Dipeptidyl peptidase 8/9-like activity in human ... - Semantic Scholar

Uncorrected Version. Published on February 7, 2007 as DOI:10.1189/jlb.0906546

Dipeptidyl peptidase 8/9-like activity in human leukocytes Marie-Berthe Maes,* Ve´ronique Dubois,* Inger Brandt,* Anne-Marie Lambeir,* Pieter Van der Veken,† Koen Augustyns,† Jonathan D. Cheng,‡ Xin Chen,§ Simon Scharpe´,* and Ingrid De Meester*,1 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

Abstract: The proline-specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9-specific activities, and DPPII- and DPPIV-specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30-fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or -9, including inhibition by allo-Ile-isoindoline and affinity for immobilized Lys-isoindoline. J. Leukoc. Biol. 81: 000 – 000; 2007. Key Words: DPP8 䡠 DPP9 䡠 fibroblast activation protein 䡠 vildagliptin

INTRODUCTION An increasing number of biological processes appear to be regulated by Pro-specific N-terminal processing. The prolinespecific dipeptidyl peptidases (DPPs), such as DPPIV, fibroblast activation protein ␣ (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. 3.4.1.4.5) has been studied most extensively [2]. Some DPPIV inhibitors (vildagliptin, sitagliptin) are under U.S. Food and Drug Ad0741-5400/07/0081-0001 © Society for Leukocyte Biology

ministration 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 upregulated 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 DPPIVlike 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 (see Fig. 1) and allow a better understanding of their role in physiology

1 Correspondence: Laboratory of Medical Biochemistry, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. E-mail: ingrid. [email protected] Received September 4, 2006; revised December 14, 2006; accepted January 15, 2007. doi: 10.1189/jlb.0906546

Journal of Leukocyte Biology Volume 81, May 2007

Copyright 2007 by The Society for Leukocyte Biology.

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penicillin, 100 ␮g/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 ␮l) in lysis buffer [LB; 1% octylglucoside, 10 mM EDTA, and 70 ␮g/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 (Supplementary Fig. 1 and Supplementary Table 1; 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 ⫻ 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 Fig. 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).

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-oxo4-(1-piperidinyl)-1,3-(S)-butanediamine dihydrochloride (UAMC00039) [12, 13]. For inhibiting DPPIV activity, the well-studied, reversible inhibitor (2S)-{[(3-hydroxyadamantan1-yl)amino]acetyl}pyrrolidine-2-carbonitrile (NVP-LAF237; vildagliptin, Galvus威) [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).

Enzyme activities were determined kinetically in a final volume of 200 ␮l 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 ␮mol pNA from the substrate/ min under assay conditions. DPPII activity was determined using Lys-AlapNA (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 ␮g/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 ␮g/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 ␮M). 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 ␮g/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 ␮l; 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 GlyPro-pNA at pH 8.3 as described above.

MATERIALS AND METHODS

Enrichment procedure of DPP8/9-like activity

Materials

Lys-isoindoline-Sepharose 4 Fast Flow affinity column preparation

The DPPII inhibitor UAMC00039, the DPP8/9 inhibitors UAMC00132 and (S)-2,6-diamino-1-(isoindolin-2-yl)hexan-1-one (Lys-isoindoline; UAMC00071), and the DPPIV inhibitor AB192 were synthesized as described [8, 12, 16]; the DPPIV inhibitor NVP-LAF237 was custom-synthesized by GLSynthesis Inc. (Worcester, MA, USA; see Fig. 1). DPPII and DPPIV were purified from human seminal plasma [17, 18]. Recombinant human (rh)DPP8 and murine FAP were expressed and purified as described [19, 20]. Buffy coats were obtained from the Antwerp Blood Transfusion Center (Belgium).

The affinity gel was prepared based on ref. [17] with slight modifications. A 4⫻ molar excess of Lys-isoindoline (compound 3; see Fig. 1) was coupled to 20 ml normal human serum-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 (3⫻) 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.

Cells

Enrichment of DPP8/9-like activity

Human U937 and Jurkat cells (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI-1640 medium supplemented with 100 U/ml

The cytosol of Jurkat cells (2.2⫻109 cells) was loaded onto a Con A-Sepharose column, equilibrated, and washed with 0.02 M HEPES buffer, pH 7.0, con-

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taining 1 mM CaCl2, 1 mM MnCl2, 14 ␮g/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 flowthrough 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 Lineair 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 Lineair 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 ␮l Lys-isoindoline affinity gel, prepared and washed as described above. Exactly the same amount of sample was applied onto 100 ␮l of the dummy affinity gel. After incubation for 1 h at room temperature, the gel was washed (3⫻) 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. MichaelisMenten 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.

RESULTS Selective DPP assays and inhibitors DPP activity could be demonstrated in the lysates of all cell types tested using Ala-Pro-pNA at pH 7.0 and Gly-Pro-pNA at pH 8.3 (Table 1). The cleavage of Ala-Pro-pNA at neutral pH can be catalyzed by DPPII, DPP8, DPP9, DPPIV, and FAP, but the cleavage of Gly-Pro-pNA at pH 8.3 is a result of DPP8, DPP9, DPPIV, and FAP but not DPPII, as DPPII is not active at this pH. DPP8, DPP9, and particularly FAP cleave these substrates at reduced catalytic efficiency compared with DPPIV [21, 26]. A proline on the second position of these substrates prevents the sequential cleavage by a number of aminopeptidases. The use of selective DPP inhibitors (Fig. 1 and Table 2) was crucial for proper analysis and interpretation of leukocyte DPP activities. In the cell lysates, DPPII activity was determined through inhibition of the Ala-Pro-pNA-cleaving activity by 100 nM UAMC00039. Although DPPII was

TABLE 1.

Specific activity Lymphocytes Monocytes Jurkat cells U937 cells

Specific DPP Activities in Leukocytes Ala-Pro-pNA, pH 7.0 (mU/mg)

Gly-Pro-pNA, pH 8.3 (mU/mg)

19 ⫾ 3 7.0 ⫾ 0.4 5.7 ⫾ 0.3 9.3 ⫾ 0.5

13 ⫾ 5 3.3 ⫾ 0.5 3⫾0 3.8 ⫾ 0.3

The specific activities of Ala-Pro- and Gly-Pro-pNA-cleaving activities in the different cell lysates are given as the mean ⫾ SEM of three to four separate experiments.

inhibited completely at this concentration, DPP8 and DPPIV remained unaffected. Potent and selective inhibitors of DPP8 and DPP9 have not yet been reported; only dual DPP8/9 inhibitors, such as UAMC00132, are known [8]. As a result of similarities in enzyme mechanism and substrate selectivity and the lack of selective inhibitors, it has proven difficult to discriminate between DPP8 and DPP9 activities in our experiments. As can be seen in Table 2, the DPPIV inhibitors NVP-LAF237 and AB192 also substantially inhibited some of the other DPPs tested, although with reduced efficiency. As DPPII also cleaves Ala-Pro-pNA at neutral pH [17], UAMC00039 was added to exclude the involvement of DPPII. FAP has been reported not to be present in normal tissues; therefore, we did not anticipate interference by FAP in our assays of human leukocytes. At the concentrations used to inhibit DPPII, DPP8/9, and DPPIV completely, UAMC00039 (100 nM), UAMC00132 (5 ␮M), and AB192 (25 ␮M) did not affect rFAP activity (Table 2). In the presence of these three inhibitors at the concentrations mentioned above, no residual Ala-Pro-pNA-cleaving activity was measured in lysates of human leukocytes, Jurkat, and U937 cells (data not shown). Therefore, we concluded that there is no interference from FAP in our experimental setup.

Presence of the different DPPs in leukocytes Based on the specific DPP activities (Table 1) and inhibition profiles (Fig. 2) in cell lysates, the contribution of the different DPPs to substrate cleavage was determined. In lysates of leukocytes, 30 –50% of the Ala-Pro-pNA-cleaving activity was attributed to DPPII, as indicated by UAMC00039-mediated inhibition. As was already suggested in a previous study [13], the monocytes and U937 cells appeared to contain the highest DPPII versus “non-DPPII” DPP activity (DPPIV/8/9). Selective inhibition of DPPII during the assay at pH 7 or measurement of DPP activity at pH 8.3 probed DPPIV and DPP8/9 activity. The degree of inhibition of the DPP activity by UAMC00132 (5 ␮M and 2.5 ␮M in the assays at pH 7.0 and pH 8.3, respectively; Fig. 2) allowed us to deduce the DPP8/9 activity. Using Ala-Pro-pNA at pH 7.0, the PBMC had a similar DPP8/9-specific activity of 3– 4 mU/mg (Fig. 3). Statistically, a slightly higher DPP8/9-specific activity was found in the Jurkat and U937 cells. With Gly-Pro-pNA at pH 8.3, DPP8/9-specific activity ranged between 2 and 3 mU/mg. The remaining activity (after inhibition of DPPII and DPP8/9) was assigned to DPPIV (Fig. 3). Lymphocytes exhibited proportionally the highest DPPIV activity (Ala-Pro-pNA: 40%; Gly-ProMaes et al. DPP8/9 activity in human leukocytes

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TABLE 2. IC50 UAMC00039 UAMC00132 NVP-LAF237 AB192

Selectivity of the DPP Inhibitors

DPPII

DPP8

DPPIV

FAP

⬍0.5 nM 29 ⫾ 1 ␮M ⬎1000 ␮M ⬎1000 ␮M

142 ⫾ 27 ␮M 0.12 ⫾ 0.01␮M 9.0 ⫾ 0.1 ␮M 56 ⫾ 18 ␮M

165 ⫾ 9 ␮M 90 ⫾ 4 ␮M 0.12 ⫾ 0.01 ␮M 5.6 ⫾ 2.7 ␮M

⬎1000 ␮M ⬎1000 ␮M 12 ⫾ 1 ␮M 130 ⫾ 30 ␮M

IC50 values were determined in our laboratory under comparable conditions at substrate concentrations near the K M (refs. [13, 15] and unpublished results).

Fig. 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.

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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.

Enrichment of DPP8/9-like activity and characterization

Fig. 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.

pNA: 80%), followed by the monocytes (Ala-Pro-pNA: 15%; Gly-Pro-pNA: 35%). As can be deduced from the left panels in Figure 2, it was not possible to choose a concentration of NVP-LAF237, which was able to discriminate clearly between DPPIV and DPP8/9. Upon subcellular fractionation of lymphocytes and U937 cells, DPP8/9 activity was found predominantly in the cytosol fraction and DPPIV activity, in the membrane fraction (Fig. 4). Activity attributed to DPPII was concentrated in the fraction containing lysosomes and mitochondria.

Different purification steps involving lectin-affinity chromatography, ion-exchange chromatography, hydrophobe interaction chromatography, and a new inhibitor-based affinity chromatography were used for the enrichment and characterization of DPP8/9-like activity from the cytosol of Jurkat cells. The Con A-Sepharose column removed DPPII and DPPIV activity. Enrichment of the DPP8/DPP9-like activity was achieved merely by ion exchange and hydrophobic chromatography. During both of these purification steps, the DPP activity eluted as one single peak. The specific DPP activity increased 30-fold, and no DPPII or DPPIV activity could be measured in the enriched preparation. Kinetic studies about the active fractions of the HiTrap Phenyl chromatography showed that the preparation had a Km value for Ala-Pro-pNA somewhat lower than the one for DPP8. The IC50 value of UAMC00132 for the sample is in the same range (40 – 60 nM) as reported for rDPP8 and rDPP9. The Jurkat cytosol enzyme preparation exhibited a high affinity for the inhibitor Lys-isoindoline immobilized onto Sepharose and not for the dummy control. So far, we have not been able to elute active enzyme from this affinity matrix. The same was seen with the rDPP8.

DISCUSSION In conclusion, using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9, is present in human peripheral blood leukocytes (lymphocytes and monocytes) and in Jurkat and U937 cells. The different leukocyte types tested were shown to contain similar DPP8/9-specific activities, and

TABLE 3.

Immunocapturing CD26/DPPIV

Sample

As shown in Table 3, no DPP activity was present in lysates of Jurkat and U937 cells, as captured by the immobilized anti-CD26 mAb. Essentially, all activity was recovered in the supernatant and was ⬃90% inhibited by 2.5 ␮M 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

Lymphocytes Monocytes Jurkat cells U937 cells Pure DPPIV Pure DPP8

Capturing DPPIV onto sCD26 ELISA Plates Inhibition by 2.5 ␮M UAMC00132 (%)

Bound Gly-PropNA-cleaving activity (%)

Cell lysate/pure enzyme sample

Supernatant

35 10 0 0 70 0

25 75 92 85 4 97

40 81 91 88 0 98

The percentage of total Gly-Pro-pNA-cleaving activity of the different cell lysates bound onto immobilized sCD26 mAB (Bender MedSystems) and the inhibition (%) by 2.5 ␮M UAMC00132 (DPP8/9 inhibitor) in the lysate or pure enzyme sample and the supernatant after immunocapture are given.

Maes et al. DPP8/9 activity in human leukocytes

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Fig. 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.

DPPII- and DPPIV-specific activities varied considerably. Subcellular fractionation enriched the DPP8/9 activity in the cytosol and DPPIV in the membrane fractions. The results were confirmed by DPPIV/CD26 immunocapture experiments. The DPP-enriched preparation from Jurkat cytosol showed characteristics different from DPPII and DPPIV, and it is compatible with what is known of DPP8/9.

ACKNOWLEDGMENTS This work was supported by the Fund for Scientific Research– Flanders (Belgium; F.W.O.–Vlaanderen, Grant No. G041005). 6


M-B. M. is a research assistant, and P. V. d. V. is a postdoctoral fellow of F.W.O.–Vlaanderen. We are grateful to Mrs. Nicole Lamoen for her excellent technical assistance.

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