Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cysteine-374 for 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, 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.
Departments of Medicine and Neuroscience, University of California, San Diego, La Jolla, California 92093, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology*; D004357:Drug Synergism; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D019163:Reducing Agents / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D008301:Maleimides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D019163:Reducing Agents; D015843:Serpins; C412320:bis(maleimido)ethane; C412321:endopin 2; D010196:Pancreatic Elastase; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol