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2004 | 51 | 1 | 81-92
Article title

Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B.

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Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P5-P1 part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'1-P'2 dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'1-S'2 sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P4 is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P2 causes an increase of the substrate preference towards this activity.
Physical description
  • Faculty of Chemistry, University of Gdańsk, J. Sobieskiego 18, 80-952 Gdańsk, Poland
  • Faculty of Chemistry, University of Gdańsk, J. Sobieskiego 18, 80-952 Gdańsk, Poland
  • Faculty of Chemistry, University of Gdańsk, J. Sobieskiego 18, 80-952 Gdańsk, Poland
  • Faculty of Chemistry, University of Gdańsk, J. Sobieskiego 18, 80-952 Gdańsk, Poland
  • Faculty of Chemistry, University of Gdańsk, J. Sobieskiego 18, 80-952 Gdańsk, Poland
  • Barlos K, Chatzi O, Gatos D, Stavropoulos G. (1991) 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int J Pept Protein Res.; 37: 513-20.
  • Barrett AJ, Kembahavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada K. (1982) L-Trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J.; 201: 189-98.
  • Berdowska I, Siewiński M. (2000) Rola katepsyn cysteinowych oraz ich inhibitorńw w procesach fizjologicznych i nowotworowych. Post Biochem.; 46: 73-84 (in Polish).
  • Blumberg S, Schechter I, Berger A. (1970) The purification of papain by affinity chromatography. Eur J Biochem.; 15: 97-102.
  • Bodanszky M, Bodanszky A. (1984) The Practice of Peptide Chemistry, pp 143. Springer-Verlag, New York.
  • Dörner B, White P. (2000) Synthesis Notes. In Novabiochem Catalog. (a) pp P33, (b) pp S15. CN Bioscience, Inc., An Affiliate of Merck KgoA, Darmstadt, Germany.
  • Grubb A. (2000) Cystatin C - properties and use as diagnostic marker. Adv Clin Chem.; 35: 63-99.
  • Grzonka Z, Jankowska E, Kasprzykowski F, Kasprzykowska R, Łankiewicz L, Wiczk W, Wieczerzak E, Ciarkowski J, Drabik P, Janowski R, Kozak M, Jaskólski M, Grubb A. (2001) Structural studies of cysteine proteases and their inhibitors. Acta Biochim Pol.; 48: 1-20.
  • Henskens YMC, Veerman ECI, Amerongen AV. (1996) Cystatins in health and disease. Biol Chem Hoppe-Seyler.; 377: 71-86.
  • Hilaire Ph, Willert M, Juliano M, Juliano L, Meldal M. (1999) Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity. J Comb Chem.; 1: 509-23.
  • Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort J. (1997) Role of the occluding loop in cathepsin B activity. J Biol Chem.; 272: 1197-202.
  • Jutras I, Reudelhuber TL. (1999) Prorenin processing by cathepsin B in vitro and in transfected cells. FEBS Lett.; 443: 48-52.
  • Katunuma N, Matsunaga Y, Matsui A, Kakegawa H, Endo K, Inubushi T, Saibara T, Ohba Y, Kakiuchi T. (1998) Novel physiological functions of cathepsins B and L on antigen processing and osteoclastic bone resorption. Adv Enzyme Regul.; 38: 235-51.
  • Keppler D, Sameni M, Moin K, Mikkelsen T, Diglio CA, Sloane BF. (1996) Tumor progression and angiogenesis. Biochem Cell Biol.; 74: 799-810.
  • Lalmanach G, Serveau C, Brillard-Bourdet M, Chagas J, Mayer R, Juliano L, Gauthier F. (1995) Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases. J Protein Chem.; 14: 645-53.
  • Liberek B, Kasprzykowska R. (1987) Oxidation products of L-lysine derivatives as starting materials for peptide synthesis and for the preparation of homoglutamic acid, homoglutamine and homoisoglutamine. Int J Peptide Protein Res.; 30: 522-32.
  • Mort JS, Buttle DJ. (1997) Cathepsin B. Int J Biochem Cell Biol.; 29: 715-20.
  • Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, Popovic T, Turk V, Towatari T, Katunuma N, Bode W. (1991) The refined 2.15 Å X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J.; 10: 2321-30.
  • Nägler D, Storer A, Portaro F. Carmona E, Juliano L, Menard R. (1997) Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts. Biochemistry.; 36: 12608-15.
  • Nägler D, Tam W, Storer A, Krupa J, Mort J, Ménard R. (1999) Interdependency of sequence and positional specificities for cysteine proteases of the papain family. Biochemistry.; 38: 4868-74.
  • Nawrocka M, Tokmina M, Wiczk W, Stachowiak K. (2001) Intramolecularly quenched fluorogenic peptide substrates for cathepsin B. In Peptides 2000, Proc. of 26th European Peptide Symposium. Martinez J, Fehrentz JA, eds, pp 566-7. EDK, Paris.
  • Neises B, Steglick W. (1978) Simple methods for the esterification of carboxylic acids. Angew Chem Int Ed Engl.; 17: 523-54.
  • Nycander M, Estrada S, Mort JS, Abrahamson M, Björk I. (1998) Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop. FEBS Lett.; 422: 61-4.
  • Otto HH, Schirmeister T. (1997) Cysteine proteases and their inhibitors. Chem Rev.; 97: 133-72.
  • Pavlova A, Krupa J, Mort J, Abrahamson M, Bjork I. (2000) Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration by release of loop anchoring through mutation of His110. FEBS Lett.; 487: 156-60.
  • Pohl J, Davinic S, Blaha I, Strop P, Kostka V. (1987) Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B. Anal Biochem.; 95: 228-35.
  • Portlando F, Santos A, Cezari M, Juliano M, Juliano L, Carmona E. (2000) Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates. Biochem J.; 347: 123-9.
  • Quraishi O, Nägler D, Fox T, Sivaraman J, Cygler M, Mort J, Storer A. (1999) The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide. Biochemistry.; 38: 5017-23.
  • Stachowiak K, Tokmina M, Kasprzykowska R, Kasprzykowski F, Wiczk W. (2001) Inhibition of cathepsin B by aldehyde derivatives of cystatins N-terminal binding fragments. In Programme and abstracts of IPS International Conference on Protease Inhibitors, p 185. Munich, Germany.
  • Szabelski M, Stachowiak K, Wiczk W. (2001) Influence of Me2SO and incubation time on papain activity studied using fluorogenic substrates. Acta Biochim Pol.; 48: 995-1002.
  • Schechter I, Berger A. (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun.; 27: 157-162.
  • Turk D, Guncar G, Podobnik M, Turk B. (1998) Revised definition of substrate binding sites of papain-like cysteine proteases. Biol Chem.; 379: 137-47.
  • Vogel A. (1984) Textbook of Practical Organic Chemistry. pp 611-612, WNT, Warsaw (in Polish).
  • Warwas M, Haczyńska H. (1998) Rola cystatyn w procesie nowotworowym i jego diagnostyce. Postepy Hig Med Dosw.; 52: 515-26 (in Polish).
  • Wenschuh H, Beyermann M, Winter R, Bienert M, Ionescu D, Carpino LA. (1996) Fmoc amino acid fluorides in peptide synthesis - extension of the method to extremely hindered amino acids. Tetrahedron Lett.; 37: 5483-5.
  • Wieczerzak E, Drabik P, Łankiewicz L, Ołdziej S, Grzonka Z, Abrahamson M, Grubb A, Bromme D. (2002) Azapeptides structurally based upon inhibitory sites of cystatins as potent and selective inhibitors of cysteine proteases. J Med Chem.; 45: 4202-11.
  • Wijffels GL. (1998) Cathepsin B. In Handbook of Proteolytic Enzymes. Barrett AJ, Rawlings ND, Woessne, JF. eds, pp 609-17. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto.
  • Yamamoto A, Tomoo K, Hara T, Murata M, Kitamura K, Ishida T. (2000) Substrate specificity of bovine cathepsin B and its inhibition by CA074, based on crystal structure refinement of the complex. J Biochem.; 127: 635-43.
  • Zore I, Krasovec M, Cimerman N, Kuhelj R, Werle B, Nielsen H, Brunner N, Kos J. (2001) Cathepsin B/cystatin C complex levels in sera from patients with lung and colorectal cancer. Biol Chem.; 382: 805-10.
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