Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked byAPMA ((4-aminophenyl)mercuric acetate)

Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matr ix metalloproteinases (MMPs). The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological condition s. SL-1 like the other MMPs can be activated in vitro by the stepwise remov al of the propeptide that contains a single unpaired cysteine which coordin ates the active site zinc. Other residues in the propeptide also play a rol e in maintaining the latency of the enzymes. Deletion mutants and single-si te amino acid replacements within the propeptide of a carboxyl-terminally t runcated stromelysin-1 (mini-SL-1) were constructed and expressed in Escher ichia coli to further examine what amino acids within the propeptide of SL- 1 are important for maintaining latency. While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower M- r in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E. coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide. Mutant Delta 63 as w ell as other mutants in which most of the propeptide had been deleted no lo nger responded to exposure to the organomercurial APMA by accelerated autol ytic processing. Rather, APMA inhibited the autolytic processing in these m utants, further confirming the complexity of the action of this organomercu rial in the activation of pro-MMPs.