Solid-phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1-93 by tetanus toxin L chain.
A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain). This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis. We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not. A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide. A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family. Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency. Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration. Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations. Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain.
Cornille
F
F
Département de Pharmacochimie Moléculaire et Structurale, CNRS URA D1500-INSERM U266, Université Paris V, France.
Goudreau
N
N
Ficheux
D
D
Niemann
H
H
Roques
B P
BP
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000187:drug effects; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000187:drug effects; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D050683:R-SNARE Proteins; D051381:Rats; D011994:Recombinant Proteins; D013744:Tetanus Toxin / Q000494:pharmacology*
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D013744:Tetanus Toxin