Journal of Biological Chemistry
Volume 282, Issue 42, 19 October 2007, Pages 30889-30900
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Glycobiology and Extracellular Matrices
Klotho-related Protein Is a Novel Cytosolic Neutral β-Glycosylceramidase*

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Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a β-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each kcat/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6Å resolution revealed that KLrP is a (β/α)8 TIM barrel, in which Glu165 and Glu373 at the carboxyl termini of β-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.

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The atomic coordinates and structure factors (code 2E9L and 2E9M) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This research was supported in part by CREST, Japan Science and Technology Agency (to M. I.), and Basic Research B 19380061 from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (to M. I.). This work was also supported in part by a grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y. K.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.