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Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock¹

Stacey L. Mueller-Ortiz^*, Dachun Wang^*, John E. Morales^*, Li Li, Jui-Yoa Chang^, and Rick A. Wetsel^2,*,

^* Research Center for Immunology and Autoimmune Diseases and Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX 77030; and Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX 77030

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1^–/– mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1^–/–/C5aR^–/– but not in CPN1^–/–/C3aR^–/– mice. Moreover, CPN1^–/– mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1^–/– mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research was supported by National Institutes of Health Public Service Grants R01 AI025011 and R01 HL074333 (to R.A.W.). J.Y.C. acknowledges the support of the Robert Welch Foundation.

² Address correspondence and reprint requests to Dr. Rick A. Wetsel, University of Texas Health Science Center at Houston, Brown Foundation Institute of Molecular Medicine, 1825 Pressler Street, Houston, TX 77030. E-mail address: Rick.A.Wetsel{at}uth.tmc.edu

³ Abbreviations used in this paper: CPN, carboxypeptidase N; SDF-1, stromal-derived factor-1; WT, wild type; ES cell, embryonic stem cell; FA, furylacryloyl; C3aR, C3a receptor; C5aR, C5a receptor; CVF, cobra venom factor; CPU, carboxypeptidase U; CPR, carboxypeptidase R; TAFI, thrombin activatable fibrinolysis inhibitor.