Journal of Protein Chemistry

, Volume 14, Issue 8, pp 645–653

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Authors

  • Gilles Lalmanach
    • Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334Université François Rabelais, Faculté de Médecine de Tours
  • Carole Serveau
    • Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334Université François Rabelais, Faculté de Médecine de Tours
  • Michèle Brillard-Bourdet
    • Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334Université François Rabelais, Faculté de Médecine de Tours
  • Jair R. Chagas
    • Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334Université François Rabelais, Faculté de Médecine de Tours
    • Departamento de BiofisicaINFAR, Escola Paulista de Medicina
  • Roger Mayer
    • Laboratoire de Biochimie des Glycoconjugués et des Lectines EndogènesCentre de Biophysique Moléculaire, CNRS
  • Luiz Juliano
    • Departamento de BiofisicaINFAR, Escola Paulista de Medicina
  • Francis Gauthier
    • Laboratoire d'Enzymologie et Chimie des Protéines, CNRS-URA 1334Université François Rabelais, Faculté de Médecine de Tours
Article

DOI: 10.1007/BF01886903

Cite this article as:
Lalmanach, G., Serveau, C., Brillard-Bourdet, M. et al. J Protein Chem (1995) 14: 645. doi:10.1007/BF01886903

Abstract

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzedAbz-QVVAGA-EDDnp andAbz-LVGGA-EDDnp at about the same rate, with specificity constants in the 107M−1 sec−1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 105 M−1 sec−1 range due to lowerkcat values, with theKm's being identical to those with papain. OnlyAbz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN2 was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k+0= 1,100,000 M−1 sec−1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.

Key words

Cysteine proteinasecystatindiazomethylketonepeptidyl fluorogenic substrate
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Copyright information

© Plenum Publishing Corporation 1995