Negative selectivity and the evolution of protease cascades: the specificity of plasmin for peptide and protein substrates Original Research Article

Abstract Background: Understanding the networks of selective proteolysis that regulate complex biological systems requires an appreciation of the molecular mechanisms used to maintain substrate specificity. Human plasmin, a serine protease that promotes the dissolution of blood clots and is essential in maintaining normal hemostasis, is usually described as having broad substrate specificity. Recent evidence that plasmin also plays a key role in a variety of other important biological and pathological processes, however, has suggested that this description might need to be re-evaluated. Results: We used substrate phage display to elucidate optimal subsite occupancy for substrates of plasmin. We identified a peptide substrate that is cleaved 710,000-fold more efficiently by plasmin than a peptide containing the activation sequence of plasminogen. Plasmin achieves this unexpected, large differential activity even though both target sequences possess an arginine residue in the P1 position. We also demonstrate that proteolysis by plasmin can be targeted to an engineered protein substrate and that introduction of substrate sequences identified by phage display into plasminogen increases plasmin-mediated cleavage of the mutant 2000-fold. Conclusions: The specificity of plasmin is more tightly controlled than previously recognized; interactions with substrates at all subsites between S4 and S2′ contribute to catalysis. Furthermore, in contrast to most enzymes that exhibit positive selectivity for substrate, the evolution of substrate specificity by plasmin has apparently been dominated by a strong negative selection against development of autoactivation activity. This ‘negative selectivity’ avoids short-circuiting regulation of the fibrinolytic system and other important biological processes, and might be an important general mechanism for controlling protease cascades. Article Outline