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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Effect of Prolyl Endopeptidase on Digestive-Resistant Gliadin Peptides in Vivo

Departments of Chemistry (C.K.), Chemical Engineering (J.L.P., C.K.), and Medicine (G.M.G.), Stanford University, Stanford, California

Received March 13, 2004; accepted May 12, 2004.

	Abstract

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Many gluten peptides elicit proliferative responses from T cells from Celiac Sprue patients, influencing the pathogenesis of this small intestinal disorder. These peptides are Pro- and Gln-rich in character, suggesting that resistance to proteolysis promotes their toxicity. To test this hypothesis, we analyzed the digestive resistance of a panel of - and -gliadin peptides believed to induce toxicity via diverse mechanisms. Most were highly resistant to gastric and pancreatic protease digestion, but they were digested by intestinal brush-border peptidases. In some instances, there was accumulation of relatively long intermediates. Control peptides from gliadin and myoglobin revealed that digestive resistance depended on factors other than size. Prolyl endopeptidase (PEP) supplementation substantially reduced the concentrations of these peptides. To estimate a pharmacologically useful PEP dose, recombinant PEP was coperfused into rat intestine with the highly digestive-resistant 33-mer peptide LQLQPF(PQPQLPY)₃ PQPQPF (PEP: peptide weight ratio 1:50 to 1:5). PEP dosing experiments indicate significant changes in the average residence time. The in vivo benefit of PEP was verified by coperfusion with a mixture of 33-mer and partially proteolyzed gliadin. These data verify and extend our earlier proposal that gliadin peptides, although resistant to proteolysis, can be processed efficiently by PEP supplementation. Indeed, PEP may be able to treat Celiac Sprue by reducing or eliminating such peptides from the intestine.

Although the adaptive immune response is often considered to be the primary trigger of the flattened mucosa that characterizes Celiac Sprue (Sollid, 2002), other hypotheses of Celiac Sprue pathogenesis have been presented (Deem et al., 1991; Tucková et al., 2000; Novák et al., 2002; Maiuri et al., 2003). Regardless of the primary mechanism, the essential prerequisite is the initiating role of the digestive-resistant intact gliadin peptide fragments, which must persist in the intestinal lumen to have deleterious effects. Previous in vitro studies have suggested digestive resistance of gliadin in Celiac Sprue patients (Cornell and Rivett, 1995; Cornell, 1998). More recent studies have demonstrated that a region of -gliadin harboring tandem repeat sequences of PQPQLPY is resistant to digestion by gastric, pancreatic, and intestinal brush-border peptidases, and harbors multiple copies of highly immunogenic epitopes (Hausch et al., 2002; Shan et al., 2002). This led to the proposal that proteolytic resistance of gliadin peptides contributes to their toxicity by allowing their intestinal luminal accumulation. In this study, we provide the first quantitative data that support this hypothesis. By comparing the in vitro and in vivo digestion of a panel of structurally diverse - and -gliadin-derived peptides that are believed to induce toxic effects in Celiac Sprue, we were able to quantify their relative resistance to digestion. The experimentally measured parameters were used to develop a quantitative model for the luminal content of gliadin peptides in the human intestine. Our findings indicate that these gliadin peptides persist 4 to 8 times longer in the intestine than physiologically relevant controls from low-proline fractions of gliadin or from myoglobin. Quantitative analysis of in vivo prolyl endopeptidase (PEP) supplementation demonstrated that resistant gliadin peptides could be effectively proteolyzed at rates comparable with nonimmunogenic control peptides.

	Materials and Methods

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Materials. A summary of the peptides used in the following experiments, the source, and peptide identifiers can be found in Table 1. Throughout this report, peptides are identified by their sequences or numerical peptide identifiers. Purity of all peptides was verified by reverse phase C18 HPLC. LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was found to contain the related 32-mer devoid of the N-terminal Leu residue, QLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, as well as smaller quantities of pyro-QLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, and consequently occurs as a dimer in HPLC traces.

All of the enzymes were purchased from Sigma-Aldrich (St. Louis, MO) except Flavobacterium meningosepticum prolyl endopeptidase, which was purchased from United States Biological (Swampscott, MA). Alternatively, the same enzyme was prepared from recombinant Escherichia coli, and its activity was comparable with commercially available PEP.

In Vitro Digestion of Gliadin Peptides. The in vitro digestion of each peptide was performed as follows: 75 µM (final concentration) of peptide was combined with 32 µl of 0.03 M HCl, pepsin (1:100 w pepsin/w peptide), and water to give a volume of 104 µl. The reaction was carried out for 30 min at 37°C and was stopped by the addition of 40 µl of 1 M sodium phosphate/sodium bisphosphate, pH 7.04. Chymotrypsin [1:100 (w/w)], trypsin [1:100 (w/w)], elastase [1:200 (w/w)], and carboxypeptidase A [1:100 (w/w)] were then added to give a final reaction volume of 168 µl. The reaction mixture was incubated at 37°C for the indicated time and then stopped by heating at 90°C for 5 min. Alternatively, the peptide (75 µM final concentration) was combined with rat intestinal brush-border enzymes, where the final concentration of brush-border aminopeptidase N was 100 µU/µl. The rat intestinal brush border was prepared as described previously (Ahnen et al., 1982), and the activity was measured by assaying continuously at 30°C in 0.1 M Tris · HCl, pH 8.0, containing 1 mM Leu-pNA [extinction coefficient at 410 nm (₄₁₀) = 8800 M^–1/cm^–1] in 1% dimethyl sulfoxide to improve solubility. In some reactions, prolyl endopeptidase was also added at a final concentration of 100 µU/µl. The reaction mixture was analyzed by reverse phase HPLC on a Vydac 218MS54 column (4.6 mm inner diameter x 15 cm).

In Vivo Rat Intestinal Perfusion. Our procedure for in vivo intestinal perfusion of peptides in rats was modified from the Smithson and Gray procedure (Smithson and Gray, 1977) as follows: Sprague-Dawley rats, 250 to 350 g, were anesthetized with isoflurane, the abdomen opened, the ligament of Treitz (duodenal-jejunal junction) identified, and polyethylene catheters (2.5 mm outer diameter x 1 mm inner diameter) were installed with the proximal infusion catheter placed 5 cm beyond the ligament of Treitz and the collection catheter positioned 20 cm distally. Animal core temperature was monitored to be at 37°C by rectal thermometer.

Peptides for intraintestinal perfusion were 1 mM GLGG, 0.1 mM P3, 0.1 mM P4, 0.1 mM P5, 0.1 mM P8, 0.1 mM P9, and 0.1 mM P10. The peptides were dissolved in 154 mM NaCl and perfused at 0.3 to 0.4 ml/min for 15 min to achieve a steady state for collection at the distal catheter. For each PEP-supplemented digestion, a solution of 50 µU/µl PEP in 154 mM NaCl and a separate 0.2 mM solution of the appropriate peptide were perfused through separate tubes at 0.2 ml/min and then combined at the infusion port, producing a total flow rate of 0.4 ml/min to achieve 0.1 mM peptide and 25 µU/µl PEP. The residence time of the perfusate in the intestinal lumen was approximately 10 min. Additional experiments were performed to determine dose dependence of P5 digestion by perfusing 0.05 mM P5 with recombinant PEP at concentrations from 5.6 to 41.7 µg/ml (25–190 µU/µl). In a separate experiment, pepsin-, trypsin-, and chymotrypsin-digested gliadin (Sigma-Aldrich) was perfused at 2 mg/ml along with 0.05 mM P5 and 41.7 µg/ml recombinant PEP (190 µU/µl) to determine the effect of smaller, more accessible peptide fragments on P5 digestion. The perfusion effluent was collected, immediately frozen, and subsequently analyzed by reverse phase HPLC on a Vydac 218MS54 column (4.6 mm inner diameter x 15 cm). The rate of substrate disappearance for the peptides was calculated as detailed previously (Smithson and Gray, 1977).

It is important to note that the in vivo experiments presented here are designed to represent the physiological assimilation of gliadin peptides that are end products of pancreatic protease digestion. Consequently, our perfusion model has been simplified to exclude the effects of endogenous pancreatic enzymes and pancreatic protein secretions. We recognize that pancreatic enzymes are likely to degrade orally administered PEP, and additional intestinal luminal proteins are likely to compete with the PEP's ability to proteolyze P5. However, our intention at this stage of the work was not to establish the stability of PEP to pancreatic digestion but instead to understand quantitatively the resistance of potentially toxic gliadin peptides to brush-border proteolysis, as well as the effect of PEP supplementation on this process under ideal intraintestinal conditions.

Mathematical Modeling of Intestinal Content of Gliadin Peptides. The in vivo data presented in this report was used to estimate the luminal content of plugs of gliadin peptides P4 and P5 as they progress through the small intestinal tract. The beneficial effect of PEP supplementation on P5 processing was also analyzed in a similar manner. In this analysis, the length and diameter of the human small intestine were assumed to be 5 m and 3.8 cm, respectively, and the transit time of a peptide plug across this distance was estimated as 4 h. For analytical purposes, the intestine was divided into 240 segments of 2.1-cm length each, such that each segment corresponded to the distance traversed per minute. To estimate the peptide concentration, C_i, that exits each segment i, it is first necessary to determine the rate constant, k, for intestinal brush-border membrane-catalyzed proteolysis of each peptide. The rate constant k was assumed to be first order, which would be the case if the Michaelis-Menten parameter for brush-border membrane (BBM) peptidases have K_M » [S]. This assumption was validated for GLGG (a peptide easily digested by intestinal brush-border peptidases) in an earlier study (Smithson and Gray, 1977).

The rate constant k was calculated in an iterative manner for each peptide. The correct k value was that at which the calculated surface rate of peptide proteolysis (i.e., moles peptide per unit surface area per unit time) was four times that observed experimentally in the rat intestinal perfusion loop. (The surface area in the human small intestine is 300-fold greater than that of a smooth cylindrical tube, whereas the surface area of the rat small intestine is 75-fold higher than that of a smooth tube (Fisher and Parsons, 1950; Ganong, 1979; Ferraris et al., 1989).) It should be noted that these surface rates of peptide proteolysis are substantially greater (>8 times) than the rate of movement across the epithelium (Matysiak-Budnik et al., 2003). Consequently, the flux of peptide across the epithelium could be safely ignored in this study without affecting its conclusions.

	Results

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Digestive Resistance of Gliadin Peptides and Effect of PEP. The gliadin peptides P2-P8 (Table 1) were incubated with pepsin, trypsin, chymotrypsin, carboxypeptidase A, and elastase and purified rat intestinal BBMs as detailed previously (Shan et al., 2002) to assess their resistance to digestion. Each peptide is derived from - or -gliadin sequences and has been shown to exhibit toxicity to the small intestine or to evoke an immune response. Peptides P2, P3, and P5 contain one or more epitopes from -gliadin, whereas P7 and P8 carry -gliadin epitopes. P4 is believed to stimulate the innate immune system (Maiuri et al., 2003), and P6 has macrophage-stimulating activity (Tucková et al., 2000; Novák et al., 2002).

The results summarized in Table 1 clearly indicate an unusual degree of resistance to digestion by gastric and pancreatic enzymes for all of the peptides tested except P6. In contrast, exposure to the BBMs revealed substantial cleavage for some of the peptides, but very little or no digestion for others. With the exception of P5 and P8, a recently identified resistant fragment from -gliadin (L. Shan, unpublished data), all of the peptides were digested by the peptidases of the intestinal surface (BBM) to levels below 20% of the starting material, but 4 h of exposure to brush-border membrane preparations was required for processing of most of the gliadin peptides. Such a prolonged exposure to the brush-border surface may not occur under typical physiological conditions.

Given the stability of gliadin peptides to intestinal peptidases and initial results indicating PEP is active against -gliadin epitopes (Hausch et al., 2002; Shan et al., 2002), the efficacy of in vitro digestion with PEP was examined. To quantify the incremental effect of PEP, we incubated the gliadin peptides with purified brush-border membranes, supplemented with 100 µU/µl PEP for 1 or 4 h at 37°C. Some of the gliadin peptides (most notably P2 and P6) were processed efficiently by the BBM peptidases; in these cases, the incremental effect of adding PEP was marginal (Table 1). In contrast, a dramatic supplemental effect of PEP was observed on the digestion of the highly immunogenic P5 peptide and the newly discovered P8 peptide.

It should be noted that these analyses of parent peptide disappearance only provide data on the initial step of processing, and it is important to also consider the putative toxic capacity of the smaller proteolysis products. HPLC traces of the in vitro digestion indicate brush-border peptidases are capable of removing only the C-terminal Phe residue from P5, illustrating that there is enhanced digestion of P5 by PEP to smaller peptides (Fig. 1). The intermediates for P5 were determined by liquid chromatography-tandem mass spectrometry to be the nontoxic peptides PQPQP and QPQLPYP or QLPYPQP. Thus, PEP seems to facilitate the digestion of brush-border-resistant gliadin peptides by generating smaller products that are either nontoxic or that are then substrates for the brush-border peptidases.

View larger version (24K): [in this window] [in a new window]   Fig. 1. A, HPLC traces for in vitro digestion of P5. B, cleavage of the terminal phenylalanine yields the main digestive product for P5. C, addition of PEP enhances the digestive process, and small nonimmunogenic peptides, PQPQP and QPQLPYP or QLPYPQP, begin to accumulate. D, artifact of the system that occurred during blank runs as well.

Disappearance of Gliadin Peptides in Intact Rat Intestine: A Process Independent of Peptide Length. To verify the in vitro stability of some gliadin-derived peptides to digestion by rat intestinal surface peptidases, we performed a series of in vivo peptide digestion experiments. Peptides were perfused at physiologically relevant concentrations via a catheter placed 5 cm distal to the ligament of Treitz, and samples were collected from a catheter positioned 20 cm distally. The peptide GLGG, known to be efficiently assimilated by the small intestine (Smithson and Gray, 1977), was used as a control to determine variations between rats and as a standard to compare with previously published data. GLGG was perfused at the beginning and end of each experiment to determine any loss of in vivo digestive capacity during the in vivo procedure. The rate of GLGG disappearance was determined to be 74 ± 7 pmol/cm² s at the beginning of the perfusion experiment, and this disappearance rate did decline at the end of the experiment to 49 ± 5 pmol/cm² s.

Figure 2 shows the rate of disappearance for the gliadin-derived peptides P3, P4, and P5, as determined by three different peptide perfusion experiments taking into account the decline, assumed to be linear, in intestinal capacity over the period of the experiment, as detailed above. Also included in Fig. 2 are the rate data for P8. P10 (a product of pepsin and pancreatic protease digestion of myoglobin) and P9 (a product of pepsin and pancreatic protease digestion of -gliadin that lacks immunogenicity) were used as physiologically relevant controls to determine whether the size of the peptide plays a role in digestion. There currently are no known physiologically relevant controls available for comparison with P5 and P8 peptides. The proteolysis rates for P10 and P9 were 18 and 21 pmol/cm² s, respectively. All data were collected under conditions where <50% of each peptide disappeared, thereby allowing for calculation of steady-state rates of proteolysis. Together with the in vitro data in Table 1, these results establish that immunogenic gliadin peptides such as P4, P5, and P8 are not only resistant to digestion with gastric and pancreatic enzymes but also display a relative resistance to digestion by the brush-border enzymes compared with nontoxic dietary peptides such as P10 and P9.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Rate of digestion for in vivo rat intestinal perfusion of P3, P4, P5, and P8. GLGG, P9, and P10 were measured at 74, 21, and 18 (picomoles per square centimeter second), respectively. Light gray columns, no prolyl endopeptidase and dark gray columns, with prolyl endopeptidase. The standard errors associated with measuring P3, P4, and P8 are ±25, ±25, and ±5%, respectively. Experiments for P8 were performed only once.

To further explore the capacity of PEP to enhance gliadin digestion, we measured PEP supplemental proteolysis of the gliadin peptides in vivo by coperfusing the gliadin peptides with PEP, 25 µU/µl, under the same conditions as detailed above. The rate of digestion was increased significantly, 50–100%, for each gliadin-derived peptide, as shown in Fig. 2, but there was no detectable change in the rate of the control peptide (GLGG) digestion (data not shown).

HPLC analysis verified the increased digestion of the otherwise resistant gliadin peptides. Digestion of P3 produced the resistant intermediate PQPQLP (Hausch et al., 2002). No other digestive intermediates were identified for the other gliadin peptides.

To determine the amount of PEP required to eliminate P5 under the perfusion conditions described under Materials and Methods, the concentration of recombinant PEP coperfused with 50 µM P5 was systematically increased (Fig. 3). A dose-dependent reduction of P5 was observed in this experiment with virtually all of the starting material eliminated in the presence of 41.7 µg/ml PEP (190 µU/µl). This dose corresponds to ca. 1:5 weight ratio for PEP:P5. Under these conditions, three intermediates were observed to accumulate to 10% of the starting material. Thus, supplementation of exogenous recombinant PEP in the jejunum can result in rapid digestion of highly proteolysis-resistant gliadin peptides such as P5.

View larger version (27K): [in this window] [in a new window]   Fig. 3. In vivo PEP dose-dependent digestion of P5. Top, the entire HPLC trace. The amount of remaining P5 is shown below: 0.0 (A), 25 µU/µl (B), 65 µU/µl (C), 125 µU/µl (D), 155 µU/µl (E), and 190 µU/µl recombinant PEP (F).

To verify that PEP has adequate specificity so as to target P5 when present as a component of a mixture of gliadin peptides, the intestinal perfusion experiment was repeated by coperfusing 41.7 µg/ml recombinant PEP (190 µU/µl) and P5 along with 2 mg/ml gliadin that had been pretreated with pepsin, chymotrypsin, and trypsin. As seen in Fig. 4, P5 was efficiently eliminated by PEP, accumulation of the nontoxic peptide products PQPQP and QPQLPYP or QLPYPQP occurs as shown in Fig. 1. Thus, efficient cleavage of P5 was observed, even in the presence of a large number of smaller gliadin fragments that might compete for the PEP active sites.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of competing substrates on in vivo PEP-supplemented digestion of P5 (A). Pepsin-, trypsin-, and chymotrypsin-digested gliadin were perfused with P5 to compete for PEP digestive activity.

A Model for Intestinal Content of Toxic Gliadin Peptides. The intestinal content for P4 and P5 during intestinal transit was calculated using the in vivo data collected in this work (Fig. 5A). The model not only indicates the importance of intestinal digestion in these gastric and pancreatic resistant fragments but also highlights the relative resistances to proteolysis by the brush border enzymes. The model predicts that the concentration for the control peptide myoglobin should be reduced to <1% starting material within 100 cm (the human upper small intestine). However, P4 persists >375 cm (the human lower small intestine) and P5 declines only to 5% after passing through the entire small intestine. In addition, data previously collected for P4 and P5 were used to determine the effect of transport across the epithelium on the concentration profile (Matysiak-Budnik et al., 2003). The overall effect on the luminal content was minimal, altering the average retention times of P4 and P5 by <7% (data not shown). On the other hand, coperfusion of 41.7 µg/ml recombinant PEP (190 µU/µl) with P5 effectively reducing the luminal content to <1% with 75 cm, an 8-fold reduction in average retention time (Fig. 5B).

View larger version (27K): [in this window] [in a new window]   Fig. 5. A, calculated model for 100 µM P5 (A), 100 µM P4 (B), and 100 µM P10 (C) luminal intestinal digestion using in vivo rat intestinal perfusion results. B, concentration profile model was adapted to illustrate the effective proteolysis of 50 µM P5 when supplemented with increasing concentrations of PEP: 0.0 (A), 25 µU/µl (B), 125 µU/µl (C), 155 µU/µl (D), and 190 µU/µl (E) recombinant PEP.

	Discussion

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Wheat-, rye-, and barley-derived peptides (named gliadins, secalins, and hordeins, respectively) are enriched in glutamine (35%) and proline (20%) residues (Wieser et al., 1983). Recent experiments have led to the hypothesis and verification that the high proline content renders portions of these proteins resistant to proteolysis by gastric, pancreatic, and intestinal brush-border enzymes (Hausch et al., 2002; Shan et al., 2002). Because Celiac Sprue has been linked to the presence of gliadin peptides in the intestinal lumen, defining a mechanism for further efficient processing of these gliadin peptides into smaller nontoxic fragments could possibly prevent the toxic interaction and prevent the intestinal damage in Celiac Sprue. Notably, PEP treatment of gliadin peptides has in fact been shown to reduce the immunostimulatory capacity of gliadin peptides (Shan et al., 2002). The work presented here quantifies the relative proteolysis resistance of several gliadin peptide segments and quantifies the in vivo role of prolyl endopeptidase as a potential enzymatic supplement to digestion of these toxic gliadin peptides. The luminal content model for gliadin peptide proteolysis illustrates the relative resistance of these gliadin peptides and the hydrolytic effects of PEP on luminal peptide content.

Incubating the panel of - and -gliadin-derived peptide fragments with proteases that they are typically exposed to in the stomach and upper intestine (pepsin, trypsin, chymotrypsin, carboxypeptidase A, and elastase) revealed, with the exception of P6, that the peptides were almost completely resistant to proteolysis by these enzymes (90% remaining after 4 h; Table 1). Certainly, there is a variable resistance of gliadin fragments to surface digestion, and some of the gliadin peptides are highly resistant to BBM processing. Because P6 is readily digested, it is unlikely to accumulate in the intestine to have a toxic effect due to macrophage stimulation (Tucková et al., 2000; Novák et al., 2002). The BBM was able to proteolyze some of the gliadin peptides, reducing them to 10% of the starting material, although this required 4 h of exposure under physiologically relevant conditions (Table 1). In particular, both P5 and P8 remained at 70% of the starting material even after 4 h. These results indicate that the brush-border enzymes are capable of cleaving many potentially toxic gliadin peptides to smaller nontoxic fragments, but at relatively slow rates that can be expected to allow the parent peptide to reside for an extended period within the upper intestinal cavity to evoke a toxic response in the Celiac Sprue. Notably, the BBM action on P5, the 33-mer known to be highly immunogenic by its capacity to stimulate T cell replication, is minimal (Fig. 1). Only the C-terminal Phe residue is removed, leaving the still toxic 32-mer peptide as the product. (Although the in vitro BBM digestion data qualitatively represents the in vivo processes, the quantity of surface membrane peptidases provided by the intact intestine at the surface in vivo cannot be achieved even in high concentration BBM enzyme stocks. This obviates direct quantitative comparison of in vivo and in vitro brush-border studies.) The addition of PEP cleaves it further to 4- to 7-mers that are substrates for subsequent surface digestion by the BBM oligopeptidases. Whereas some peptides are degraded into easily assimilated fragments, relatively long intermediates accumulate in the lumen in other instances (Fig. 1). These two points seem to indicate the possibility of two categories of digestive-resistant toxic peptides. The first category consists of highly resistant gliadin peptides such as P5 and P8; notably, P5 has already been shown to be a highly potent trigger of T cell proliferation from Celiac patients (Shan et al., 2002). The second category is composed of the less digestive-resistant, but still toxic, gliadin peptides that may be processed to nontoxic products in Celiac patients. Notably, the latter category of gliadin peptides may play a role only after the intestinal Celiac lesion has become well established with consequent reduction in brush-border enzyme expression (Mercer et al., 1990), thereby allowing the less resistant gliadin peptides to persist in the intestinal lumen.

At a low dose of PEP (25 µU/µl), the processing rates of the gliadin peptides were enhanced by 50% (Fig. 2), but not to the high levels of digestion seen for the control peptides. When PEP concentration was increased to 190 µU/µl, in the in vivo perfusion experiments, P5 (33-mer) could be shown to be completely cleaved to small nontoxic peptides (Fig. 3). These dosing levels (1:50 to 1:5; PEP to peptide) compare favorably to dosing levels currently in use for treating lactose intolerance (1:100 to 1:10; lactase to lactose), indicating that these dosing levels are in a reasonable range. Also, despite the possible competitive effect of other gliadin peptide fragments that would be released by pancreatic protease action on gluten within the intestinal lumen, perfusion of gluten pretreated with pepsin and pancreatic proteases along with P5 had minimal effect on the efficient processing of the peptide to its nontoxic products (note that 85% of PEP's P5 digestive capacity remained; Fig. 4). These data, along with digestive data for P5 with PEP alone, suggest that P5 is a high-affinity substrate for PEP (Shan et al., 2002). Currently, it is not known what quantities of intact peptide are required to initiate and maintain the Celiac inflammatory response. The compact nongliadin peptide GLGG is efficiently digested, and comparison of proteolysis for the gliadin peptides to those for GLGG, P10, and P9, may provide insight concerning the elimination of toxic gliadin in the intestine (Smithson and Gray, 1977). The in vivo rate data indicate that toxic gliadin peptide digestion is an order of magnitude slower than that for GLGG and is reduced by >50% compared with the digestion of the nongliadin peptides. It has been suggested that PEP is capable of accelerating the proteolysis of resistant gliadin peptides. In the work presented here, PEP-supplemented digestion of P5 and P8 showed significant increases in proteolysis rates in both in vitro and in vivo assays. Although there are some peptides, such as P7, that are resistant to PEP digestion, PEP addition was capable of decreasing the in vitro concentrations of the majority of gliadin peptides tested to below 10% of those persisting after maximal gastric and pancreatic protease action. Furthermore, in vivo PEP increases the rate of proteolysis by >50% (Table 1), additional PEP dosing indicates that luminal gliadin concentration of these peptides can be reduced or be potentially eliminated. The quantitative analysis here indicates that PEP has activity for large peptides, including the 33-mer (P5). However, this activity could in fact be enhanced by prior removal of terminal residues. As is evident from Fig. 1, PEP seems to completely process the 32-mer (indicated as B), whereas the relative effect on the intact 33-mer is relatively slower. This indicates that removal of the terminal Phe may be an enhancing factor in the digestion through PEP supplementation. Using the quantitative data collected here, profiles of the luminal content for P4 and P5 were calculated. These profiles clearly illustrate the proteolysis resistance of these gliadin peptides with the gliadin peptides persisting 4 to 8 times longer than a physiologically relevant peptide from myoglobin (Fig. 5A). The profile for luminal content was recalculated using previously collected data for flux of P4 and P5 across the epithelium, which determined a minimal effect on the luminal content (Matysiak-Budnik et al., 2003). Further support for the beneficial effects of PEP supplementation is gained by analyzing the PEP dosing experiment presented in a similar manner (Fig. 5B). These results clearly illustrate the dramatic effect of PEP on luminal peptides.

The work presented here has demonstrated the resistance of several immunogenic gliadin peptides to gastric, intestinal brush-border proteolysis. Furthermore, we have determined that there seem to be two levels of resistance, one at the gastric and luminal (pancreatic) protease level and one at the brush-border peptidase level. The in vivo rate data have shown that the reduced rate of proteolysis is not solely due to size and that PEP can function in concert with the brush-border peptidases by providing the initial key cleavages on a number of peptides so as to increase the overall rate of peptide processing. Dosing experiments have also confirmed that highly resistant peptides such as P5 can effectively be eliminated with PEP supplemented digestion with minimal accumulation of products. Perfusion of pepsin-, trypsin-, and chymotrypsin-digested gliadin with P5 has demonstrated that PEP is capable of digesting P5 even in the presence of smaller, more accessible targets that would be present under physiological conditions (Fig. 4). Digestion of P5 under these conditions is at least comparable with that for the other gliadin digestive resistant fragments released after completed processing of gluten by pepsin, trypsin, and chymotrypsin, further suggesting that PEP has appreciable potential as a digestive supplement. Luminal profiles of gliadin content illustrate the relative proteolysis resistance of these gliadin peptides compared with physiologically relevant myoglobin peptide and that flux across epithelial cells only plays a minor role in determining luminal gliadin content (Fig. 5A). Furthermore, the abundance of toxic peptides calculated from the PEP dosing experiments establishes a dramatic effect of PEP on luminal concentration with a 3-fold increase in rate, resulting in an 8-fold reduction in average retention time (Fig. 5B).

These experiments have further advanced the hypothesis of PEP-enhanced digestion of gliadin as a means of therapeutic intervention for Celiac Sprue. To continue making progress in this area, it is important to continue whole gliadin proteolysis studies, to determine other potentially resistant fragments. This should allow the identification, quantification, and the stimulatory capacity of PEP-resistant peptides to be defined. Given the importance of understanding the digestion of these gliadin peptides and of determining the means to detoxify these peptides, it is important to elucidate the in vivo situation of a complex organ such as the intestine. Using the rat intestinal perfusion setup as our model system of digestion, more physiologically relevant experiments can now be explored with pepsin-digested gluten in the intact duodenum where luminal protein digestion is very active. Although PEP cannot be expected to cleave intact gluten, it does cleave the gliadin peptides that are released by pancreatic enzyme digestion. Perfusion through the duodenum will determine the stability of PEP to endogenous pancreatic enzymes and determine whether PEP can effectively eliminate the toxic gliadin peptides, thereby protecting the upper regions of the small intestine. Perfusion experiments will also allow the continued study of PEP as a potential detoxifying agent, providing insight into the key events in the digestion and uptake of these gliadin peptides in order to prevent the cascade of events that follows in Celiac Sprue.

	Acknowledgements

 
We thank Lu Shan for sharing unpublished results and providing recombinant prolyl endopeptidase.

	Footnotes

 
doi:10.1124/jpet.104.068429.

ABBREVIATIONS: HLA, human leukocyte antigen; PEP, prolyl endopeptidase; HPLC, high-performance liquid chromatography; BBM, brush-border membrane; PTCtCE, pepsin, trypsin, chymotrypsin, carboxypeptidase A, and elastase.

Address correspondence to: Dr. C. Khosla, Departments of Chemistry and Chemical Engineering, 380 Roth Way, Keck Science Bldg., Rm. 389, Stanford, CA 94305-5025. E-mail: ck{at}chemeng.stanford.edu

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