or inhibitors of dipeptidyl peptidase IV

Biochimie 86 (2004) 31–37 www.elsevier.com/locate/biochi

Hemorphins: substrates and/or inhibitors of dipeptidyl peptidase IV Hemorphins N-terminus sequence influence on the interaction between hemorphins and DPPIV M. Cohen, I. Fruitier-Arnaudin *, J.M. Piot Laboratoire de Génie Protéique et Cellulaire, EA3169, Bâtiment Marie-Curie, avenue Michel-Crépeau, 17042 La Rochelle cedex 1, France Received 25 June 2003; accepted 10 November 2003

Abstract Hemorphins are endogenous peptides belonging to the family of “atypical” opioid peptides released from sequentially hydrolyzed hemoglobin. In this paper, we report an inhibitory effect of these peptides on dipeptidyl peptidase IV (DPPIV) activity, known to be involved in regulatory functions such as the activation or inactivation of peptides. The structure activity research revealed that hemorphins N-terminus sequence influences nature of the interaction between hemorphins and DPPIV. Kinetic studies conducted with purified DPPIV demonstrated that hemorphin-7 (H7) constitutes a good substrate (Kcat/Km of 137 mM–1 s–1) for this enzyme but could also act as a selective competitive inhibitor by substrate binding site competition. These blood-derived peptides could represent endogenous regulators of this enzyme activity. © 2003 Elsevier SAS. All rights reserved. Keywords: Hemorphin; Dipeptidyl peptidase IV, DPPIV; Kinetic study; Inhibition study; Peptide hydrolysis

1. Introduction Proteolysis of some functional proteins in vitro leads to the generation of peptides exhibiting an opioid like activity when they have a Tyr-Pro N-terminus sequence. In contrast to “classical” bioregulator peptides, this group of peptides can be generated from hydrolysis of proteins having wellestablished functions in vivo. Thus b-casomorphins [1] are released from b-casein, and hemorphins [2] from hemoglobin by in vitro peptic hydrolysis. The members of the hemorphin family include peptides from 4 to 10 amino acids, which are generated by proteolytic degradation of the (32–41) segment of human hemoglobin b-chain or (31–40) segment of the bovine hemoglobin b-chain [3]. These peptides were also isolated in vivo from tissues (hypothalamus [4], pituitary [5]) or fluids [6–9] (plasma, serum, cerebrospinal, gingival crevicular, bronchoalveolar lavage fluid). The composition and the content in hemorphins seem to be characteristic for each given organ or tissue [10]. * Corresponding author. Tel.: +33-5-46-45-85-62/86-44; fax: +33-5-46-45-82-47. E-mail address: [email protected] (I. Fruitier-Arnaudin). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2003.11.001

The hemorphins could be released in the organism during physiological or physiopathological hemoglobin proteolysis [5–9], but the precise mechanism of their generation in vivo remains unknown. It was hypothesized that proteolytic degradation of hemoglobin by endogenous lysosomal proteases could give rise to hemorphins [11]. Among these proteases, cathepsin D could be the first enzyme implied in the release of two hemorphins from hemoglobin: LVV-Hemorphin-7 (LVVYPWTQRF) and VV-Hemorphin-7 (VVYPWTQRF) [12,13]. The other hemorphins could come from LVV and VV-Hemorphin-7 further degradation. In comparison with proteolytic degradation of host hemoglobin by shistosomes, it seems that there are many proteases which could be involved in the degradation of hemoglobin to give hemorphins [14]. These enzymes would act in an ordered way: the polypeptides released after the digestion by one of these proteases becoming substrates for other proteases. Many in vivo physiological effects have been studied with regard to hemorphins. They were shown to exhibit coronaroconstrictory [15], anti-tumorous [16], and immunoregulatory activities [17]. Their potential role in the renin angiotensin system (RAS) was also studied [18,19]. In fact, it was demonstrated that hemorphins could interact at various levels of the RAS by inhibiting angiotensin converting enzyme and

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M. Cohen et al. / Biochimie 86 (2004) 31–37

Angiotensinogen

Hemoglobin

Renin/Cathepsin D

Cathepsin D

Angiotensin I ACE

inhibition

Hemorphins

Angiotensin II Aminopeptidase A

inhibition

Angiotensin III

Table 1 Hemorphin peptides sequence Sequence Hemorphin-7 peptides LVV-Hemorphin-7 VV-Hemorphin-7 Hemorphin-7

LVVYPWTQRF VVYPWTQRF YPWTQRF

Other hemorphin peptides VV-Hemorphin-6 VV-Hemorphin-5 Hemorphin-5 Hemorphin-4

VVYPWTQR VVYPWTQ YPWTQ YPWT

Aminopeptidase B

Angiotensin IV Aminopeptidase N

Angiotensin IV degradation

Fig. 1. Known interactions of hemorphins with the RAS.

aminopeptidase N activity (Fig. 1). Moreover, some hemorphins (LVV-Hemorphin-7 and VV-Hemorphin-7) behave, like angiotensin IV, as receptor binding competitors [20,21]. The putative interactions of hemorphins in the RAS led us to investigate their renal metabolism. In a recent study, we have already demonstrated that LVVH7 has a very short half life time, comparable with Angiotensin IV (AgIV), in the rat renal cytosol (<2 min, [22]). The major products of LVVH7 degradation by renal cytosol are hemorphins of type 7 and 6, suggesting both aminopeptidase and carboxypeptidase activities which could operate according to an ordered process, since addition of prolyl endopeptidase inhibitor in the incubation mixture allows an increase of the half life time of LVVH7 and involves a modification of the products obtained. In the other subcellular fractions of rat kidney (mitochondrial, microsomal and nuclear), the degradation of LVVH7 and its products is too rapid to observe a kinetic (half life time of LVVH7 <30 s). So it was interesting to research peptidasic or proteolytic activities present in the kidney and able to inactivate hemorphin peptides. In this context, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) seems a good candidate to interact with hemorphins. DPPIV is a serine protease, mainly extracellular, expressed in a variety of cells in many mammalian species [23]. In the kidney, this enzyme is exceptionally concentrated, located primarily in the cortex and found in the brush border and microvillus fractions. It is known to be involved in a number of cell functions such as cell-to-cell communication, cell activation, proliferation, adhesion, metastazing [23]. It is also implicated in regulatory functions such as the activation or inactivation of peptides, e.g., peptide hormone, various cytokines and growth factors. DPPIV is a post-proline cleaving enzyme, and consequently

any natural peptide bearing the Tyr-Pro motif at the N-terminus should be regarded as an ideal substrate unless the amino acid behind the bond to be split is also proline. So, it was very probable that hemorphins structure could be appropriate to interact with DPPIV. In this study, we studied what kind of interaction could be involved between DPPIV and hemorphins. We tested hemorphin potentiality to be substrate and/or inhibitor of DPPIV. The influence of hemorphins N-terminus and C-terminus sequence on interaction with DPPIV was also examined.

2. Materials and methods 2.1. Materials Hemorphins (LVVH7, VVH7, H7, VVH6, H5 and H4, Table 1) were synthesized by Altergen (Illkirch, France). Porcine kidney dipeptidyl peptidase IV, bovine serum albumin (BSA), kits for marker enzymes (ALP, procedure 245; ACP, procedure 435; LDH, procedure 500), were from Sigma. The DPPIV substrate, Gly-Pro-pNA and diprotin A were from Bachem. The liquid chromatographic system consisted of a Waters Alliance 2690 automated module equipped with a photodiode array detector. Millenium software was used to plot, acquire and analysis chromatographic data. All the chromatographic processes were carried out with a Delta Pak C18 column (3.9 mm × 300 mm, 15 µm). Chemicals and solvents were of analytical grade and acetonitrile of high performance liquid chromatography (HPLC) grade. Aqueous HPLC eluents and samples were filtered prior to use on 0.22 µm Millipore filters and degassed with helium, prior and during analysis. 2.2. Metabolism studies of LVVH7 in rat kidney microsomal subcellular fraction 2.2.1. Preparation of rat kidney microsomal subcellular fraction Eight male Wistar rats weighing 250–300 g were starved overnight and the next morning anesthetized with ethyl car-

M. Cohen et al. / Biochimie 86 (2004) 31–37

bamate (40 mg). The kidneys were isolated from each rat, washed in ice-cold buffer solution (SPB-10: 0.25 M sucrose and 10 mM potassium phosphate buffer, pH 7.4), trimmed of adhering tissue, and cut into small pieces. After the tissue pieces were weighed, pooled homogenates from the eight rats were prepared. Five milliliter of phosphate buffer (40 mM, pH 7.4) were used per gram of tissue. A pooled homogenate from the eight rats was prepared, first, with Ultraturrax, modèle TP 18/10 and then with a Thomas Potter (eight up-and-down strokes). The kidney homogenate was centrifuged for 60 min at 100,000 × g. The resulting pellet was washed with SPB-10, and then, resuspended in 15 ml of SPB-10. The microsomal fraction obtained was aliquoted, immediately frozen, and stored at –20 °C until used. The protein concentration of kidney microsomal fraction was determined by the method of Lowry [24]. Marker enzymes for brush border membranes (ALP), lysosomes (ACP) and cytosol (LDH) were used to characterize the microsomal fraction. 2.2.2. Degradation of LVVH7 in microsomal subcellular fraction The degradation of LVVH7 in the presence of kidney microsomal fraction was investigated using incubation mixtures of 1 ml consisting of microsomal fraction (very low final protein concentration, 200 µg/ml, in order to allow the observation of kinetics of degradation) added with 100 nmoles of LVVH7 in phosphate buffer (70 mM, pH 7.4) containing NaCl (48 mM), KCl (5.4 mM), Na2HPO4 (59 mM), mannitol (15 mM) and D-Glucose (10 mM). Each incubation mixture was preincubated at 37 °C for 10 min, under gently shaking, before LVVH7 was added. Samples (200 µl) were withdrawn at 30, 60, 90 and 240 s, and immediately heated at 90 °C to stop the enzymatic reaction. The mixtures were then centrifuged for 10 min at 5000 rpm (Sigma 203, rotor 12012). The resulting supernatants were filtered (0.22 µm) and stored at –20 °C until chromatographic analysis. Control incubations of LVVH7 (100 nmoles) or microsomal fraction (final protein concentration 200 µg/ml) were performed separately according to the same experimental procedure. In order to demonstrate the putative interaction of DPPIV with hemorphins, diprotin A (50 µM) was added in the incubation mixture. Each hydrolysis was performed in triplicate. 2.2.3. Chromatographic analysis The degradation products from each incubation medium were resolved as follows: a sample (100 µl) of each hydrolysate was injected in the Delta Pak C18 column. The mobile phase comprised 10 mM ammonium acetate buffer, pH 6 as solvent A and acetonitrile as solvent B. The flow rate was 1 ml/min and the absorbance was recorded at 280 nm. The linear gradient applied was 15–40% B in 30 min after an initial isocratic step (15% acetonitrile) over a period of 5 min. Standard hemorphins (LVVH7, LVVH6, VVH7, VVH6, H7)

33

were chromatographed under the same conditions and stored into the Millenium library. During chromatographic acquisition, detection and quantitation of hemorphins were carried out by performing continuous UV-spectral scans between 190 and 300 nm. Spectrum matching results (i.e. comparison between spectrum of each eluted peak and a library of spectra from hemorphins available in our laboratory) were expressed as match angle and match threshold. Such spectral scan procedure has been extensively and routinely used in previous studies [25,26]. 2.3. Hemorphins enzymatic conversion studies Forty nanomoles of peptide (H7, H5, H4) were incubated 2 h at 37 °C in Eppendorf tubes with 11 mUI of DPPIV in a final volume of 500 µl of 0.2 M Tris–HCl at pH 8. The reactions were stopped by the addition of 250 µl of 0.2 M HCl. Assays were stored at –20°C until chromatographic separations. The degradation products from different hemorphins were resolved as follows: a sample (90 µl) of each DPPIV hydrolysate corresponding to about 4.8 nmoles of initial peptide was injected in the Delta Pak C18 column. The mobile phase comprised 10 mM ammonium acetate buffer, pH 6 as solvent A and acetonitrile as solvent B. The flow rate was 1 ml/min and the absorbance was recorded at 280 nm. The linear gradient applied was 15–40% B in 30 min after an initial isocratic step (15% acetonitrile) over a period of 5 min. Standard peptides were chromatographed under the same conditions. 2.4. Determination of the potentiality of H7, H5 and H4 to be substrate of DPPIV Five concentrations of H7, H5 and H4 (10–80 µM) were incubated in 0.2 M Tris–HCl buffer, pH 8 (final volume of 500 µl) with 20 mUI of purified DPPIV, at 37 °C, for 30 min. The reactions were stopped by the addition of 250 µl of 0.2 M HCl. Assays were stored at –20 °C until chromatographic separations as described above. Each hydrolysis was performed in triplicate. 2.5. Studies of kinetics of inhibition The enzymatic assay used for the determination of DPPIV activity is based on the cleavage of a specific substrate, Gly-Pro-pNA (Km = 0.125 mM), whose resulting product absorbs at 400 nm. The rate of hydrolysis of the substrate was determined by monitoring the absorbance increase at 400 nm for 3 min. Hemorphins inhibitory potential is determined with a substrate concentration of 0.15 mM, peptide concentration of 30 µM and DPPIV activity of 2 mUI at 37 °C, in a final volume of 1 ml of 0.2 M Tris–HCl buffer, pH 8, for 3 min. For each test, triplicate assays were achieved and inhibition of DPPIV was expressed as mean percentage of the control without hemorphin.

M. Cohen et al. / Biochimie 86 (2004) 31–37

To achieve the inhibitory mechanism, five substrate concentrations between 0.15 and 0.40 mM, and six concentrations of each hemorphin between 0 and 80 µM in 0.2 M Tris–HCl buffer, pH 8 were tested with respect to DPPIV activity. The components were incubated in a final volume of 1 ml for 3 min at 37 °C with a fixed activity of 2 mUI DPPIV. For each test, triplicate assays were achieved and inhibition of DPPIV was expressed as mean percentage of the control without hemorphin. The slope between 0 and 2 min was calculated by the software and was considered as the initial velocity. Ki values and type of inhibition were determined from Lineweaver–Burk plots of the reciprocal of reaction versus reciprocal of substrate concentration.

3. Results–discussion 3.1. Influence of DPPIV inhibitor on LVVH7 metabolism in kidney microsomal fraction In a previous study, we have shown that rat kidney cytosolic metabolism of LVVH7 liberates mainly VVH7, H7 and LVVH6 [22]. However, degradation kinetics of LVVH7 in the other subcellular fractions of rat kidney (microsomal, mitochondrial and nuclear) could not be observed due to the rapid degradation of LVVH7 and its products. That is why LVVH7 was incubated with a very low protein concentration of microsomal fraction. As shown in Fig. 2, the major products released from this incubation were VVH7, H7 and LVVH6. Due to the specificity and the mainly localization of DPPIV, it was very probable that this enzyme could interact with H7 (Tyr-Pro-Trp-Thr-Gln-Arg-Phe). In order to verify this hypothesis, diprotin A, an inhibitor of DPPIV, was added in the incubation mixture. As a result, a diminution in H7 0.018

VVH7

0.016 0.014

LVVH7

AU

0.012 0.010

H7

0.008

LVVH6

0.006

12

diprotin A control

10

H7 (nmoles)

34

8 6 4 2 0 0

50

100

150

200

250

t (s)

Fig. 3. Liberation of H7 during metabolism of LVVH-7 incubated with kidney microsomal fraction and diprotin A (black triangle) or without inhibitor (square) at 37 °C. Data are means ± S.E.M. from three experiments. Results are expressed in terms of nmoles of H7 apparition.

degradation rather than an increase in H7 release was observed (Figs. 2 and 3). On the other hand, it seems that the level of LVVH7 degraded or VVH7 and LVVH6 liberated are not influenced by DPPIV activity. 3.2. Determination of the potentiality of H7 to be a substrate of DPPIV In order to confirm the potentiality of H7 to be an endogenous substrate of DPPIV, the kinetics of its degradation was investigated in vitro and compared with other hemorphins with a similar N-terminus sequence (H4 and H5). Five hemorphin concentrations (10–80 µM) were hydrolyzed by 20 mUI DPPIV to determine different kinetic parameters: Km and Kcat. The rate Kcat/Km permits to determine the best DPPIV substrate. Although it seems that C-terminus sequence influences the affinity and Kcat of DPPIV for these peptides (Table 2), it appears that kinetics of degradation of these peptides are very similar (Fig. 4) and that they could represent good substrates for DPPIV with a similar specificity constant evaluated to around 150 mM–1 s–1 (Table 2), comparable with other endogenous neuropeptides [27] (Table 3). Considering this observation, it can be envisaged that hemorphins may constitute DPPIV inhibitors by substrate binding site competition.

0.004

3.3. Inhibiting potential of the hemorphins for DPPIV

0.002 0.000 10

12

14

16

18

20

22

24

26

28

30

Minutes

Fig. 2. UV-absorbance profile (280 nm) of HPLC chromatogram of LVVH7 (100 nmoles) products after 30 s of incubation with rat kidney microsomal fraction (200 µg/ml of proteins) at 37 °C in absence (black line) or presence of diprotin A (grey line). One hundred microliters of hydrolysates were applied to a C18 column as described in Section 2. LVVH7, VVHemorphin-7 (VVH7), LVV-Hemorphin-6 (LVVH6) and Hemorphin-7 (H7) are identified. An UV-second order spectroscopy analysis was performed on each UV-absorbance peak by a spectroscopic analysis method [25,26]. AU: absorbance unit.

Considering that H7, H5 and H4 are good substrates for DPPIV, different hemorphins (LVVH7, VVH7, H7, VVH6, VVH5, H5, H4) were tested on DPPIV activity in the presTable 2 Kinetic constants of H4, H5 and H7 for DPPIV Kcat (s–1) Km (mM) Kcat/Km (mM–1 s–1)

H4 1774.4 13.5 131

H5 823.7 6.4 129

H7 5.3 38.7 × 10–3 137

M. Cohen et al. / Biochimie 86 (2004) 31–37

20

16 14 12

1/V (min/Do)

peptide (%)

VVH7=0 VVH7=45µM VVH7=15µM VVH7=20µM VVH7=30µM VVH7=60µM

H4 H5 H7

18

35

10 8 6

100 90 80 70 60 50 40 30

4

20

2

10

0 2,8

3

3,2

3,4

3,6

0 -6

log t

-4

-2

0

2

4

6

8

1/S (m M -1) Fig. 4. A comparison of kinetic degradation of H4, H5 and H7 peptides by DPPIV. Forty nanomoles of peptide (H7, H5, H4) were hydrolyzed by 11 mUI of DPPIV at 37 °C and pH 8 during 2 h.

Table 3 Steady state kinetics for neuropeptide truncation by DPPIV from Lambeir et al. [27] Kcat/Km (mM–1 s–1)

VIP 12 ± 2

VIP(3-28) 1.7 ± 0.05

H4

H5

GRP NPY 1800 ± 300 765 ± 50

PACAP38 24 ± 4

35

inhibition %

30 25 20 15 10 5 0 H7

LVVH7

VVH7

VVH6

VVH5

Fig. 5. Comparative inhibition effects of seven hemorphins on DPPIV activity. DPPIV enzyme activity was measured at 37 °C in the presence of 0.15 mM of Gly-Pro-pNA, hemorphins concentration of 30 µM and 2 mUI of DPPIV. Inhibition (%) was expressed relative to the control value without inhibitory peptides. Data were the means ± S.D. of three different experiments.

ence of 0.15 mM Gly-Pro-pNA in order to evaluate their inhibitory potential. The results presented in the Fig. 5, revealed that all hemorphins seem able to inhibit DPPIV activity, and particularly VVH7 and H7 which present 30% of inhibiting activity for DPPIV in these conditions. It means that the hemorphin C-terminus sequence would play a very significant role to inhibit DPPIV activity. Moreover, the pres-

Fig. 6. Lineweaver–Burk plots of Gly-Pro-pNA concentration effect on the activity of purified DPPIV in absence or presence of VVH7. The concentration of DPPIV was 2 mUI/1 ml. Temperature was maintained at 37 °C. DPPIV activity was measured in the presence of increasing concentration of VVH7 and a double-reciprocal plot was drawn. Experiments were performed in triplicates; results are given as mean ± S.D.

ence of leucine in N-terminus sequence of LVVH7 seems to reduce the inhibitory activity of this hemorphin for DPPIV. So only hemorphins of type 7, excepted LVVH7 are able to inhibit DPPIV activity efficiently. In order to understand this mechanism of inhibition, a kinetic study was performed with both the substrate Gly-PropNA (Kcat estimated at 52.3 s–1 in very similar conditions [28]) and/or VVH7 or H7 in the presence of DPPIV. The mechanism of inhibition and the Ki value were determined from Lineweaver–Burk linearization. As shown on the Fig. 6, H7 (data not shown) and VVH7 seem to be competitive inhibitors for DPPIV activity with Ki values evaluated at 29.9 ± 3.9, and 33.4 ± 3.2 µM for VVH7 and H7, respectively. So these hemorphins seem to have the same inhibiting potential for DPPIV. As shown by the study of substrate binding of DPPIV with diprotin A [29], the proline in position P1 of hemorphins like H7 will fit optimally into the pocket of the active site of the enzyme. Taking into account the interactions study of the active site of DPPIV with diprotin A [29], we could suggest that the apparent competitive inhibition caused by a low turnover rate of H7 (Kcat = 5.3 s–1) could be due to hydrophobic interactions made by the phenylalanine residue in C-terminal position, and the existence of charged amino acids (glutamine and arginine) within the sequence which would enable it to reach the active site easily and to be stabilized there in an unsuitable conformation for the progress of the reaction. Like microbial peptides diprotin A or diprotin B [30], these peptides could represent competitive substrates that are slowly hydrolyzed and act as selective competitive inhibitors for DPPIV at micromolar range. On the basis of these results one may speculate that these hemorphins could regulate DPPIV activity. Indeed, specific DPPIV inhibitors acting at low concentrations are of special

36

M. Cohen et al. / Biochimie 86 (2004) 31–37

interest for physiological investigations and for potential clinical applications. For example peptides like VVH7 and H7 inhibiting both DPPIV and angiotensin converting enzyme (ACE) could limit large degradation of the substance P [31].

[5]

4. Conclusion

[7]

Hemorphins are bioactive peptides derived from hemoglobin hydrolysis. Their mechanism of generation is still not well understood but it is very likely that one of the first steps of their in vivo generation (LVVH7 and VVH7) could be the result of cathepsin D activity [12,13]. The other hemorphins could be released from LVV and VV-Hemorphin-7 further degradation by aminopeptidase or carboxypeptidase activities. However, it seems that these activities could be different in each tissue since the composition and content in hemorphins appear to be characteristic for each given organ or tissue [10]. In this context, we were particularly interested to investigate potential peptidasic activities responsible for the degradation of hemorphins in the kidney. In a previous study, it was already shown that prolyl endopeptidase activity could constitute an important step for LVVH7 degradation in rat kidney cytosol [22]. In the present study, we were interested in another proline peptidase activity, DPPIV activity, with regard to the released products from LVVH7 degradation in kidney microsomal fraction. It was shown that H7 released from LVVH7 incubation with microsomal fraction could be degraded by DPPIV present in this fraction since the addition of diprotin A in the incubation mixture could involve a four times increase in the H7 level released after 4 min of hydrolysis. The kinetic study with purified DPPIV permits to confirm that H7 constitutes a good substrate for this enzyme independently of its C-terminus sequence with a specificity constant of 137 mM–1 s–1. If in many cases hemorphins could represent endogenous substrates of DPPIV, it can be also envisaged that some of them may also constitute DPPIV inhibitors by substrate binding site competition. Two hemorphins, H7 and VVH7, could act as selective competitive inhibitors for DPPIV at micromolar range and may represent consequently endogenous regulators of this enzyme activity.

[6]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

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