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J. Biol. Chem., Vol. 283, Issue 17, 11253-11259, April 25, 2008

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Autoproteolytic Cleavage and Activation of Human Acid Ceramidase^*

Nataly Shtraizent¹, Efrat Eliyahu, Jae-Ho Park, Xingxuan He, Ruth Shalgi, and Edward H. Schuchman²

From the Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029 and the Department of Cell and Developmental Biology, Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into and β subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of self-cleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis.

Received for publication, November 8, 2007 , and in revised form, January 24, 2008.

^* This research was supported by Grant R01 DK54830 from the National Institutes of Health. 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 Portions of this work were performed in partial fulfillment of the requirements of Tel Aviv University for a Ph.D. thesis.

² To whom correspondence should be addressed: Dept. of Genetics and Genomic Sciences, Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029. Tel.: 212-659-6711; Fax: 212-849-2447; E-mail: Edward.Schuchman{at}mssm.edu.

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