty milligrams (0.35 mmol) of 6, 259 mg (0.66 mmol) of fluorescein isothiocyanate, and 1 eq (49 ~1) of triethyl amine dissolved in 3 ml of dimethylformamide were stirred at 0°C for 1 h and at room temperature overnight. The solution was then evaporated in uacuo. The residue, after chromatography using a silica gel column with solvent mixture C, gave 164.9 mg (yield 65%) of 7, as a yellowish-red to red powder at 155°C: R, (C) 0.55. N-[5-Fluoresceinyl]-N’-[6-(3-mercapto-2-benzyl-l-o~opropyl)amino-1 -hexyl] thiocarbamide (8) (FTI). Fifty milligram (0.07 mmol) of 7 was dissolved in 5 ml of H,O/MeOH (l/l) at 0-5°C and 10 eq of 1 M NaOH (700 ~1) was added with stirring under nitrogen atmosphere. After stirring at 0-5°C for 40 min, the reaction was stopped by addition of 700 ~1 of 1 M HCl. The mixture was evaporated to dryness in vacua and the residue twice washed with water and dried to give 13.8 mg (50%) of a yellowish-red powder (red powder at 135°C) (8): R, (C) 0.1; HPLC (CH,CN/TFA, 0.05%, 50/50) 9.5 min. NMR assignments of the final compound, measured in (CD,),SO, were as follows (chemical shifts given in ppm from hexamethyldisiloxane): FTI: CH, diaminohexane (C, to C,) 1.13 to 1.52, SH 2.08, HP, Ha (Phe), and CH, diaminohexane (C,) 2.54 to 3, CH, diaminohexane (C,) 3.44, C, (phenyl) and C, to C, (xanthene) 6.56 to 6.71, CH (Phe) 7.09, C, (phenyl) 7.27, C, (xanthene) 7.69, NH-CO (inh) 7.78, C, (phenyl) 8.11, NHCS (inh) 8.20, NH-CS (fluo) 9.85, OH 10.07. Cell Lines Several leukemic cell lines were used for this study. Reh6 (24) and KM3 are derived from early stages of the B cell lineage and, like Daudi (25), a Burkitt cell line, express the NEP. Nalm6 (26) is a pre-B cell line (the variant used in this study does not express a significant level of NEP as judged by immunological studies). The antigen is also not detectable by flow cytometry on the surface of K562, an erythroid cell line derived from a chronic myeloid leukemia blastic crisis (27), and HEL, a megakariocytic cell line (28). Its level varies from undetectable to weakly expressed on the surface of CEM, a T lymphoid cell line (29). These cell lines have been maintained in the laboratory of one of us (C.B.) for several years and checked repeatedly for various surface

OF NEUTRAL

59

ENDOPEPTIDASE m

- (CH,),

- C+ N

1 IBoc-RR-[@Xi.).-C=N a %*

w/c

J Boc

- NH

- [CH,),

- NH,

2 cl&-co-s-C~-cfH-CooH a =no

I CHs-CO-S-CH2-YH-CO-NH-(CH,),-NH-Box c=a@

= WA

J CH3-CO-S-CH,-CH-CO-NH-(CH&-NlQ+.TFA-

I

1) FITC

2) NaOH 3) HCl

8 II R-NH-C-NH

B

R=-WH&-NH-CO-CH-CH*-SH

I--@

mTlc:muorescehlmthlocyanate

FIG.

1.

Scheme of the synthesis for the NEP fluorescent inhibitor

FTI.

markers and caryotype. The NEP-expressing MDCK (Madin-Darby canine kidney) cell line (30), transfected with a vector containing the cDNA for rabbit enzyme, was a generous gift from P. Crine (Universite de Montreal). The cell lines growing in suspension were cultured in RPM1 medium and MDCK cells in a medium consisting of 50% Dubecco’s modified Eagle’s medium and 50% Ham F12, both supplemented with 10% fetal calf serum and antibiotics. Assay for NEP and APN (aminopeptidase

N) Activity

NEP activity was measured using 1 nM purified rabbit kidney enzyme (30) with 20 nM D-Ala2[tyrosyl-[3H]leucinel-enkephalin as substrate in 100 ~1 of 50 m M Tris-HCl, pH 7.4, at 25°C for 15 min. The reaction was stopped by adding 10 ~1 of 0.5 M HCl. The metabolite [3H]tyrosylglycylglycine was separated using Porapak Q beads as previously described (31). To measure the NEP enzymatic activity of the cell lines, cell suspensions were preincubated for 10 min in Hank’s medium with 10 pM bestatin and 1 pM captopril (inhibitors of APN and angiotensin converting enzyme (ACE), respec-

60

MILHIET

ET AL.

APN activity was measured following the same procedure and using purified enzyme (Boehringer-Mannheim) with [3H]leucine-enkephalin as substrate at a final concentration of 10 nM. IC,, values were determined as already described (33) and the correlation of enzymatic activity with fluorescence was calculated using the Sigma Plot program. 450

so0

s9l

WAvELENGrH (Ml) FIG. 2. Fluorescence excitation spectra (dashed line) and emission spectra (solid line) of 10 pM FTI. The excitation was monitored at 494 nm and the emission at 515 nm.

tively). One micromole of the selective NEP inhibitor retrothiorphan (32) was included in the controls. Incubation times were chosen in order to obtain less than 10% substrate degradation.

Angiotensin

Converting

Enzyme Activity

ACE activity was measured using 50 m M Cbz-PheHis-Leu as substrate. Membrane preparations of rabbit kidney cortex (14 mg/ml), prepared as described (8), were incubated at 37°C in a total volume of 250 ~1 of 50 m M Tris-HCl, pH 8.0, containing 1% (w/v) NaCl and 0.1 m M ZnCl,. The enzymatic reaction was stopped by heating at 100°C for 5 min and the concentration of the His-Leu metabolite was determined by the fluorimetric assay of Piquillaud et al. (34) using a Perkin-Elmer

FIG. 3. Fluorescence of FTI-labeled NEP on MDCK cells in suspension. MDCK cells were incubated with 0.1 FM FTI in the presence (C) or absence (A) of 10 jiM HACBOGly as describedunder Materials and Methods. Cells were visualized alternatively by fluorescence (A and C) and phase (B and D, respectively) microscopy. The cellular fluorescence obtained with FTI was essentially located on plasma membrane (A) and the signal completely displaced by HACBOGly, a nonfluorescent NEP inhibitor (C). Bar, 10 pM.

FLUORESCENCE

DETECTION

m /Cmtrd

61

ENDOPEPTIDASE

fluorescence measurement brated fluorescent beads.

was

checked

using

cali-

Photomicroscopy

HJ -FTI+HACBOGly y3 8 e

OF NEUTRAL

LA-

-F-l-I (and Cmtrol or ALBl)

NEP-expressing MDCK cells were observed using 70-pm coverslips and phase or epifluorescence microscopy. In this system, the Olympus IMT2 microscope was coupled to a video camera SIT (LHESA, France) and the video images were analyzed with a Biocom 500 associated to an Hamamatsu Argus 10. Trypsinized cells were used and the specificity of the labeling obtained with 1 PM FTI was controled by co-incubation or displacement of the fluorescent probe with 10 PM HACBOGly. RESULTS

AND

DISCUSSION

Synthesis The synthesis of the fluorescent NEP inhibitor FTI, shown in Fig. 1, was based on the coupling of 3-ace-

log fluorescence intensity

FIG. 4. Analysis of NEP expression on Reh6 and HEL cell lines by flow cytometry. A significant shift of fluorescence curve was observed on the human leukemia B cell line (Reh6) after 1 pM FTI or ALBl incubation compared to control (nontreated cells or 1 pM FTI-10 HIM HACBOGly co-incubation). No difference with control cells was observed on a CALLA-negative cell line HEL after FTI or ALBl incubation.

MPF 44A spectrofluorimeter (excitation, 365 nm; emission, 500 nm). NEP activity was inhibited by including Controls contained either 1 PM 0.1 PM retrothiorphan. of the ACE inhibitor captopril or membrane preparations previously heated at 100°C for 5 min. Cell Surface Labeling

and Flow Cytometry

For FTI labeling, a one-step procedure was used in which lo5 cells were incubated at 4°C with successive dilutions of the probe (from 1O-5 to 10-l’ M) in the presence or absence of the unlabeled competitive inhibitor HACBOGly at 10e5 M (22). After washing, the cells were analyzed. For immunological labeling, the CD10 mAb ALBl and the secondary antibody (F(ab)H) goat antimouse IgG labeled with FITC (GAM-FITC) were used at saturating concentrations following a previously described procedure (35). Fluorescence analysis of cell surface molecules was carried out on a profile II flow cytometer (Coultronics, France) at 488 nm excitation with a laser power at 15 mW. A 525-nm bandpass filter was used for FITC fluorescence. Scatter gates were set to eliminate analysis of debris. Fluorescence signals were processed by using a log amplifier with a four-log-scale representation. Results were expressed as mean fluorescence intensity (MFI) converted to a linear value. The linearity of the

$J ;

100

% 8 s &so :5 4 3 0 c w

FIG. 5. Correlation between NEP expression measured by flow cytometry using 0.1 pM FTI and ALBl mAb (10 pg/ml) (A) or enzymatic activity (B). A FITC-labeled secondary antibody (F(ab)‘) goat antimouse IgG was used under saturating concentration for the detection of ALBl binding. The confidence intervals obtained with the curves were 95% (A) and 99% (B). A 95% confidence interval is displayed by the two lines drawn on either side of the regression line.

62

MILHIET

tylthio-2-benzylpropanoic acid, a precursor used in the synthesis of thiol inhibitors of zinc metallopeptidases (l&36), with fluorescein isothiocyanate. In order to preserve the physical and biological properties of each moiety, a diaminohexyl linker was introduced between the carboxyl-terminal part of the thiol inhibitor precursor and the isothiocyanate group of FITC. Thus, 3-acetylthio-2-benzylpropanoic acid was first condensed with the N-Boc monoprotected 1,6-diaminohexane linker. After deprotection of the amino group with TFA, the product was condensed with FITC and provided the new conjugate FTI after saponification of the thioacetyl group. FTI was characterized by its mass and NMR spectra and its purity was checked by HPLC analysis as described under Materials and Methods. Spectral Properties The fluorescence spectral shape of FTI (Fig. 2) is identical to that of FITC with optimal excitation and emission wavelengths occuring at 494 and 515 nm, respectively. The maximal fluorescence intensities obtained with 1 pM FTI or 1 pM FITC were equivalent. A fluorescence signal was detected at concentration as low as 0.1 nM FTI dissolved in water and using a PerkinElmer MPF 44A spectrofluorimeter. Inhibitory

Properties

of FTI

FTI was a potent inhibitor of the pure rabbit kidney enzyme with a value of 10 nM IC,, . The condensation of a bulky group on the C-terminal part of the thiorphan derivative did not therefore dramatically decrease the inhibitory potency for NEP. A similar result has already been observed when a glycinamide group was condensed on the carboxyl group of thiorphan (IC,, = 2 nM) (37), this compound displaying an IC,, value of 35 nM for NEP. The selectivity of the FTI probe was tested by measuring its inhibitory potency for angiotensin converting enzyme (32 pM) and aminopeptidase N (>lOO PM), two widely distributed zinc-peptidases for which NEP inhibitors often show some degree of cross-reactivity (38,39). The high selectivity factors of 8930 and 2860 exhibited by FTI for NEP, as compared to APN and ACE, respectively, allow the probe to be used at concentrations as high as 1 pM without any significant cross-reactivity with either APN or ACE. Microscopic

Visualization

Given the affinity and specificity of FTI for NEP, several biological applications could be considered. The ability of FTI to label membrane-bound cellular NEP was tested using suspended NEP-transfected MDCK cells and an episcopic fluorescence microscope as described under Materials and Methods. The MDCK cell

ET AL.

line was chosen because this type of cell line has often provided excellent experimental systems for investigating cellular protein traffic. Moreover, these cells express large levels of enzyme after transfection (40). Specific labeling of membrane NEP was obtained using 0.1 or 1 pM FTI after 30 min incubation of trypsinized cells at 37°C (Fig. 3A); as expected for NEP-transfected epithelial cells, the labeling was essentially localized to plasma membranes (40). The specificity of FTI labeling was controlled by co-incubation with 10 pM HACBOGly, a potent and specific inhibitor of NEP (IC,, = 1.4 nM), which completely displaced the probe (Fig. 3C). Specific labeling was also observed using Reh6, a leukemia NEP-positive cell line (results not shown). Thus, FTI appears to be a good probe for studying the fate of the inhibitor-enzyme complex in cultured cells, which is important considering the therapeutic interests of inhibitors (10). This or similar inhibitors might also contribute to better characterize different NEP modulations, obtained using neutrophils or transfected epithelial cells after phorbol 12-myristate 13-acetate (41) or glucocorticoid treatment (42), respectively.

Flow Cytometry Analysis of Membrane Correlation with Enzymatic Activity

Fluorescence

and

The analysis of NEP expression by flow cytometry was first investigated using two cell lines growing in suspension: Reh6, a NEP-positive cell line, and HEL, which normally appears to be immunologically negative (43). Treatment of Reh6 cells with 1 pM FTI leads to an important shift in comparison to the reference curve, which was obtained using untreated cells or cells co-incubated with 1 pM FTI/lO pM HACBOGly (Fig. 4). At this concentration of the probe, a slight residual nonspecific binding was observed on Reh6 cells, which was not observed at 0.1 yM FTI. No labeling could be detected at 0.1 nM. HEL cells were not labeled by FTI (1 pM) or by ALBl (10 pg/ml), an anti-CD10 mAb (Immunotech, Marseille) (44) (Fig. 4). Labeling of other human cell lines with different concentrations of FTI in the presence or absence of HACBOGly also showed that a specific binding to NEP-expressing cells could be obtained (results not shown). In order to establish the correlation between the different methods of measuring cellular NEP, equivalent samples of cells from the seven human leukemic cell lines were processed simultaneously for analysis of NEP using the mAb ALBl revealed by a secondary FITC-labeled antibody and the FTI fluorescent probe, both by flow cytofluorometry, or the determination of enzymatic activity. The laser power and voltage of the photomultiplier collecting green fluorescence (FITC) were kept at the same values for all cell lines in order to compare the results. The relative fluorescence intensity

FLUORESCENCE

DETECTION

was obtained by subtracting the MFI of the control (labeling with GAM-FITC only) from the MFI obtained with ALBl (10 pug/ml) and GAM-FITC. With the fluorescent probe FTI, the relative fluorescence intensity was expressed as the difference between the MFI obtained with FTI alone at concentrations varying from 10 PM to 0.1 nM and the MFI obtained with FTI at the same concentrations in the presence of 10 PM HACBOGly. Enzymatic activity was measured as described under Materials and Methods. A relationship between enzymatic activity and cell sorter fluorescence was established. Figure 5B shows the high correlation obtained between the NEP activity of intact cells, expressed in fmol/cell/min, and the fluorescence of cells treated with 0.1 @M FTI, both measured in Hank’s buffer (r = 0.979 with a 99% confidence interval); 1 I.LM FTI treatment leads to an analog type of correlation with a 95% confidence interval, which validates its use in flow cytometry. A good correlation was also established between the cellular fluorescence obtained with 0.1 PM FTI and 10 pg/ml ALBl mAb (Fig. 5A). The responses given by the two probes can be correlated with a confidence interval of 95%. The fluorescent inhibitor can therefore be used in flow cytometry for species in which no mAb is available. However, it should be noted that the method using FTI is less sensitive than immunological labeling due to the amplification of signal obtained with a secondary antibody bearing three or four FITC per molecule. Until the present, immunological labeling has been the sole method for detecting NEP using flow cytometric techniques. Even though this technique is convenient and sufficiently sensitive for many applications, one of its major limitation is its species restriction linked to the epitope recognition. As an example, out of 20 CD10 mouse mAbs, produced against human antigen, none cross-reacted with the mouse antigen (unpublished data). This limitation can be overcome by the use of FTI or analogous probes which are directed against the active site of the molecule which has been highly conserved during evolution. FTI labeling could therefore be suitable for studying mouse models of acute leukemias derived from the B lymphoid cell lineage.

CONCLUSION

The fluorescent inhibitor of mammalian neutral endopeptidase provides a very potent probe for investigating different processes involving NEP, since this bifunctional molecule maintains high inhibitory potency and selectivity for the enzyme as well as the fluorescence properties of the fluorochrome. The fluorescein-conjugated inhibitor FTI is now under study in different experimental systems. Its ubiquitous recognition of NEP and its good sensitivity could

OF NEUTRAL

63

ENDOPEPTIDASE

be very useful in determining hematopoietic cells.

the role of the antigen

on

REFERENCES 1. Kerr, M. A., and Kenny, A. J. (1974) Biochem. J. 137,489-495. 2. Letarte, M., Vera, S., Tran, R., Adis, J. B. L., Onizuka, R. J., Quackenbush, E. J., Jongeneel, C. V., and McInnes, R. R. (1988) J. Exp. Med. 168, 1247-1253. 3. Greaves, M. F., Brown, G., Rapson, N. T., and Lister, T. A. (1975) Clin. Immunol. Immunopathol. 4, 67. 4. Le Bien, T. W., and McCormack, R. T. (1989) Blood 73,625-635. 5. Kenny, A. J. (1986) in Cellular Biology of Ectoenzymes (Kreutzberg, G. W., Reddington, M., and Zimmerman, H., Eds.), Springer-Verlag, Berlin. M. C., and Roques, 6. Waksman, G., Hamel, E., Fournie-Zaluski, B. P. (1986) Proc. N&l. Acad. Sci. 83, 1523-1527. 7. Connely, J. C., Skidgel, R. A., Schulz, W. W., Johnson, A. R., and Erdos, E. G. (1985) Proc. Natl. Acad. Sci. 82,8737-8741. 8. Beaumont, A., Brouet, J. C., and Roques, B. P. (1989) Biochem. Biophys. Res. Commun. 160, 1323-1329. A. J. (19871 in Neuropeptides and Their Peptidases 9. Turner, (Turner, A. J., Eds.), pp. 181-201, Ellis Horwood, Chichester. 10. Roques, B. P., and Beaumont, A. (1990) TIPS 11, 2455249. 11. Devault, A., Lazure, C., Nault, C., Moual, H. L., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322. 12. Malfroy, B., Shoffield, P., Kuang, W. J., Seeburg, P. H., Masson, A. J., and Henzel, W. J. (1987) Biochem. Biophys. Res. Commun.

144,59-66. 13. Malfroy, B., Kuang, W. J., Seeburg, P. H., Mason, A. J., and Shoffield, P. (1988) FERS Lett. 229, 206-210. 14. Salles, N., Charnay, Y., Zajac, J. M., Dubois, P. M., and Roques, B. P. (1989) Chem. Neuroanat. 2, 179-188. 15. Mailleux, P., Przedborski, S., Beaumont, A., Verslipje, M., Dupierreux, M., Levivier, M., Kitabgi, P., and Vanderhaegen, J. J. (1990) Peptides 11, 1245-1253. 16. Helene, A., Milhiet, P. E., Haouas, H., Boucheix, C., Beaumont, A., and Roques, B. P. (1992) Biochem. Pharmacol. 43,809-814. K. J., and 17. Pesando, J. M., Ritz, J.. Lazarus, H., Tomaselli, Schlossman, S. F. (1981) J. Zmmunol. 126, 540-544. 18. Roques, B. P., Fournie-Zaluski, M. C., Soroca, E., Lecomte, J. M.. Malfroy, B., Llorens, C., and Schwartz, J. C. (1980) Nature 288, 286-288. 19. Taylor, D. L., and Wang, Y. (1980) Nature 284,405-410. 20. Hazum, E., Chang, K. J., Shechter, Y., Wilkinson, S., and Cuatrecasas, P. (1979) Biochem. Biophys. Res. Commun. 88, 841-846. 21. Rigaudy, P., Garbay-Jaureguiberry, C., Jacquemin-Sablon, A., Le Pecq, J. B., and Roques, B. P. (1987) Znt. J. Peptide Protein Res. 30, 347-355. 22. Cushman, D. W., Cheung, H. S., Sabo, E. F., and Ondetti, M. A. (1977) Biochemistry 16, 5484-5491. M. C., 23. Bouboutou, R., Waksman, G., Devin, J., Fournie-Zaluski, and Roques, B. P. (1984) Life Sci. 35, 1023-1030. 24. Rosenfeld, C., Goutner, A., Choquet, C., Venuat, A. M., Kayibauda, M., and Picot, J. L. (1977) Nature 267, 841-843. 25. Klein, E., Klein, G., and Nadkarni, J. S. (1968) Cancer Res. 28, 1300. 26. Hurwitz, R., Hozier, J., Bien, T. L., Minowada, J., Gajl-Peczalska, K., Kubonishi, I., and Kersey, J. (1979) Znt. J. Cancer 23, 174180.

64

MILHIET

27. Lozzio, C. B., and Lozzio, B. B. (1975) Blood 45,321-334. 28. Martin, P., and Papayannopoulou, T. (1982) Science 216, 1235.

1233-

29. Foley, G. E., Lazarus, H., Farber, S., Uzman, B. G., Boone, B. A., and McCarthy, R. F. (1965) Cancer l&522-529. 30. Aubry, M., Berthelot, A., Beaumont, A., Roques, B. P., and Crine, P. (1987) Biochem. Cell. Biol. 65, 398-404. 31. Vogel, Z., and Alstein, M. (1977) FEBS L&t. 80, 332-336. 32. Roques, B. P., Lucas-Soroca, E., Chaillet, P., Costentin, J., and Fournie-Zaluski, M. C. (1983) Proc. N&l. Acad. Sci. 80, 317% 3182. 33. Blumberg, S., Vogel, Z., and Alstein, M. (1981) Life Sci. 28, 301306. 34. Piquillaud, Y., Reinharz, A., and Biophys. Acta 206, 136-142.

Roth,

M.

(1970)

35. Boucheix, C., Perrot, J. Y., Mirshahi, M., Bernadou, senfeld, C. (1983) J. Immunol. Methods 57, 145.

Biochem.

A., and Ro-

36. Ondetti, M. A., Condon, M. E., Reid, J., Sabo, E. F., Cheung, H. S., and Cushman, D. W. (1979) Biochemistry 188, 1427-1430.

ET AL. 37. Roques, B. P., Fournie-Zaluski, M. G., Sassi, A., Chaillet, P., Collado, Life Sci. 31,1749-1752. 38. Fournie-Zaluski, M. C., Lucas, E., B. P. (1984) Eur. J. Biochem. 139,

C., Florentin, D., Waksman, H., and Costentin, J. (1982) Waksman,

G., and Roques,

267-274.

39. Sullivan, J., and Johnson, A. R. (1989) Biochem. Biophys. Res. Commun. 162,300-307. 40. Jalal, F., Lemay, G., Zollinger, M., Berthelot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19857. 41. Erdos, E. G., Wagner, B., Harbury, C. B., Painter, R. G., Skidgel, R. A., and Xiang-Guang, F. (1989) J. Biol. Chem. 264, 1451914523. 42. Borson, D. B., and Gruenert, D. C. (1991) Am. J. Physiol. 260, L83-L89. 43. Knapp, W., D&ken, B., Rieber, E. P., Stein, H., Gilks, W. R., Schmidt, R. E., and von dem Borne, A. E. G. (1989) Leucocyte Typing. IV. White Cell Differentiation Antigens, Oxford Univ. Press, Oxford. 44. Boucheix, C., Perrot, J. Y., Mirshahi, M., Gianonni, F., Billard, M., Bernadou, A., and Rosenfeld, C. (1985) Leuk. Res. 9,597-604.

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)...
2MB Sizes 0 Downloads 0 Views