Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 dithiothreitol-sensitive inhibition of elastase

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, sug gesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory act ivity. To test these predicted properties, mutagenesis and chemical modific ation of recombinant endopin 2 were performed to examine the influence of d ithiothreitol (DT), a reducing agent, on endopin 2 activity. Endopin 2 inhi bited elastase in a DTT-dependent manner, with enhanced inhibition in the p resence of DTT. The stoichiometry of inhibition in the presence of DTT occu rred at a molar ratio of endopin 2 to elastase of 8/1, resulting in complet e inhibition of elastase. However, a higher molar ratio (25/1) was required in the absence of DTT. DTT enhanced the formation of SDS-stable complexes of endopin 2 and elastase, a characteristic property of serpins. Site-direc ted mutagenesis of endopin 2, with substitution of Ala for Cys-232 or Cys-3 74, demonstrated that Cys-374 (but not Cys-232) was required for the DTT-se nsitive nature of endopin 2. Chemical modification of Cys-374 by bis(maleim ido)ethane also reduced inhibitory activity. Modified electrophoretic mobil ities of mutant endopin 2 suggested the presence of intramolecular disulfid e bonds; in addition, chemical modification suggested that Cys-374 influenc es the electrophoretic and conformational properties of endopin 2. Moreover , the reducing agent glutathione enhanced endopin 2 activity, suggesting th at glutathione can function as an endogenous reducing agent for endopin 2 i n vivo. These findings demonstrate the importance of Cys-374 for DTT-sensit ive inhibition of elastase by endopin 2.