Klotho-related protein is a novel cytosolic neutral β- glycosylceramidase

Yasuhiro Hayashi, Nozomu Okino, Yoshimitsu Kakuta, Toshihide Shikanai, Motohiro Tani, Hisashi Narimatsu, Makoto Ito

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

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 k cat/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6 Å resolution revealed that KLrP is a (β/α)8 TIM barrel, in which Glu165 and Glu 373 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.

Original languageEnglish
Pages (from-to)30889-30900
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number42
DOIs
Publication statusPublished - Oct 19 2007

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Lactase-Phlorizin Hydrolase
Glucosylceramides
Proteins
Enzymes
Substrates
Glucosidases
Glucose
Derivatives
Palmitic Acid
Nucleophiles
Zebrafish
Oleic Acid
klotho protein
Recombinant Proteins
Cytosol
Small Interfering RNA
Fibroblasts
Cats
Embryonic Structures
Rats

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Klotho-related protein is a novel cytosolic neutral β- glycosylceramidase. / Hayashi, Yasuhiro; Okino, Nozomu; Kakuta, Yoshimitsu; Shikanai, Toshihide; Tani, Motohiro; Narimatsu, Hisashi; Ito, Makoto.

In: Journal of Biological Chemistry, Vol. 282, No. 42, 19.10.2007, p. 30889-30900.

Research output: Contribution to journalArticle

Hayashi, Yasuhiro ; Okino, Nozomu ; Kakuta, Yoshimitsu ; Shikanai, Toshihide ; Tani, Motohiro ; Narimatsu, Hisashi ; Ito, Makoto. / Klotho-related protein is a novel cytosolic neutral β- glycosylceramidase. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 42. pp. 30889-30900.
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abstract = "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 k cat/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6 {\AA} resolution revealed that KLrP is a (β/α)8 TIM barrel, in which Glu165 and Glu 373 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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AB - 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 k cat/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6 Å resolution revealed that KLrP is a (β/α)8 TIM barrel, in which Glu165 and Glu 373 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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