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Stability of mutant serpin/furin complexes: Dependence on pH and regulation at the deacylation step

Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4

(RECEIVED April 28, 2004; FINAL REVISION October 6, 2004; ACCEPTED October 6, 2004)

Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of ₁-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and ₁-antichymotrypsin (ACT) mutants by modifying the P₇–P₁ region of their RSLs. The biochemical properties of these variants were assessed by evaluating their propensity to establish SDS-resistant complexes with furin in a variety of conditions (pH 6.0–9.0) and by measuring their association rate constants. The effect of pH during the initial steps of complex formation was minimal, suggesting that the acylation step is not rate-limiting. The decrease in stoichiometry of inhibition (SI) values observed in AT variants at high pHs was a result of the reduced pH-dependent deacylation rate, which is rate-limiting in this mechanism and which suggests increased complex stability. Conversely, the SI values for ACT mutants had a tendency to be lower at acidic pH. Transiently transfecting HEK293 cells with these mutants abolished processing of the pro-von Willebrand factor precursor but, interestingly, only the ACT variants were secreted in the media as uncleaved forms. Our results suggest that reengineering the reactive site loops of serpins to accommodate and target furin or other serine proteases must take into account the intrinsic physicochemical properties of the serpin.

Keywords: furin; serpin mechanism; convertases; serine proteases; ₁-antitrypsin; ₁-antichymotrypsin; protease inhibitors

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04843305.

Reprint requests to: Richard Leduc, Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4; e-mail: Richard.Leduc{at}USherbrooke.ca; fax: (819) 564-5400.

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