Published online before print February 7, 2007
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Laboratories of
* Medical Biochemistry and
Medicinal Chemistry, University of Antwerp, Wilrijk, Belgium;
Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA; and
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Nankang, Taipei, Taiwan, Republic of China
1 Correspondence: Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. E-mail: ingrid.demeester{at}ua.ac.be
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Key Words: DPPIV • DPP8 • DPP9 • fibroblast activation protein • vildagliptin
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(FAP), DPPII, DPP8, and DPP9, because of their preference for cleavage after X-Pro in vitro, are likely to be involved in many of these processes. These DPPs are emerging as an important protease family with roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an interesting therapeutic potential, particularly in diabetes, oncology, and hematology [1
]. Among the DPPs able to cleave post-Pro-bonds, DPPIV (E.C. 3.4.14.5) has been studied most extensively [2
]. Some DPPIV inhibitors (vildagliptin, sitagliptin) are under U.S. Food and Drug Administration review for the treatment of Type 2 diabetes, having demonstrated improved hypoglycemic control through this unique mechanism [3
]. Clinical studies in cancer patients are also ongoing using Val-boro-Pro (Talabostat), a nonselective DPP inhibitor. The target in these studies was suggested to be FAP [4
]. Several immunological functions have also been attributed to DPPIV (identified as the surface antigen CD26). Among them are T cell costimulatory activation and regulation of chemokine biology through truncation at their N terminus [2
]. In 2000 and 2002, two new DPPIV homologues, DPP8 and DPP9, were cloned and expressed [5
, 6
]. The functions of these DPPs are unknown. Because of their similar substrate specificity and tissue distribution, it is tempting to speculate participation of these recently discovered DPPs in biological functions, which were attributed to DPPIV previously. For example, in several reported studies, DPPIV inhibitors elicited similar responses in cells and animals, irrespective of DPPIV expression [7
]. Recently, DPP8/9 inhibitors were suggested to attenuate T cell activation in human in vitro models, properties that were attributed previously to inhibition of DPPIV [8
]. Furthermore, only recently, a dual mechanism of action in cancer was suggested for the DPP inhibitor Talabostat. Besides targeting FAP, the compound was suggested to stimulate innate and acquired immunity through inhibition of DPP8/9 [9
]. Based on mRNA expression, DPP8 was shown to be up-regulated in activated T cells and expressed in several B and T cell lines [5 ]. Northern blots showed DPP9 was also expressed in peripheral blood leukocytes [10 ]. However, only recombinant forms of DPP8 and DPP9 have been characterized previously, and their enzymatic activity has not been demonstrated in or purified from any natural source. Cellular DPPIV-like enzymatic activity represents the sum of the hydrolytic activities of several DPPIV homologues. Discriminating between these enzymes of the DPPIV family is difficult because of their similar substrate selectivity on X-Pro-derived synthetic substrates at neutral pH. However, selective inhibitors for the new DPPs have been developed (Fig. 1 ) and allow a better understanding of their role in physiology and pathology [8 , 11 ]. The potent, dual DPP8/9 inhibitor (2S,3R)-2-amino-1-(isoindolin-2-yl)-3-methylpentan-1-one (allo-Ile-isoindoline; UAMC00132), used by Lankas et al. [8 ], was synthesized and applied in our study as well as the selective DPPII inhibitor N-(4-chlorobenzyl)-4-oxo-4-(1-piperidinyl)-1,3-(S)-butanediamine dihydrochloride (UAMC00039) [12 , 13 ]. For inhibiting DPPIV activity, the well-studied, reversible inhibitor (2S)-{[(3-hydroxyadamantan-1-yl)amino]acetyl}pyrrolidine-2-carbonitrile (NVP-LAF237; vildagliptin, Galvus®) [14 , 15 ] and the irreversible inhibitor Bis(4-acetamidophenyl) 1-((S)-prolyl)pyrrolidine-2(R,S)-phosphonate hydrochloride (AB192) [16 ] were used. As inhibition of DPP8/9 activity was suggested to be toxic [8 ], the natural distribution of DPP8/9 activity deserves investigation. Here, we investigated the presence of DPP8/9 activity in human leukocytes. Based on inhibition profiles, we demonstrate DPP activity, attributable to DPP8/9, in human peripheral blood leukocytes (lymphocytes and monocytes), in Jurkat cells (a human T cell line), and in U937 cells (a human monocytic cell line).
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Figure 1. Structures of the DPP inhibitors used. The DPPII inhibitor UAMC00039 (1), the DPP8/9 inhibitors UAMC00132 (2) and UAMC00071 (3), and the DPPIV inhibitors NVP-LAF237 (4) and AB192 (5).
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Cells
Human U937 and Jurkat cells (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) heat-inactivated FBS at 37°C in 5% CO2. Human PBMC were isolated by Ficoll-Paque density gradient centrifugation of buffy coats, washed three times in RPMI, and incubated overnight in complete medium at 37°C in 5% CO2 before use (nonadherent cells: lymphocytes; adherent cells: monocytes).
Enzyme activity and protein content were determined after lysing the cells. Washed cells were suspended (
107 cells/100 µl) in lysis buffer [LB; 1% octylglucoside, 10 mM EDTA, and 70 µg/ml aprotinin in 0.05 M cacodylic acid-NaOH buffer, pH 5.5 (LB, pH 5.5), or in 0.05 M HEPES buffer, pH 7.0 (LB, pH 7.0)], incubated for 1 h, and centrifuged for 10 min at 12,000 rpm at 4°C. The resulting supernatant was used as cell lysate. Cells were lysed at neutral pH as a result of the instability of purified DPP8 at acidic pH (Supplemental Fig. 1 and ref. [21
]). However, cell lysis at pH 7.0 made it difficult to measure DPPII activity at pH 5.5 as a result of the precipitation of proteins during the assay.
Subcellular fractionation was performed according to ref. [22 ]. Cells were ruptured by a single freeze-thaw cycle, followed by 3 x 15 s sonication on ice. Fractions were lysed in LB, pH 7.0.
Protein content was determined according to Bradford [23 ] with BSA as a standard.
Enzyme assays
Enzyme activities were determined kinetically in a final volume of 200 µl for 10 min at 37°C by measuring the initial velocities of pNA release (405 nm) from the substrate using a Spectramax plus microtiterplate reader (Molecular Devices, Sunnyvale, CA, USA). One unit enzyme activity was defined as the amount of enzyme that catalyzes the release of 1 µmol pNA from the substrate/min under assay conditions. DPPII activity was determined using Lys-Ala-pNA (1 mM) at pH 5.5 [17
]. The substrates Ala-Pro-pNA (1 mM in 0.05 M HEPES buffer, pH 7.0, containing 10 mM EDTA, 14 µg/ml aprotinin, and 0.1% Tween 20) and Gly-Pro-pNA (0.5 mM in 0.05 M Tris buffer, pH 8.3, containing 10 mM EDTA and 14 µg/ml aprotinin) were used to probe DPPIV, DPP8/9, and/or FAP activity.
Inhibition assays
Cell lysates and pure enzyme samples diluted in LB, pH 7.0, containing 10 mg/ml BSA were preincubated for 15 min at 37°C with a wide range of inhibitor concentrations of UAMC00039 (0.1–1000 nM), NVP-LAF237 (50–50,000 nM), UAMC00132 (5–5000 nM), or AB192 (25 µM). DPP activities were determined as described above.
CD26/DPPIV immunocapture assay
In brief, MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with mouse anti-hDPPIV antibodies (anti-TA5.9 mAb, 20 µg/ml in PBS) [24
, 25
] overnight at 4°C and blocked with 0.5% BSA, 0.05% Tween 20, in PBS. Alternatively, a coated ELISA plate recognizing human soluble (hs)CD26 from Bender MedSystems (Vienna, Austria) was used. The lysates or the standards (100 µl; pure CD26/DPPIV) were added to each well and incubated for 3 h at room temperature under gentle agitation. After washing three times using 0.05% Tween 20 in PBS and a final wash step with 0.05 M Tris, pH 8.3, bound CD26/DPPIV was detected by measuring the enzymatic activity using Gly-Pro-pNA at pH 8.3 as described above.
Enrichment procedure of DPP8/9-like activity
Lys-isoindoline-Sepharose 4 Fast Flow affinity column preparation
The affinity gel was prepared based on ref. [17
] with slight modifications. A 4x molar excess of Lys-isoindoline (compound 3; seeFig. 1
) was coupled to 20 ml N-hydroxysuccinimide-activated Sepharose 4 Fast Flow, using propan-2-ol as a coupling solvent. Blocking and washing conditions were as described in ref. [17
]. After washing (3x) with 0.05 M HEPES buffer, pH 7.0, containing 0.15 M NaCl at room temperature, the Lys-isoindoline affinity gel was ready for use. A "dummy" affinity gel was prepared in parallel exactly as described above except for the presence of Lys-isoindoline during the coupling.
Enrichment of DPP8/9-like activity
The cytosol of Jurkat cells (2.2x109 cells) was loaded onto a Con A-Sepharose column, equilibrated, and washed with 0.02 M HEPES buffer, pH 7.0, containing 1 mM CaCl2, 1 mM MnCl2, 14 µg/ml aprotinin, and 0.15 M NaCl. Elution was performed with 1 M methyl--D-glucopyranoside (Sigma Chemical Co., St. Louis, MO, USA) in the same buffer. Active fractions of the flow-through were brought to pH 7.4 and applied to a HiTrap Q HP column, equilibrated and washed with 0.02 M Tris buffer, pH 7.4. Elution was performed with a Linear 0–1 M NaCl gradient in equilibration buffer. (NH4)2SO4 (0.5 M) was added to fractions containing DPP activity. The sample was incubated for 1 h on ice and subsequently centrifuged for 90 min at 10,000 rpm at 4°C. The supernatant was loaded onto a 1-ml HiTrap Phenyl HP column, equilibrated, and washed with 0.02 M Tris buffer, pH 7.4, containing 0.5 M (NH4)2SO4. Elution was performed with a Linear 0.5–0 M (NH4)2SO4 gradient in 0.02 M Tris buffer, pH 7.4, followed by 0.02 M Tris buffer, pH 7.4. Active fractions were concentrated on a Microcon YM-50 centrifugal concentrator (Millipore, Bedford, MA, USA) and diluted in 0.05 M HEPES buffer, pH 7.0, containing 0.15 M NaCl. One part of the sample was applied to 100 µl Lys-isoindoline affinity gel, prepared and washed as described above. Exactly the same amount of sample was applied onto 100 µl of the dummy affinity gel. After incubation for 1 h at room temperature, the gel was washed (3x) with 0.05 M HEPES buffer, pH 7.0, containing 0.1% Tween 20 and increasing concentrations of NaCl. A mixture of DPPII, DPPIV, and 1 mg BSA, diluted in HEPES buffer, pH 7.0, containing 0.15 M NaCl, underwent the same procedure for the Lys-isoindoline affinity chromatography.
Kinetic and inhibition parameters of the enriched DPP preparation
Pure DPP8 and DPPIV were diluted in 0.02 M Tris buffer, pH 7.4, containing 0.125 M (NH4)2SO4 and 0.1 mg/ml BSA and were incubated overnight at 4°C, similar to the active fractions obtained from HiTrap Phenyl HP chromatography. Activity toward Ala-Pro-pNA was assayed as described above. Michaelis-Menten constant (Km) values were determined by plotting the initial velocities of product formation against the different Ala-Pro-pNA concentrations used (at least six different concentrations) and fitting the data to the Michaelis-Menten equation by nonlinear regression analysis using GraFit Version 5.
IC50 values of UAMC00132 for DPP8 diluted in 0.02 M Tris buffer, pH 7.4, containing 0.125 M (NH4)2SO4 and 0.1 mg/ml BSA and for the enriched preparation eluted from the HiTap Phenyl HP column were obtained with substrate concentration near the Km value (DPP8) and at least 10 different inhibitor concentrations. IC50 values were calculated using GraFit software.
Statistical analysis
Data are expressed as mean ± SEM. For the statistical analysis, the SPSS statistical package (SPSS for Windows, v. 12.0, SPSS, Chicago, IL, USA) was used. Differences between groups were assessed using one-way or univariate ANOVA, followed by the Dunnett test. A value of P< 0.05 was considered significant. n represents the number of experiments.
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Table 1. Specific DPP Activities in Leukocytes
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Table 2. Selectivity of the DPP Inhibitors
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Figure 2. Inhibition of DPP activities in different leukocytes. DPP activities in the cell lysates were measured in the presence of different concentrations of the DPPIV inhibitor NVP-LAF237 and the DPP8/9 inhibitor UAMC00132 using Ala-Pro-pNA at pH 7.0 in the presence of 100 nM UAMC00039 (A) and Gly-Pro-pNA at pH 8.3 (B). For comparison, inhibition of the purified enzymes DPP8 and DPPIV was also measured. Relative activities (Vi/V0) are represented as the mean ± SEM of three to seven separate experiments. Vi, initial rate in presence of inhibitor; V0, initial rate in absence of inhibitor.
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Figure 3. DPP-specific activity in different leukocytes. DPP activity in the cell lysates was calculated based on the inhibition profiles using UAMC00039 for DPPII, UAMC00132 for DPP8/9, and residual activity as DPPIV. Specific activities (mU/mg) are represented as the mean ± SEM of three or four separate experiments. (A) Ala-Pro-pNA, pH 7.0. (B) Gly-Pro-pNA, pH 8.3. The differences between the activity groups were assessed with one-way ANOVA, followed by the Dunnett test. ***, P< 0.001; **, P < 0.01; *, P < 0.05, versus lymphocytes.
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Figure 4. Subcellular fractionation of lymphocytes and U937 cells. After lysis of the fractions, DPP8/9- and DPPIV-specific activities were determined based on the inhibition profiles using UAMC00039 and UAMC00132. Specific activities (mU/mg) are represented. (A) Lymphocytes: Ala-Pro-pNA, pH 7.0. (B) Lymphocytes: Gly-Pro-pNA, pH 8.3. (C) U937 cells: Ala-Pro-pNA, pH 7.0. (D) U937 cells: Gly-Pro-pNA, pH 8.3.
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90% inhibited by 2.5 µM UAMC00132, confirming the predominance of DPP8/9 in total cell lysates. Lymphocytes and monocytes did contain activity attributable to DPPIV. Thirty-five percent of the Gly-Pro-pNA-cleaving activity in the lymphocyte lysate could be captured by immobilized anti-CD26 mAb. Based on the inhibition profiles, a maximal binding of 70% could be expected. However, as the DPPIV standard also captured only 70% under these conditions (Table 3)
, we assumed that the DPPIV content was probably higher, as was predicted based on the inhibition profiles. The conclusion that lymphocytes contained the highest percentage of DPPIV activity remained valid. Immunocapture onto plates with immobilized TA5.9 mAb produced similar results. None of the anti-CD26 mAb used in our study cross-reacted with DPP8. |
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Table 3. Capturing DPPIV onto sCD26 ELISA Plates
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Received September 4, 2006; revised December 14, 2006; accepted January 15, 2007.
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-Amino-substituted analogues of 1-[(S)-2,4-diaminobutanoyl] piperidine as highly potent and selective dipeptidyl peptidase II inhibitors J. Med. Chem. 47,2906-2916[CrossRef][Medline] J. Biol. Chem. 280,19441-19444This article has been cited by other articles:
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