Expression and Mutagenesis of the Novel Serpin Endopin 2 Demonstrates a Requirement for Cysteine-374 for Dithiothreitol-Sensitive Inhibition of Elastase

• Published in: Biochemistry (Easton) 2000-08, Vol.39 (30), p.8944-8952
• Main Authors: Hwang, Shin-Rong, Steineckert, Brent, Hook, Vivian Y. H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Cysteine - metabolism; Dithiothreitol - pharmacology; Drug Synergism; Glutathione - pharmacology; Humans; Maleimides - pharmacology; Molecular Sequence Data; Mutagenesis, Site-Directed; Pancreatic Elastase - antagonists & inhibitors; Protease Inhibitors - pharmacology; Protein Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Reducing Agents - pharmacology; Sequence Homology, Amino Acid; Serpins - biosynthesis; Serpins - genetics; Serpins - pharmacology; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9929655

The primary sequence of the serpin endopin 2 predicts a reactive site loop (RSL) region that possesses high homology to bovine elastase inhibitor, suggesting inhibition of elastase. Moreover, endopin 2 possesses two cysteine residues that implicate roles for reduced Cys residue(s) for inhibitory activity. To test these predicted properties, mutagenesis and chemical modification of recombinant endopin 2 were performed to examine the influence of dithiothreitol (DTT), a reducing agent, on endopin 2 activity. Endopin 2 inhibited elastase in a DTT-dependent manner, with enhanced inhibition in the presence of DTT. The stoichiometry of inhibition in the presence of DTT occurred at a molar ratio of endopin 2 to elastase of 8/1, resulting in complete 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-directed mutagenesis of endopin 2, with substitution of Ala for Cys-232 or Cys-374, demonstrated that Cys-374 (but not Cys-232) was required for the DTT-sensitive nature of endopin 2. Chemical modification of Cys-374 by bis(maleimido)ethane also reduced inhibitory activity. Modified electrophoretic mobilities of mutant endopin 2 suggested the presence of intramolecular disulfide bonds; in addition, chemical modification suggested that Cys-374 influences the electrophoretic and conformational properties of endopin 2. Moreover, the reducing agent glutathione enhanced endopin 2 activity, suggesting that glutathione can function as an endogenous reducing agent for endopin 2 in vivo. These findings demonstrate the importance of Cys-374 for DTT-sensitive inhibition of elastase by endopin 2.