Cathepsin B inhibitory peptides derived from β-casein

Peptides 21 (2000) 807– 809

Cathepsin B inhibitory peptides derived from ␤-casein夞 Hyun Sook Lee, Kye Joon Lee* Department of Microbiology, College of Natural Sciences, and Research Centre for Molecular Microbiology, Seoul National University, Seoul 151-742, Korea Received 24 January 2000; accepted 7 April 2000

Abstract Two cathepsin B inhibitory peptides were isolated from a commercial pancreatic digest of casein. The peptides were identified as the Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile corresponding to the sequence 61– 66 and 203–207 of bovine ␤-casein. These peptides showed competitive inhibition for cathepsin B with the K i values of 2.31 and 3.30 mM, respectively. Two related analogues, Tyr-ProPhe-Pro-Gly-Pro-Ile and Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile, were synthesized but their cathepsin B inhibitory activity was not detected. © 2000 Elsevier Science Inc. All rights reserved. Keywords: ␤-Casein; Cathepsin B; Inhibitor; Bioactive peptides; K i value

1. Introduction Milk proteins, in particular caseins, are currently the main source of biologically active peptides. Such bioactive peptides can be released during in vivo and in vitro hydrolysis of milk proteins by gastric and/or pancreatic enzymes [14] and may exert different biological activities, such as opioid agonistic and antagonistic activities, angiotensin converting enzyme (ACE) inhibitory activity, immunomodulatory effects, antithrombotic activity, antimicrobial activity, and mineral binding properties [11]. In this study, cathepsin B inhibitory peptides were found to be present in the bovine ␤-casein sequence. Cathepsin B (EC 3.4.22.1) is one of the lysosomal cysteine proteases and has been implicated in intracellular proteolysis [2,13] and a variety of pathological processes including dysregulated protein turnover such as muscular dystrophy [5], bone resorption [4], and tumor metastasis [8,16,17]. Therefore, cathepsin B inhibitory peptides may serve as therapeutic drugs for disease treatment or health-enhancing nutraceuticals for disease prevention. Although several natural product inhibitors of cathepsin B have been identified, foodderived cathepsin B inhibitor has not yet been reported. 夞This work was supported by G7 grant (G7, 1995–1998) from the Ministry of Science and Technology in Korea. * Corresponding author. Tel.: ⫹82-2-880-6705; fax: ⫹82-2-882-9285. E-mail address: [email protected] (K.J. Lee).

In the course of a screening program to discover cathepsin B inhibitor [9], we isolated cathepsin B inhibitory compounds from the culture broth of Streptomyces chromofuscus SMF28. Two of them were identified to be ProPhe-Pro-Gly-Pro-Ile and Gly-Pro-Phe-Pro-Ile by mass spectrometry, amino acid analysis, and amino acid sequencing. The sequences of these peptides correspond to the sequence 61– 66 and 203–207 of bovine ␤-casein [6] and are also present in the ovine and buffalo ␤-casein. Thus, it was suggested that these peptides were derived from commercial casitone, a pancreatic digest of casein, which was used as a nitrogen source in the culture medium. To investigate whether these peptides are components of casitone or fragments of casitone digested by microbial protease(s), the isolation of peptides from casitone was attempted in the present study. In addition, cathepsin B inhibitory activity of two peptides and their derivatives were investigated.

2. Methods Peptides were isolated from casitone as follows: 50 g of Bacto-casitone (Difco Laboratories Inc., Detroit, USA) was dissolved in 500 ml of distilled water and filtered. This solution was fractionated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC) on a JAIGEL-GS310 (Japan Analytical Industry Co., Ltd., Tokyo, Japan) GFC hydrophilic column (20 ⫻ 500 mm).

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Peptides were eluted with 40% methanol containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 5 ml/min. Runs were conducted at room temperature using a LC-908-G30 HPLC system (Japan Analytical Industry Co., Ltd., Tokyo, Japan) with UV detection at 214 nm. Fractions corresponding to the each peak was separately pooled and lyophilized. The dried materials were dissolved in 10% acetonitrile in 20 mM sodium phosphate buffer (pH 6.0) and applied to a semipreparative Hypersil H5ODS (Hichrom Co., Novato, USA) C18 column (10 ⫻ 250 mm). Peptides were eluted with a linear gradient of 10 to 40% acetonitrile in 20 mM sodium phosphate buffer (pH 6.0) for 60 min at a flow rate of 2.5 ml/min. A few peaks showing the similar retention time as those of the Pro-Phe-Pro-Gly-Pro-Ile and Gly-ProPhe-Pro-Ile purified from the culture broth of S. chromofuscus SMF28 were separately pooled and lyophilized. These materials were assayed for cathepsin B inhibitory activity and some of them were subjected to mass spectrometry, amino acid analysis, and amino acid sequencing. Cathepsin B inhibitory activity was measured using BzDL-Arg-2-naphthylamide (Sigma Co., St. Louis, USA) as a substrate by the method of Barrett [1]. For the studies of inhibitor kinetics, initial velocities of amidase activity of cathepsin B were determined by continuous spectrophotometric assay using N-benzyloxycarbonyl-L-arginyl-L-arginine-p-nitroanilide (Z-Arg-Arg-pNA; Bachem AG, Bubendorf, Switzerland) as a chromophore substrate. Peptides were preincubated with enzyme for 10 min and then reactions were started by addition of the substrate. p-Nitroaniline release from substrate was monitored at 400 nm at 30°C in an UV-160 spectrophotometer (Shimadzu Co., Kyoto, Japan). The K i values for peptides were determined from double-reciprocal Lineweaver–Burk plot [10]. Five or six concentrations of the substrate that gave evenly distributed values of reciprocal values were used. The fast atom bombardment mass (FAB-MS) spectra were recorded on JMS AX505WA mass spectrometer (Jeol Ltd., Tokyo, Japan) using glycerol as a matrix. Peptides were hydrolyzed in 6 N HCl at 110°C for 24 h in evacuated sealed tubes. The liberated amino acids were analyzed using an automatic PicoTaq amino acid analyzer (Waters, Milford, USA). Amino acid sequences of the purified peptides were analyzed by Milligen 6600/Procise 491 protein sequencer (Perlin–Elmer, Foster City, USA). Peptides were synthesized using 9050 PepSynthesizer (Perlin–Elmer) by Fmoc method.

3. Results Several peptides were purified from casitone by a semipreparative RP-HPLC column and appeared as pale yellow powder (2 to 23 mg). Cathepsin B inhibitory activity was detected in two peaks, peak A and peak B, with the same retention times as those of the Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile, respectively (Fig. 1). Amino acid

Fig. 1. Reversed-phase HPLC of the peptides from casitone on a Hypersil H5ODS column. Absorbance of the eluate was monitored at 214 nm. Individual fractions as indicated by the arrow were assayed for cathepsin B inhibitory activity and their structures were identified.

analysis revealed that each peptide was composed of Pro: Phe:Gly:Ile (3:1:1:1) and Pro:Phe:Gly:Ile (2:1:1:1). The FAB-MS spectra of these peptides exhibited molecular ion peaks, [M⫹H]⫹, at m/z 627 and 530, respectively, and the kinds of amino acids were shown by the fragmentation patterns. Thus, it was assumed that neither N- nor C-terminals of these peptides attached to other molecule. Amino acid sequence analysis identified the primary structures of these peptides to be Pro-Phe-Pro-Gly-Pro-Ile and Gly-ProPhe-Pro-Ile, respectively, which corresponded to residues No. 61– 66 and 203–207 of bovine ␤-casein. Although peak C eluted behind two peaks on a column did not show the cathepsin B inhibitory activity, it was subjected to further characterization. The molecular weight of peak C compound was determined to be 888 Da by FAB-MS analysis and its amino acid composition was shown to be Val:Tyr:Pro:Phe:Gly:Ile (1:1:3:1:1:1). The chemical structure of peak C compound was identified to be [Val0]-␤-casomrphin-7 (Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile) corresponding to the position 59 – 66 of the ␤-casein by amino acid sequence analysis. Based on the results, the Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile isolated from the culture broth of S. chromofuscus SMF28 were components of casitone, that is, products of ␤-casein hydrolyzed by pancreatic enzymes. The K i values of peptides for cathepsin B were determined using synthetic peptides with the corresponding se-

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Table 1 Inhibition of cathepsin B by four peptides derived from ␤-casein Peptide

Residue positiona

Inhibition mode

K i (mM)

Pro-Phe-Pro-Gly-Pro-Ile Gly-Pro-Phe-Pro-Ile Tyr-Pro-Phe-Pro-Gly-Pro-Ile Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile

61–66 203–207 60–66 59–66

Competitive Competitive — —

2.31 3.30 NIb NI

a b

Residue positions are indicated by the number of the N- and C-terminal amino acids in the bovine ␤-casein sequence. NI means that no inhibition was detected up to the concentration of 4 mM.

quences. The structurally related ␤-casomrphin-7 (Tyr-ProPhe-Pro-Gly-Pro-Ile) was also synthesized to estimate the cathepsin B inhibitory activity. The K i values determined by Lineweaver–Burk plot are listed in Table 1. The Pro-PhePro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile showed competitive inhibition with K i values of 2.31 and 3.30 mM, respectively. The Tyr-Pro-Phe-Pro-Gly-Pro-Ile and the ValTyr-Pro-Phe-Pro-Gly-Pro-Ile did not inhibit cathepsin B up to the concentration of 4 mM. These observations suggest that the addition of one or two residues at N terminus of Pro-Phe-Pro-Gly-Pro-Ile may not be appropriate for enzyme subsite recognition.

4. Discussion The Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-ProIle peptides have never been detected as biologically active peptides from food proteins. The Pro-Phe-Pro-Gly-Pro-Ile was only reported to be isolated from the incubated medium of Pseudomonas syringae pv. mori as a phytopathogenic substance [7]. The opioid activity [3] and bitter taste [15] of synthetic Pro-Phe-Pro-Gly-Pro-Ile has been compared with that of active fragments of ␤-casein such as morphiceptin and ␤-casomorphin-7. The effect of Gly-Pro-Phe-Pro-Ile was initially investigated in this study. The potency of these peptides toward cathepsin B was relatively low as compared with that of cathepsin B inhibitors developed so far. For example, CA030 and CA074 showed inhibition with K i values of a nM order [18]. Because two peptides had the common sequence, -Pro-PhePro-, it was suggested that this sequence might contribute to the cathepsin B inhibitory activity. Thus, chemical synthesis of the peptide with the corresponding sequence was attempted. However, it was not possible to obtain Pro-PhePro tripeptide using the Fmoc method. In the case of [Val0]␤-casomrphin-7, it contained X-Pro-Phe-Pro-Y structure but did not show cathepsin B inhibitory activity.

Acknowledgments We thank Dr Yung-Hee Kho and Dr Myung-Chul Chung (KRIBB) for discussions and Dr Jin-Hee Han (Seoul National University) for a critical reading of the manuscript.

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