From the
Metabolic Disorders,
Medicinal Chemistry, ¶Comparative Medicine, and ||Applied Computer Science and Mathematics, Merck Research Laboratories, Rahway, New Jersey 07065
Received for publication, December 4, 2002 , and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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A DP-IV inhibitor improves glucose tolerance in GLP-1 receptor-deficient mice, but not in DP-IV-deficient mice (7), indicating that substrates beyond GLP-1 are important for efficacy in glucose control. One likely candidate is GIP, another incretin in the glucagon family of peptides, which is also regulated by DP-IV in vivo (3, 14). DP-IV has also been implicated in functions beyond glucose homeostasis; specifically, DP-IV-deficient Fischer rats gain less weight than wild type animals on a high fat diet (15), and DP-IV-deficient mice are also resistant to diet-induced obesity,2 suggesting that the enzyme may be involved in the regulation of additional bioactive peptides that control feeding behavior. In this regard, a number of peptides have been shown to be efficiently processed in vitro, including glucagon family peptides, neuropeptides, and chemokines.
To better understand the biological role of DP-IV and to determine the potential therapeutic benefit and safety issues that may be associated with DP-IV inhibition, a thorough understanding of endogenous substrate specificity is required. Numerous strategies have been devised to determine protease substrate specificity and provide insight into their biological roles. For serine- and cysteine-type proteases, "positional scanning" combinatorial libraries of peptide substrates are often effective in identifying residue specificities proximal to the scissile amide bond and revealing consensus amino acid sequences for substrate recognition (16, 17). These data can facilitate the identification of potential substrates from protein sequence databases, although rapid assays to quantitatively measure cleavage efficiencies have been lacking to date.
We have been evaluating mass spectrometry (MS) as the method of choice for developing sensitive and quantitative assays of protease activity. MS techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (18, 19) and electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) (20) can facilitate quantitative measurements of polypeptide cleavage based upon the specific changes in m/z that accompany each proteolytic event (21). MS is of particular utility in assays of amino/carboxypeptidase or dipeptidase activity involving the cleavage of only one or two residues, where the quantitation of substrate and product peptides can be limited by the poor resolution and low sensitivity of conventional separation techniques such as high performance liquid chromatography and gel electrophoresis. Here, we describe a general MS-based approach for screening libraries of bioactive polypeptides against a target protease, measuring specificity constants of cleavage (kcat/Km) from a single kinetic run, and evaluating the effects of genetic ablation (or inhibition) of protease activity on the in vivo metabolism of exogenously administered candidate substrates in mice. These studies have led to the identification of oxyntomodulin and growth hormone-(143) as novel in vitro substrates for DP-IV and provide compelling evidence that PACAP38 is regulated by this enzyme in vivo.
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EXPERIMENTAL PROCEDURES |
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LC/MS Analysis of in Vitro SubstratesThe MS instrumentation consisted of an Agilent 1100 HPLC and autosampler connected to a Thermo Finnigan LCQ ion trap mass spectrometer equipped with a standard electrospray ionization (ESI) source. Sample aliquots containing 2080 pmol of each peptide analyte were injected on a 100 x 1 mm HAISIL 300 C18 column (Higgins Analytical) at a flow rate of 150 µl/min (solvent A = water, 0.03% trifluoroacetic acid, solvent B = acetonitrile, 0.03% trifluoroacetic acid, initial equilibration at 5% B, linear gradients of 3.75% B/min over 315 min and 2.1% B/min over 1537 min after injection). The flow was diverted to waste for 5.2 min before the column eluate was introduced to the electrospray source. All samples were analyzed in positive ion mode with the source voltage set at 4500 V and the capillary voltage at 5 V. The heated capillary was maintained at 200 °C. Full scan data were collected as centroid spectra (4001800 m/z). Extracted ion chromatograms (Fig. 1) were generated for each peptidyl species using prominent charge states that were empirically determined for each substrate and product species (typically [M+3H]+3 and/or [M+4H]+4, see Table I). MS acquisition, peak integration, and data analysis were performed using Xcalibur software version 1.2 (Thermo Finnigan).
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Measurement of Response FactorsQuantitative measurements of substrate conversion were made by ratiometric analysis of substrate (S) and product (P) peak areas in the extracted ion chromatograms of each species. For an accurate analysis, instrument response factors were determined for every substrate-product pair. Peptide standards corresponding to the DP-IV cleavage products of each identified in vitro substrate were obtained by incubating the latter (1 µM in 528 µl of assay buffer) with excess enzyme (100 nM, 192 milliunits) at 37 °C for 3 h. The reactions were subsequently quenched with 10 µl of 0.2 N HCl. Under these conditions, even relatively inefficient substrates of DP-IV (kcat/Km < 104M1s1) were cleaved to >95% completion. Peptide mixtures containing 20 pmol of each substrate and 2080 pmol of the corresponding DP-IV product in 100 µl of assay buffer were prepared using a grouping of peptides identical to that of the in vitro screen. Aliquots of these mixtures (90 µl) were analyzed by ESI-LC/MS. The m/z of predominant charge states for each substrate and product peptide (see Table I) was used to generate extracted ion chromatograms from which integrated peak areas were measured. The ratio of the product:substrate peak areas was plotted against the known ratio of product:substrate in the mixtures (see Fig. 2). The slope of the resulting linear plot was used as a response factor to correct the measured peak area of product peptide in the kinetic assays (Pcorr = (Pobs)/(slope)).
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Calculation of Specificity ConstantsProgress curves for the cleavage of individual substrates (see Fig. 3) were generated from extracted ion chromatograms using peak areas of substrate and product, percent conversion = ((Pcorr)/(Pcorr + Sobs)) x 100. For data obtained from repeat assays, the standard error of this measurement was typically <15% of the mean. The specificity constant (kcat/Km) for cleavage of a particular substrate was directly calculated from a first order fit of the enzyme kinetics: kobs = (kcat)/(Km) · Eo, where kobs is the first order rate constant and Eo is the total enzyme concentration (22).
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In Vivo Metabolism of PACAP38 Animal procedures were approved by the Merck-Rahway Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals (47). The generation of DP-IV-deficient (CD26/) C57Bl/6 mice has been reported previously (7). These animals were obtained from INSERM, Paris, France. A solution of PACAP38 was prepared in saline (0.4 mg/ml) for intravenous dosing. Two groups of naive 9-week-old male C57Bl/6mice (wild type and DP-IV-deficient, respectively) were administered an intravenous bolus of PACAP38 (2 mg/kg). Blood samples were collected by cardiocentesis after CO2 euthanasia from 46 animals at each time point (15 min after dose). Blood from individual animals was placed immediately into chilled EDTA-coated tubes containing a DP-IV inhibitor (2S,3S-isoleucyl thiazolidide hemi-fumarate salt, 100 µM, obtained from Heumann Pharma) and aprotinin (0.04 mg/ml, purchased from Sigma) to prevent further degradation of PACAP38 and its metabolites. The samples were centrifuged at 6000 rpm for 10 min at 4 °C. Plasma was transferred to a 96-deep-well plate and stored at 70 °C until extraction and analysis.
Plasma Extraction and Sample PreparationPACAP38 and related peptides were extracted from mouse plasma using Oasis HLB 10 mg 96-well solid phase extraction plates (Waters). Plasma aliquots (100 µl) were diluted with 100 µl of water, acidified with 100 µl of 0.1 N acetic acid, and loaded on the extraction plate (preconditioned as per the manufacturer's instructions). Plasma samples of known peptide concentrations (for calibration curves) were prepared by spiking mouse plasma with 25 µl of a stock solution containing equimolar mixtures of PACAP-(138) and PACAP-(338) (synthesized by Invitrogen) in 10% acetonitrile, 0.1% trifluoroacetic acid. After loading all samples, the extraction plate was washed with 0.5 ml of 5% methanol. Peptides were eluted with 50% acetonitrile, 0.1% trifluoroacetic acid (2 x 0.25 ml), and the eluate was collected in a 96-well plate. The eluted samples were dried at 40 °C under a stream of nitrogen. Recoveries of PACAP-(138) and PACAP-(338) were typically 65 and 78%, respectively. Dried samples were reconstituted in 190 µl of an aqueous solution containing 5% acetonitrile, 5% 1-propanol, and 0.03% trifluoroacetic acid. The samples were transferred to 0.5-ml microcentrifuge tubes and centrifuged at 13,000 rpm for 5 min. Supernatants (180 µl) were transferred to plastic LC vials, and 45-µl aliquots were analyzed in triplicate by tandem mass spectrometry.
Quantitation of Peptides by Tandem Mass SpectrometryExtracted plasma samples were injected on a Symmetry-300 C4 column (2.1 x 50 mm, Waters) at a flow rate of 200 µl/min (solvent A = water, 0.03% trifluoroacetic acid, solvent B = 1:1 acetonitrile:1-propanol, 0.03% trifluoroacetic acid, initial equilibration at 10% B, linear gradients of 2.2% B/min over 110 min and 10% B/min over 1217 min after injection). The flow was diverted to waste for 4.5 min before introducing the column eluate to the ESI source. All samples were analyzed in positive ion mode (centroid spectra) with the source voltage at 4.5 kV, capillary voltage at 42 V, and capillary temperature at 200 °C. For the analysis of intact PACAP38, the parent ion at m/z 1134.6 (corresponding to [M+4H]+4) yielded three significant fragment ions at m/z 1045.5 , 1049.9
, and 1221.6
, with 30% relative collision energy (see Fig. 4). For the PACAP-(338) metabolite, the parent ion at m/z 1437.7 (corresponding to [M+3H]+3) yielded fragment ions at m/z 1126.4
and 1431.5 ([M-H2O+3H]+3) with 30% relative collision energy. The total integrated peak area for these major fragment ions was used to quantitate the corresponding peptides, using standard curves generated from plasma samples of known peptide concentrations (see Fig. 5). The standard error for repeat measurements was <15% of the mean.
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RESULTS AND DISCUSSION |
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Measurement of Specificity Constants by MSSince mass spectrometry measures analyze concentrations indirectly, appropriate precautions were taken to ensure that the measured ion signals reflected accurate estimates of substrate turnover. These steps included the measurement of instrument response factors for each substrate-product pair (Fig. 2 and Table I) and operation within the linear dynamic range of the assay (010,000 ng/ml). The high sensitivity of ESI-LC/MS allowed kinetic data to be collected at relatively low (nanomolar) concentrations of substrate as compared with the Michaelis constant (Km), which is typically in the high micromolar range for peptides that are cleaved by DP-IV in vitro (14, 2527). Each potential substrate could therefore effectively compete for the enzyme active site without a bias toward high affinity substrates. Under these conditions of low substrate concentrations ([S]< 0.1 Km), the specificity constant for cleavage could be directly calculated from a single progress curve for each substrate (Fig. 3) using the integrated Michaelis-Menten equation (22). Repeat analyses confirmed that kinetic measurements were unaffected by the composition of peptides in the assay mixture. Identical results were also obtained when substrates were incubated individually with the peptidase.
Efficient in Vitro Cleavage of P1-Pro/Ala-containing PeptidesTable I lists the bioactive peptides that were screened in this study, ranked in descending order of their measured specificity constants for cleavage by DP-IV. The efficient processing of GHRH-(144), GRP, GLP-1-(736), peptide histidine methionine, GLP-2, and GIP (kcat/Km ≥ 105 M1 s1) was consistent with previous reports of their in vitro processing by DP-IV (23, 25, 27, 28), which typically cleaves the amide bond after a penultimate amino-terminal Pro or Ala residue (the P1 site, according to the nomenclature of Berger and Schechter (29)). For GRP, sequential processing of the Val-Pro-Leu-Pro amino terminus produced the intermediate GRP-(327), which was further cleaved to GRP-(527) as has been reported previously (25, 27). Rate constants for these cleavages were determined from the progress curves for each species using standard equations (30). In addition, oxyntomodulin, a glucagon family peptide that inhibits food intake when administered centrally in rats (31), and the pituitary growth hormone fragment GH-(143), which exhibits insulin potentiating activity in obese or diabetic rodents (32, 33), were established as novel in vitro substrates of DP-IV.
Differential Processing of P1-Ser-containing Peptides in VitroIn general, where comparisons can be made, the specificity constants determined in this study are in good agreement with values reported previously in the literature. The exceptions in this regard are data for the P1-Ser-containing peptides glucagon, PACAP27, and PACAP38. The specificity constant determined for glucagon was 10-fold lower than that reported using MALDI-TOF mass spectrometry (34). Indeed, most P1-Ser peptides were either inefficient substrates in our assay (kcat/Km < 105 M1 s1, PACAP27, secretin) or not detectably cleaved by DP-IV (VIP, VIP-(112)). Similar results have been reported for P1-Ser-containing analogs of GHRH. Substitution of Ala2 by Ser in Ala15-GHRH-(129) led to a 400-fold decrease in kcat/Km (26), and first order rate constants for DP-IV cleavage of Ser-containing GHRH analogs were typically 25-fold lower than that of the corresponding P1-Ala substrates (35). Unexpectedly, PACAP38 and oxyntomodulin, both of which contain basic carboxyl-terminal extensions relative to PACAP27 and glucagon, respectively, were processed 15-fold and 8-fold more efficiently than their shorter isoforms in each case. Such differential processing indicates that residues remote from the scissile bond can modulate the efficiency of DP-IV cleavage, as was further demonstrated by a modest (1.5-fold) increase in kcat/Km for GHRH-(144) cleavage relative to GHRH-(129).
Differential in vitro processing of the pancreatic neuropeptides PACAP38 and PACAP27 by DP-IV has been described previously (27). The reported specificity constants of these peptides, however, were an order of magnitude lower than the values obtained in this work. Although the reason for these discrepancies is unknown, the evidence linking PACAP to metabolic control (see Ref. 38) and the requirement of an intact amino terminus for PACAP bioactivity (36, 37) prompted us to investigate the potential role of DP-IV in the in vivo catabolism of PACAP38.
In Vivo Metabolism of PACAP Peptides Monitored by Tandem Mass SpectrometryIncreased steady-state levels of PACAP38 in DP-IV-deficient mice would provide conclusive evidence of a physiological role for DP-IV in the regulation of this neuropeptide. Unfortunately, measurements of endogenous PACAP38 in mice were not possible for several reasons. First, specific immunoassays that discriminate between intact PACAP-(138) and its DP-IV metabolite PACAP-(338) were not available. Second, since PACAP38 functions as a non-cholinergic sympathoadrenal neurotransmitter (38), local stabilization of the neuropeptide at nerve ganglia in DP-IV/ mice would probably not be reflected in assays that measured circulating peptide. Finally, PACAP38 binds the plasma protein ceruloplasmin (39), which further complicates the quantitation of endogenous peptide, estimated to be 25100 pM (40). For a typical 100-µl mouse plasma sample, this low abundance of endogenous PACAP38 would require attomole sensitivity of detection, which is approximately 3 orders of magnitude lower than what can be typically achieved for extracted plasma peptides using conventional ion trap mass spectrometers. We therefore adopted an alternate approach that compared the metabolic fates of exogenously administered PACAP38 in wild type and DP-IV-deficient C57Bl/6 mice.
Measurement of intact PACAP-(138) and the inactive DP-IV metabolite PACAP-(338) in mouse plasma was accomplished using tandem mass spectrometry (MS-MS) following intravenous dosing of animals with supraphysiological levels of neuropeptide. Although the concentration of infused peptide was much higher than that of endogenous PACAP38, the metabolic fate of this neuropeptide when exposed to a milieu of competing peptidases in vivo (including DP-IV) would still be expected to provide valuable insight regarding the enzyme(s) responsible for its regulation. Indeed, similar infusion experiments using radioiodinated GLP-1 and GIP in DP-IV-positive and -negative rats provided among the first in vivo evidence for inactivation of these incretins by the enzyme (3).
The enhanced selectivity and improved sensitivity of tandem mass spectrometry is ideal for the quantitation of peptides in biological samples, where the complexity of the matrix otherwise results in unacceptable chemical noise. The MS-MS experiments for PACAP-(138) and PACAP-(338) monitored the production of characteristic fragment ions from a specific parent ion following collision-activated dissociation (Fig. 4). Despite the high abundance of ceruloplasmin in plasma (1.7 µM, (39)), an ESI-LC/MS-MS assay was developed that simultaneously measures PACAP-(138) and PACAP-(338) with a limit of quantitation of 45 nM (200 ng/ml, Fig. 5). The sensitivity and dynamic range of this assay confirmed that the protocols developed for sample preparation were effective in separating PACAP38 from bound proteins and allowed the metabolism of infused PACAP38 in C57Bl/6 mice to be monitored over a period of several minutes.
PACAP38 but Not PACAP27 Is Processed in Vivo by DP-IV As illustrated in Fig. 6, exogenous PACAP38 was rapidly degraded in wild type C57Bl/6 mice with concomitant formation of the inactive DP-IV metabolite PACAP-(338), consistent with our in vitro observations. In contrast, formation of PACAP-(338) was virtually absent in age- and gender-matched DP-IV/ mice, and clearance of intact PACAP38 from the circulation was significantly slower in these animals (presumably by renal filtration, as has been reported for GLP-1-(41)). Significantly, the total concentration of PACAP38 and its DP-IV metabolite in wild type mice was similar to levels of intact PACAP38 in DP-IV/ animals at each time point (Fig. 6), consistent with DP-IV playing a major role in the degradation of circulating PACAP38. A similar experiment monitoring the catabolism of exogenous PACAP27 in wild type C57Bl/6 mice found no evidence of the corresponding des-dipeptidyl PACAP-(327) metabolite (data not shown), consistent with the 15-fold lower specificity constant of this peptide relative to PACAP38. Nevertheless, since PACAP38 constitutes
90% of the endogenous PACAP pool, metabolic inactivation by DP-IV would be expected to regulate overall PACAP bioactivity in vivo.
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The Metabolic Effects of DP-IV Inhibition May Involve Stabilization of Multiple SubstratesImprovements in metabolic control obtained upon inhibition of DP-IV in human subjects and diabetic animals have been classically attributed to stabilization of the incretins GLP-1 and GIP. Although other bioactive peptides are cleaved by DP-IV in vitro, most research on DP-IV-mediated peptide processing has been conducted without attention to turnover efficiency (kcat/Km) and biologically relevant enzyme levels. A critical evaluation of potential and purported substrates, including members of the glucagon/VIP/secretin superfamily, was therefore undertaken to measure specificity constants in vitro under physiologically relevant conditions and to identify new candidate substrates for in vivo validation. Oxyntomodulin and GH-(143) were identified as novel in vitro substrates of DP-IV, but the unexpectedly efficient in vitro processing of the incretin PACAP38, a critical regulator of lipid and carbohydrate metabolism, led us to focus on the metabolism of this neuropeptide in vivo. Infusion studies in mice recapitulated the in vitro cleavage pattern of PACAP38 by DP-IV (except in DP-IV-deficient animals), although the substrate was exposed to multiple proteases. Furthermore, a closely related but relatively inefficient substrate (PACAP27) was not demonstrably processed by DP-IV in vivo, although infused at supraphysiological levels. These results suggest that DP-IV may participate in the inactivation of endogenous PACAP38.
Although a number of functions have been attributed to PACAP, recent studies with PACAP receptor-deficient mice and PACAP-deficient mice suggest a role for this neuropeptide in glucose control, lipid metabolism, and adaptive thermogenesis. PACAP signals via three receptor subtypes (PAC1, VPAC1, and VPAC2) (42). The PAC1 receptor is specific for PACAP; VPAC1 and VPAC2 are shared with VIP. PAC1-deficient mice display impaired glucose tolerance and fed hyperinsulinemia (43). PACAP-deficient mice have an impaired response to insulin-induced hypoglycemia and a 23-fold increase in serum triglycerides and cholesterol (44). Loss of PACAP in these animals also results in inadequate heat production due to reduced norepinephrine stimulation of brown adipose tissue (45). Our data on the in vivo degradation of exogenous PACAP38 by DP-IV therefore suggest that inhibitors of this peptidase may have therapeutic utility extending beyond improvements in glucose control, specifically in the regulation of lipid metabolism and/or adaptive thermogenesis. In this regard, we have noted that DP-IV-deficient mice maintained on a high fat diet exhibit reduced adiposity. Furthermore, chronic administration of the DP-IV inhibitor FE999011 in male Zucker diabetic fatty rats reportedly prevents a rise in circulating free fatty acid, in addition to postponing the development of diabetes (11). It is possible that increased endogenous pools of intact PACAP38 may mediate some of these effects, although stabilization of oxyntomodulin, GH-(143), or of an as yet undiscovered DP-IV substrate may also contribute to the improved metabolic control. Final confirmation of the in vivo regulation of such substrates by DP-IV will require quantitation of the corresponding endogenous peptides in the setting of enzyme inhibition or ablation. Encouragingly, rapid advances in sensitive MS instrumentation increase the feasibility of measuring picomolar plasma peptides in the near future, which should facilitate development of the requisite assays. Clearly, additional studies of substrate specificity will also be required to obtain an intimate understanding of the overall biological role of DP-IV and the prospects and liabilities of enzyme inhibition.
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FOOTNOTES |
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** Present address: ExSAR Corporation, 11 Deer Park Dr., Ste. 103, Monmouth Junction, NJ 08852.
To whom correspondence should be addressed: Dept. of Metabolic Disorders, Mail code RY50G-241, Merck Research Laboratories, Rahway, NJ 07065. Tel.: 732-594-3132; Fax: 732-594-3664; E-mail: ranabir{at}merck.com.
1 The abbreviations used are: DP-IV, dipeptidyl peptidase IV; ESI-LC/MS, electrospray ionization-liquid chromatography/mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MS-MS, tandem mass spectrometry; GH, growth hormone; GHRH, growth hormone releasing hormone; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucagon-like peptide; GRP, gastrin releasing peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; PHM, peptide histidine methionine; VIP, vasoactive intestinal peptide; S, substrate; P, product.
2 Bei Zhang, personal communication.
3 S. Jagpal, N. A. Thornberry, and R. Sinha Roy, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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