Transcellular Proteolysis Demonstrated by Novel Cell Surface-associated Substrates of Dipeptidyl Peptidase IV (CD26)
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m200798200
ISSN1083-351X
AutoresSusan Lorey, Ju ̈rgen Faust, Carmen Mrestani‐Klaus, Thilo Ka ̈hne, Siegfried Ansorge, Klaus Neubert, Frank Bu ̈hling,
Tópico(s)Signaling Pathways in Disease
ResumoProteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates. Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates. Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26).Journal of Biological ChemistryVol. 277Issue 43PreviewPage 33177: Ref. 34 is incorrect. The following reference should be substituted: Full-Text PDF Open Access dipeptidyl peptidase acetic acid ethyl ester Chinese hamster ovary middle pressure liquid chromatography petroleum ether phycoerythrin p-nitroanilide TR, Texas Red human immunodeficiency virus wild-type phosphate-buffered saline reactive anchor Dipeptidyl peptidase (DP)1 IV (CD26, EC 3.4.14.5) is a serine protease (1Hopsu-Havu V.K. Glenner G.G. Histochemie. 1966; 7: 197-201Crossref PubMed Scopus (406) Google Scholar) that is anchored in the membrane of cells via a transmembrane domain of 22 amino acids and a short cytoplasmic tail of six amino acids (2Ogata S. Misumi Y. Ikehara Y. J. Biol. Chem. 1989; 264: 3596-3601Abstract Full Text PDF PubMed Google Scholar). The enzyme occurs in most mammalian epithelial tissues such as kidney, liver (3Ikehara Y. Ogata S. Misumi Y. Methods Enzymol. 1994; 244: 215-227Crossref PubMed Scopus (22) Google Scholar), intestine (4Darmoul D. Voisin T. Couvineau A. Rouyer-Fessard C. Salomon R. Wang Y. Swallow D.M. Laburthe M. Biochem. Biophys. Res. Commun. 1994; 203: 1224-1229Crossref PubMed Scopus (65) Google Scholar), and also in bacteria (5Yoshimoto T. Tsuru D. J. Biochem. (Tokyo). 1982; 91: 1899-1906Crossref PubMed Scopus (39) Google Scholar) and plants (6Stano J. Kovacs P. Psenak M. Gajdos J. Erdelsky K. Kakoniova D. Neubert K. Pharmazie. 1997; 52: 319-321Google Scholar). DP IV catalyzes the cleavage of dipeptides from the N terminus of oligo- and polypeptides provided that the penultimate residue is proline or alanine (7Heins J. Welker P. Scho¨nlein C. Born I. Hartrodt B. Neubert K. Tsuru D. Barth A. Biochim. Biophys. Acta. 1988; 954: 161-169Crossref PubMed Scopus (120) Google Scholar). Several bioactive polypeptides such as substance P, β-casomorphins, somatoliberine, and a number of chemokines are hydrolyzed by DP IV catalysis (8Mentlein R. Regul. Pept. 1999; 85: 9-24Crossref PubMed Scopus (1151) Google Scholar). In the immune system, DP IV is expressed on activated T and B lymphocytes as well as natural killer cells (9Scho¨n E. Ansorge S. Biol. Chem. Hoppe-Seyler. 1990; 371: 699-705Crossref PubMed Scopus (38) Google Scholar, 10Bu¨hling F. Junker U. Neubert K. Ja¨ger L. Ansorge S. Immunol. Lett. 1995; 45: 47-51Crossref PubMed Scopus (99) Google Scholar, 11Bu¨hling F. Kunz D. Reinhold D. Ulmer A.J. Ernst M. Flad H.-D. Ansorge S. Nat. Immun. 1994; 13: 270-279PubMed Google Scholar). Furthermore, the enzyme has been shown to be involved in the activation and proliferation of immune cells (12Dang N.H. Torimoto Y. Deusch K. Schlossman S.F. Morimoto C. J. Immunol. 1990; 144: 4092-4100PubMed Google Scholar, 13Dang N.H. Torimoto Y. Shimamura K. Tanaka T. Daley J.F. Schlossman S.F. Morimoto C. J. Immunol. 1991; 147: 2825-2832PubMed Google Scholar). Ka¨hne et al.(14Ka¨hne T. Lendeckel U. Wrenger S. Neubert K. Ansorge S. Reinhold D. Int. J. Mol. Med. 1999; 4: 3-15PubMed Google Scholar) discussed a regulatory role for DP IV in signal transduction reflected by DP IV inhibitor-induced phosphorylation of proteins. The inhibition of DP IV by HIV-1 Tat protein reveals that the enzyme has a possible function in HIV infection (15Wrenger S. Hoffmann T. Faust J. Mrestani-Klaus C. Brandt W. Neubert K. Kraft M. Olek S. Frank R. Ansorge S. Reinhold D. J. Biol. Chem. 1997; 272: 30283-30288Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These effects may be mediated via the interaction of ligands with cell surface-associated DP IV. In addition to this, several soluble isoforms and related enzymes were found in blood plasma (16Duke-Cohan J.S. Morimoto C. Rocker J.A. Schlossman S.F. J. Biol. Chem. 1995; 270: 14107-14114Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 17Duke-Cohan J.S., Gu, J. McLaughlin D.F., Xu, Y. Freeman G.J. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11336-11341Crossref PubMed Scopus (125) Google Scholar, 18Blanco J. Nguyen C. Callebaut C. Jacotot E. Krust B. Mazaleyrat J.-P. Wakselman M. Hovanessian A.G. Eur. J. Biochem. 1998; 256: 369-378Crossref PubMed Scopus (26) Google Scholar). A number of substrates with fluorophoric groups, including 6-aminoquinoline (19Brynes P.J. Bevilacqua P. Green A. Anal. Biochem. 1981; 116: 408-413Crossref PubMed Scopus (35) Google Scholar), naphthylamine (20Gossrau R. Histochem. J. 1985; 17: 737-771Crossref PubMed Scopus (54) Google Scholar), 7-amino-4-methylcoumarine (21Kojima K. Kinoshita H. Kato T. Nagatsu T. Takada K. Sakakibara S. Anal. Biochem. 1979; 100: 43-50Crossref PubMed Scopus (70) Google Scholar), 6-aminonaphthalenesulfonamide (22Butenas S. DiLorenzo M.E. Mann K.G. Thromb. Haemostasis. 1997; 78: 1193-1201Crossref PubMed Scopus (29) Google Scholar), and cresyl violet (23Van Noorden C.J.F. Boonacker E. Bissell E.R. Meijer A.J. Van Marle J. Smith R.E. Anal. Biochem. 1997; 252: 71-77Crossref PubMed Scopus (38) Google Scholar) have been used for characterization of isolated and cell-bound enzymatic activity. The stable cellular association of the released fluorophor, which prevents a high background fluorescence, is essential for sensitive detection of cellular enzymatic activity. Rhodamine 110 is a highly fluorescent xanthene dye with an excitation wavelength at 494 nm and a quantum yield of 0.91, reflecting the quotient of emitted and absorbed photons (24Leytus S.P. Melhado L.L. Mangel W.F. Biochem. J. 1983; 209: 299-307Crossref PubMed Scopus (145) Google Scholar). It possesses two amino groups suitable for the coupling of protease substrates, which results in a colorless nonfluorescent compound. Hydrolysis of these substrate structures releases the highly fluorescent rhodamine 110 (24Leytus S.P. Melhado L.L. Mangel W.F. Biochem. J. 1983; 209: 299-307Crossref PubMed Scopus (145) Google Scholar). Earlier investigations dealt with the use of such substrates for the detection of enzymatic activities of lysosomal localized and isolated proteases (25Assfalg-Machleidt I. Rothe G. Klingel S. Banati R.B. Mangel W.F. Valet G. Machleidt W. Biol. Chem. Hoppe-Seyler. 1992; 373: 433-440Crossref PubMed Scopus (42) Google Scholar, 26Leytus S.P. Patterson W.L. Mangel W.F. Biochem. J. 1983; 215: 253-260Crossref PubMed Scopus (97) Google Scholar, 27Rothe G. Klingel S. Assfalg-Machleidt I. Machleidt W. Zirkelbach C. Banati R.B. Mangel W.F. Valet G. Biol. Chem. Hoppe-Seyler. 1992; 373: 547-554Crossref PubMed Scopus (48) Google Scholar, 28Ulbricht B. Spiess E. Schwartz-Albiez R. Ebert W. Biol. Chem. Hoppe-Seyler. 1995; 376: 407-414Crossref PubMed Scopus (14) Google Scholar, 29Hug H. Los M. Hirt W. Debatin K.-M. Biochemistry. 1999; 38: 13906-13911Crossref PubMed Scopus (111) Google Scholar, 30Liu J. Bhalgat M. Zhang C. Diwu Z. Hoyland B. Klaubert D.H. Bioorg. Med. Chem. Lett. 1999; 9: 3231-3236Crossref PubMed Google Scholar). In these studies, a stable cellular fluorescence was considered because of the intracellular hydrolysis of the substrates and the accumulation of the fluorescent cleaving products within the cells. Our previous data described rhodamine compounds of the types (Xaa-Pro)2-R110 and Xaa-Pro-R110 as sensitive substrates of isolated and cell surface-associated DP IV (31Lorey S. Faust J. Hermanns U. Bu¨hling F. Ansorge S. Neubert K. Adv. Exp. Med. Biol. 1997; 421: 157-160Crossref PubMed Scopus (4) Google Scholar). Here we describe novel fluorogenic DP IV substrates Xaa-Pro-R110-R, which represents bifunctional compounds containing the substrate structure Gly(Ala)-Pro and thiol and amino group reactive anchor R for the covalent fixation of R110 on the cell surface. These substrates provide the opportunity to determine cell surface-associated DP IV activity on single cells. Moreover, by studying these membrane-fixed substrates, it was possible to demonstrate that the ectoenzyme DP IV could be involved in transcellular proteolysis. R110 was obtained from Synthon AcMaRi GmbH & Co. (Wolfen, Germany). Boc-Gly-Pro-OH and Boc-Ala-Pro-OH were purchased from Bachem (Heidelberg, Germany). 4-Chlorobutyrylchloride, 5-chlorovalerylchloride, and 5-bromovalerylchloride were from Aldrich. 4-Maleimidobutyric acid, 6-aminocaproic acid, and 2,3-dichloromaleic acid anhydride were from Fluka Chemie AG (Buchs, Germany), 6-Maleimidocaproic acid was from Sigma, and chloroacetylchloride was obtained from Merck. RPMI 1640 medium, Iscove's modified Dulbecco's medium, Glasgow minimum essential medium, trypsin/EDTA, antibiotic mix (penicillin/streptomycin), and fetal calf serum were obtained from Invitrogen and PAA Laboratories GmbH (Linz, Austria). The antibody CD26-PE was purchased from Becton Dickinson (San Jose, CA), and sheep anti-mouse IgG1 antiserum conjugated to Texas Red (TR) was from ICN Biomedical GmbH (Eschwege, Germany). Boc-Gly-Pro-R110 and Boc-Ala-Pro-R110 were synthesized according to the carbodiimide method (32Deffner M. Deimer K.-H. Jaeger E. Stelzel P. Thamm P. Wendlberger G. Wu¨nsch E. Wu¨nsch E. Methoden der organischen Chemie: Synthese von Peptiden. Georg Thieme Verlag, Stuttgart1974Google Scholar). Boc-Xaa-Pro-OH (1 mmol; 272.3 mg of Boc-Gly-Pro-OH, 286.3 mg of Boc-Ala-Pro-OH) was dissolved in 2 ml of dimethylformamide, and the solution was cooled down to 4 °C. While stirring, 366.8 mg (1 mmol) of R110·HCl, 127 μl (1 mmol) of N-ethylmorpholine, and 191.7 mg (1 mmol) of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide·HCl were added, and the solution was stirred for 1 h at 4 °C and then for 6 h at room temperature. The solution was poured into 60 ml of ice water and extracted three times with 40 ml of AcOEt. The organic phase was collected, washed three times with saturated NaCl solution, then dried over anhydrous Na2SO4, filtered, and evaporated. The compounds obtained, Boc-Gly-Pro-R110 and Boc-Ala-Pro-R110, were purified by MPLC with AcOEt yielding 137.0 mg (28.3%) of Boc-Gly-Pro-R110 and 171.7 mg (28.7%) of Boc-Ala-Pro-R110. Boc-Gly-Pro-R110-R (where R = R2–R6) were synthesized by the acid chloride procedure (32Deffner M. Deimer K.-H. Jaeger E. Stelzel P. Thamm P. Wendlberger G. Wu¨nsch E. Wu¨nsch E. Methoden der organischen Chemie: Synthese von Peptiden. Georg Thieme Verlag, Stuttgart1974Google Scholar). A 292.2-mg (0.5 mmol) quantity of Boc-Gly-Pro-R110 was dissolved in 2 ml of dimethylformamide and cooled down to 4 °C. Then 0.5 mmol of the acid chloride of R (56.5 mg of chloroacetylchloride (R2), 70.5 mg of 4-chlorobutyroylchloride (R3), 77.5 mg of 5-chlorovaleroylchloride (R4), 99.7 mg of 5-bromovaleroylchloride (R5), and 94.5 mg of chloromethylbenzoylchloride (R6)) was added, and the solution was stirred for 1 h at 4 °C and then for 1 h at room temperature. The preparation was performed as described above. The compounds obtained, Boc-Gly-Pro-R110-R (R = R2–R6), were purified by MPLC with AcOEt yielding 206.6 mg (62.6%) of Boc-Gly-Pro-R110-R2, 201.3 mg (58.5%) of Boc-Gly-Pro-R110-R3, 203.3 mg (57.9%) of Boc-Gly-Pro-R110-R4, 183.6 mg (49.2%) of Boc-Gly-Pro-R110-R5, and 68.8 mg (18.7%) of Boc-Gly-Pro-R110-R6. Boc-Gly-Pro-R110-R (R = R7 and R8) were synthesized by the carbodiimide method as described above and purified by MPLC with AcOEt/PEth = 9:1 (v/v, R7) or AcOEt/PEth = 8:2 (v/v, R8) yielding 243.2 mg (64.9%) (where R = R2–R6) Boc-Gly-Pro-R110-R7 and 169.5 mg (43.6%) (where R = R2–R6) Boc-Gly-Pro-R110-R8. Dichloromaleic acid anhydride (2.11 g; 1 mmol) and 6-aminocaproic acid (1.31 g; 1 mmol) were dissolved in dry tetrahydrofurane and refluxed for 3 h. The solvent was removed, and the solid was dissolved in CHCl3and filtered. The filtrate was then evaporated. The residue was recrystallized from diethyl ether/PEth to give 1.81 g (86%) of 6-(2,3-dichloromaleimidyl)caproic acid (melting point 95.5–96 °C; electrospray mass spectrometry, 279; [M+H]+, calculated 279.0). To 61.4 mg (0.22 mmol) of 6-(2,3-dichloromaleimidyl)caproic acid in 2.5 ml of dimethylformamide, 119.6 mg (0.2 mmol) of Boc-Ala-Pro-R110 and 46 mg (0.24 mmol) of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide·HCl were added. The solution was stirred for 1 h at 0 °C and then for 20 h at room temperature. Then 30.7 mg (0.11 mmol) of 6-(2,3-dichloro-maleimidyl)caproic acid and 23 mg (0.12 mmol) of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide·HCl were added, and the mixture was stirred at room temperature for 6 h. The preparation was performed as described above. The crude product Boc-Ala-Pro-R110-R9 was purified by MPLC with AcOEt/PEth = 85/15 (v/v) yielding 60 mg (35%) (electrospray mass spectrometry, 860.3; [M+H]+, calculated 859.23). Boc-Gly-Pro-R110-R1was obtained by acetylation of Boc-Gly-Pro-R110. A 116.85-mg (0.2 mmol) quantity of Boc-Gly-Pro-R110 was dissolved in 2.5 ml of pyridine, and after the addition of 0.5 ml acetanhydride, the solution was left for 24 h at room temperature. The solvent was evaporated, the residue was resolved in 10 ml of toluol, and the solvent was evaporated again. This procedure was repeated three times, and then 120.6 mg (96.3%) of the product Boc-Gly-Pro-R110-R1 was precipitated by the addition of diethyl ether. The Boc protecting group of the compounds Boc-Gly-Pro-R110-R (R = R2–R8) was removed by dissolving 0.1 mmol of Boc-Gly-Pro-R110-R in 1.8 ml of CH2Cl2 and 200 μl of trifluoroacetic acid. The solution was left for 3 h at room temperature. The product Gly-Pro-R110-R was precipitated by the addition of cold diethyl ether before being filtered and dried in a vacuum. Boc-Gly-Pro-R110-R1 and Boc-Ala-Pro-R110-R9 were deprotected by dissolving 0.1 mmol in 1 ml of HCl/acetic acid (1 m HCl). The solution was left for 30 min at room temperature, and the product was precipitated as described above. R110-R6 was synthesized using the carbodiimide method as described above. The product obtained was purified by MPLC with AcOEt/PEth (1:1, v/v) yielding 137.0 mg (10.0%). The molecular weight for C28H19O4N2Cl was calculated as 482.10 (electrospray mass spectrometry, m/z483.20; [M+H]+). Gly-Pro-R110-R compounds where R = R1, R2, R3, and R6 were purified by reversed-phase HPLC. These were analyzed by TLC (butan-1-ol/acetic acid ethyl ester/formic acid/water, 1:1:1:1, v/v/v/v), HPLC, mass spectrometry, and 1H NMR (H,H-correlated spectroscopy, total correlated spectroscopy, and Rotating Frame Overhauser Effect Spectroscopy spectra). Compounds of the type Gly-Pro-R110-R appeared ascis and trans conformers with regard to the Gly-Pro peptide bond, each of them existing as mixture ofR/S stereoisomers. The analytical data are shown in Table I.Table IAnalytical data of the synthesized compoundsCompoundSummary formulaYieldMelting point[α]20420 (c) in methanolm/z ([M +H]+)trans(R/S)cis (R/S)%°C°%%Gly-Pro-R110-R R = R11-aHydrochloride.C29H26O6N460.0 201–20527.360.1 /25.914.0 R = R2C29H25O6N4Cl66.5166−59.7 (0.86)561.547.3 /39.213.5 R = R31-bTrifluoroacetate.C33H30O8N4ClF380.5159−83.9 (0.89)589.543.5 /42.514.0 R = R4C32H31O6N4Cl57.0155−68.8 (0.62)603.544.0 /41.015.0 R = R51-bTrifluoroacetate.C34H32O8N4CBrF382.6163−65.2 (1.37)649.544.7 /41.314.0 R = R6C35H29O6N4Cl69.3−60.1 (0.61)636.940.9 /40.119.0 R = R71-bTrifluoroacetate.C37H32O10N5F380.6173−54.4 (0.87)650.6 R = R81-bTrifluoroacetate.C39H36O10N5F383.1177−32.9 (0.75)678.6Ala-Pro-R110-R R = R91-aHydrochloride.C38H36O8N5Cl324.0<180−63.0 (0.84)760.01-a Hydrochloride.1-b Trifluoroacetate. Open table in a new tab DP IV with a specific activity of 51.9 units/mg (measured with Gly-Pro-pNA) at a protein concentration of 2.25 mg/ml was used. All of the enzyme assays were incubated at 30 °C in 0.04m Tris/HCl buffer (pH 7.6, I = 0.125m with KCl). The cleavage of substrates catalyzed by DP IV (pig kidney) was measured at enzyme concentrations of 6.85 × 10−10 and 1.37 × 10−9m. The hydrolysis product R110-R was monitored at 494 nm and substrate concentrations between 10−6 and 4 × 10−4m over 120 s, with at least two independent measurements. The kinetic parameters were evaluated using the software Microcal Origin 4.10 and specified by standard deviation. The molar absorption coefficients of R110-R were established via enzymatic hydrolysis and acid hydrolysis. Substrate solutions (5 × 10−6, 10−5, and 2 × 10−5m) were incubated with DP IV (10−8m) for 5 h at 30 °C. After completion of hydrolysis, the absorption was measured spectrophotometrically at 494 nm. For acid hydrolysis, the substrate solution was incubated in 6 m HCl for 8 h at room temperature. The solution was then neutralized with 6m NaOH and diluted with Tris/HCl buffer. The absorption was measured spectrophotometrically at 494 nm. U937 cells (33Sundstrom C. Nilsson K. Int. J. Cancer. 1976; 17: 565-577Crossref PubMed Scopus (1951) Google Scholar) were cultured in RPMI 1640 medium supplemented with 10% (w/v) fetal calf serum, 10−3m glutamine, and antibiotics. CHO wild-type cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% (w/v) fetal calf serum, and CD26-transfected CHO cells (34Hong W.J Piazza G.A. Hixson D.C. Doyle D. Biochemistry. 1989; 28: 8474-8479Crossref PubMed Scopus (16) Google Scholar) were cultured in Glasgow minimal essential medium with supplements (10% fetal calf serum (w/v), antibiotics, sodium bicarbonate (29.3 mm), 1% nonessential amino acids (v/v, 100×), 372.2 mm glutamine, 364.7 mmasparagine, 0.91 mm sodium pyruvate, 23.9 mmadenosine, 22.6 mm guanosine, 26.3 mm cytidine, 26.2 mm uridine, and 9.0 mm thymidine). All of the cells were cultured at 37 °C in 5% (v/v) CO2. The peripheral blood mononuclear cells were isolated from heparinized blood (10 units/ml blood) of healthy donors using density gradient centrifugation with Ficoll-Paque (Pharmacia Corp.) for 30 min at 500 × g. The cells were incubated and washed with PBS (pH 7.2), and all of the centrifugation steps were performed at room temperature. The cell suspension (100 μl, 2 × 105 U937 cells) was mixed with 100 μl of substrate solution (3.3 × 10−3mGly-Pro-pNA in PBS). Part of this mixture (100 μl) was removed immediately, and the enzymatic reaction was stopped by the addition of 400 μl of acetate buffer (1 m, pH 4.4). The remaining 100 μl were incubated for 60 min at 37 °C. The reaction was then stopped as described above. After centrifugation (2 min at 10,000 × g) the absorbance of the supernatant was measured spectrophotometrically at 390 nm. For measurements of substrate hydrolysis by cell-associated DP IV, 96-well plates were used. Substrates Xaa-Pro-R110-R (50 μl, five concentrations between 10−6 and 10−5m) were incubated with 50 μl of a suspension of U937 cells (DP IV activity determined using Gly-Pro- pNA = 25 pkat/ml) at 37 °C. Fluorescence was measured immediately and after 30 min (excitation, 490 nm; emission, 520 nm) using a fluorescence plate reader Fluorolite 1000 (Dynatech Laboratories). The specificity of the substrate hydrolysis was determined using specific DP IV inhibitors Lys[Z(NO2)]thiazolidide, Lys[Z(NO2)]piperidide, and Lys[Z(NO2)]pyrrolidide (34Hong W.J Piazza G.A. Hixson D.C. Doyle D. Biochemistry. 1989; 28: 8474-8479Crossref PubMed Scopus (16) Google Scholar). The U937 cells (50 μl, 105 cells) were incubated with the substrates (10−5m) and the inhibitors (10−6–10−4m). U937 cells (200 μl, 2 × 105 cells) were incubated with substrates Xaa-Pro-R110-R (5 × 10−6m) for 30 min at 37 °C and washed as described above. The cellular fluorescence was measured by flow cytometry (FACS Calibur, Beckton Dickinson), and 104 cells were registered for each sample. Wild-type CHO cells were incubated for 20 min with the substrate Xaa-Pro-R110-R (5 × 10−5m) at 4 °C. The cells were then washed, resuspended in 100 μl of PBS, and incubated for 20 min at 37 °C in the presence or absence of 100 μl of isolated DP IV (100 pkat/ml) or CD26-overexpressing CHO cells (100 pkat/ml). As a control the cells were incubated in presence of the DP IV-specific inhibitor Lys[Z(NO2)]thiazolidide (10−4m). The cellular fluorescence was measured by flow cytometry. Wild-type CHO cells incubated in PBS as well as incubated in the presence of the inhibitor and the substrate were used as controls. Wild-type CHO cells and CD26-overexpressing CHO cells as well as a mixture of both cell types were incubated for 20 min with the substrate Xaa-Pro-R110-R (5 × 10−6m) at 4 °C. Simultaneously, the cells were incubated with a PE-labeled anti-CD26 antibody. The cells were washed, resuspended in 200 μl of PBS, and incubated for 20 min at 37 °C. Alternatively, peripheral blood mononuclear cells were stained using the substrates Xaa-Pro-R110-R and antiCD26 antibody. The cellular fluorescence was measured by flow cytometry. The cells incubated with substrate only, PE-labeled anti-CD26 antibody, and PE-labeled IgG were used as controls. CD26-transfected CHO cells and wild-type CHO cells were grown in 8-well chambers and stained with anti-CD26 and TR-labeled secondary antibodies. After antibody staining, the cells were incubated with Gly-Pro-R110-R6 or Ala-Pro-R110-R9 (5 × 10−6m) in the presence of Lys[Z(NO2)]thiazolidide (10−4m) for 30 min at room temperature, then washed, and incubated for a further 30 min. The cellular fluorescence was observed using a conventional microscope (Axiovert 135 TV, Zeiss, Jena, Germany). We synthesized novel DP IV anchor substrates Xaa-Pro-R110-R with Xaa being Gly or Ala and R being a reactive anchor group of varying length, hydrophobicity, and reactivity (Fig.1). The compounds were colorless and nonfluorescent. The DP IV-catalyzed substrate hydrolysis resulted in the release of the highly fluorescent R110-R, which was measured at absorption maxima of 467 and 494 nm and by an emission maximum of 525 nm. The hydrolysis products R110-R were neither substrates nor inhibitors of DP IV. The molar absorption coefficients were found to be almost 30,000 m−1 cm−1 (R110-R (m−1·cm−1); R1, ε494 = 25,495; R2, ε494 = 30,687; R3, ε494 = 30,263; R4, ε494 = 29,653; R5, ε494 = 28,256; R7, ε494 = 30,237; R8: ε494 = 29,204, acid hydrolysis; R9, ε494 = 32,173, acid hydrolysis). R110-R6showed a decreased molar absorption coefficient (ε494 = 9686 m−1·cm−1) caused by the aromatic moiety. All compounds Xaa-Pro-R110-R were hydrolyzed by isolated DP IV from pig kidney; thekcat/Km values were between 1.14 × 106 (Gly-Pro-R110-R2) and 3.33 × 106 (Gly-Pro-R110-R6)m−1·s−1 (TableII). The compounds were hydrolyzed by the model of substrate inhibition in a nonhyperbolic dependence of initial velocity on substrate concentration. A longer and more hydrophobic R resulted in a decrease in the Km value.Table IIKinetic constants of the DP IV-catalyzed hydrolysis of Xaa-Pro-R110-RCompound105Kmkcat10−6kcat/Km104Kims−1m−1·s−1mGly-Pro-R110-R R = R117.40 ± 6.24235.60 ± 43.251.35 ± 0.562.95 ± 1.04 R = R26.23 ± 0.6170.84 ± 4.211.14 ± 0.132.30 ± 0.25 R = R33.77 ± 0.4067.49 ± 3.461.79 ± 0.216.05 ± 0.90 R = R43.83 ± 0.3159.65 ± 2.551.56 ± 0.142.24 ± 0.19 R = R51.54 ± 0.1742.39 ± 1.782.75 ± 0.337.87 ± 1.58 R = R61.10 ± 0.2036.64 ± 3.743.33 ± 0.691.02 ± 0.27 R = R72.67 ± 0.2160.12 ± 2.082.25 ± 0.196.32 ± 0.77 R = R81.25 ± 0.1040.41 ± 1.233.23 ± 0.283.69 ± 0.39Ala-Pro-R110-R R = R90.58 ± 0.0714.67 ± 0.922.55 ± 0.440.80 ± 0.13The measurements were performed in 40 mm Tris/HCl buffer (pH 7.6, I = 0.125) at 30 °C over 120 s. The cleavage of the substrates (between 10−6 and 4 × 10−4) was monitored by measuring the absorption of the released R110-R at 494 nm. In the assay, DP IV (pig kidney) was set between 6.85 × 10−10 and 1.37 × 10−9m. Open table in a new tab The measurements were performed in 40 mm Tris/HCl buffer (pH 7.6, I = 0.125) at 30 °C over 120 s. The cleavage of the substrates (between 10−6 and 4 × 10−4) was monitored by measuring the absorption of the released R110-R at 494 nm. In the assay, DP IV (pig kidney) was set between 6.85 × 10−10 and 1.37 × 10−9m. All compounds Xaa-Pro-R110-R were hydrolyzed after incubation with DP IV-rich U937 cells. The cleavage of the substrates Gly-Pro-R110-R depended on the steric requirements of the anchor group R. A large or a short R residue resulted in decreased fluorescence after cleavage of the substrates by cell-associated DP IV (Fig.2). Using the specific DP IV inhibitors Lys[Z(NO2)]thiazolidide, Lys[Z(NO2)]pyrrolidide, and Lys[Z(NO2)]piperidide (35Born I. Faust J. Heins J. Barth A. Neubert K. Eur. J. Cell Biol. 1994; 63 (Suppl. 40): 23Google Scholar) there was almost full inhibition of substrate hydrolysis at inhibitor concentrations of 10−5m (not shown). The suitability of the anchor group R for the stable fixation of the substrates Xaa-Pro-R110-R or the hydrolysis products R110-R on cells was examined. U937 cells were incubated with substrates Xaa-Pro-R110-R (5 × 10−6m), and the cell-associated fluorescence of the hydrolysis product R110 was measured directly and after one to four washing steps. The stability of the cell-associated fluorescence was classified according to the R residue (Table III). After hydrolysis of Gly-Pro-R110-R1 (R1 = acetyl group) and four washings, only 3% of the initially detected cleavage product remained surface-associated. On the other hand, after hydrolysis of Gly-Pro-R110-R2 (R2 = chloroacetyl anchor) and four washings, 92.5% of the initial fluorescences signal was detected on the cells. The substrates with the chloromethylbenzoyl anchor (R6) and N-carboxyalkyl-maleinimide anchors (R7, R8, and R9) were less reactive (cell-associated fluorescence was between 70 and 80%). Aliphatic halogenalkylcarbonyl residues R (R3, R4, and R5) were the most ineffective anchors with cell-associated fluorescence between 10 and 30%. Generally, the insertion of a reactive group on the R110 molecule resulted in stable fixation of the fluorophore on cells.Table IIICellular fluorescence after incubation with substrates Xaa-Pro-R110-RCompoundCell-associated fluorescence relative to the cumulative fluorescence intensityWash step 1Wash step 2Wash step 3Wash step 4%Gly-Pro-R110-R R = R110.40 ± 5.406.84 ± 3.404.62 ± 2.672.73 ± 1.28 R = R293.84 ± 4.4394.82 ± 1.2493.50 ± 0.9192.50 ± 0.78 R = R336.18 ± 7.3429.65 ± 9.0429.65 ± 9.0425.72 ± 7.48 R = R424.77 ± 5.4414.07 ± 5.3811.91 ± 4.269.91 ± 3.91 R = R542.77 ± 0.8236.94 ± 5.0235.36 ± 5.8732.90 ± 7.02 R = R675.23 ± 2.5871.81 ± 3.2971.50 ± 3.9371.03 ± 5.14 R = R789.56 ± 8.7378.09 ± 9.3971.04 ± 8.2871.04 ± 8.28 R = R888.46 ± 6.2680.36 ± 9.5174.78 ± 8.8172.42 ± 9.40Ala-Pro-R110-R R = R993.59 ± 5.4685.31 ± 9.8380.26 ± 9.6377.39 ± 9.57106 U937 cells/ml were incubated in PBS (pH 7.2) with substrates Xaa-Pro-R110-R (5 × 10−6m) at 37 °C. After 20 min, the cell-associated fluorescence was measured by flow cytometry (excitation, 488 nm; emission, 530/30 nm). The cells were washed four times with 1 ml of PBS (pH 7.2), and the cell-associated fluorescence intensity was measured by flow cytometry after each washing step a
Referência(s)