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J. Biol. Chem., Vol. 279, Issue 11, 9777-9784, March 12, 2004

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Klotho Is a Novel -Glucuronidase Capable of Hydrolyzing Steroid -Glucuronides^*

Osamu Tohyama, Akihiro Imura¶, Akiko Iwano, Jean-Noël Freund||, Bernard Henrissat**, Toshihiko Fujimori, and Yo-ichi Nabeshima

From the Department of Pathology and Tumor Biology, Graduate School of Medicine, and the ^¶Horizontal Medical Research Organization, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, the Core Research for Evolutional Science and Technology, Kawaguchi 332-0012, Japan, ^||INSERM, Unité 381, 3 avenue Molière, Strasbourg 67200, France, and ^**Architecture et Fonction des Macromolécules Biologiques, UMR6098, CNRS, and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, Marseille 13402, France

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) -glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl -D-glucuronide in contrast to bovine liver -glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of -glucuronidase. A number of natural -glucuronides were screened by competitive inhibition for KLFc -glucuronidase. We found that steroid -glucuronides such as -estradiol 3--D-glucuronide, estrone 3--D-glucuronide, and estriol 3-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel -glucuronidase and that steroid -glucuronides are potential candidates for the natural substrate(s) of Klotho.

Received for publication, November 12, 2003 , and in revised form, December 22, 2003.

^* This work was supported by grants from the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim Applied Bioscience. 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.

To whom correspondence should be addressed: Dept. of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4422; Fax: 81-75-753-4676; E-mail: nabemr{at}lmls.med.kyoto-u.ac.jp.

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