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Purification and characterization of human intestinal neutral ceramidase.

Ohlsson, Lena LU ; Palmberg, Carina; Duan, Rui-Dong LU ; Olsson, Maria LU ; Bergman, Tomas and Nilsson, Åke LU (2007) In Biochimie 89(8). p.950-960
Abstract
Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[C-14] sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase... (More)
Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[C-14] sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu2+ and Zn2+ ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut. (c) 2007 Elsevier Masson SAS. All rights reserved. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
cholesterol proteomics, intestine, neutral ceramidase, human, mass spectrometry
in
Biochimie
volume
89
issue
8
pages
950 - 960
publisher
Elsevier
external identifiers
  • wos:000248585100005
  • scopus:34447107198
ISSN
1638-6183
DOI
10.1016/j.biochi.2007.03.009
language
English
LU publication?
yes
id
8d49eda6-252c-4890-a5d5-0c4425d576c9 (old id 168467)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17475390&dopt=Abstract
date added to LUP
2007-07-10 11:08:20
date last changed
2017-01-01 04:55:17
@article{8d49eda6-252c-4890-a5d5-0c4425d576c9,
  abstract     = {Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[C-14] sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu2+ and Zn2+ ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut. (c) 2007 Elsevier Masson SAS. All rights reserved.},
  author       = {Ohlsson, Lena and Palmberg, Carina and Duan, Rui-Dong and Olsson, Maria and Bergman, Tomas and Nilsson, Åke},
  issn         = {1638-6183},
  keyword      = {cholesterol
proteomics,intestine,neutral ceramidase,human,mass spectrometry},
  language     = {eng},
  number       = {8},
  pages        = {950--960},
  publisher    = {Elsevier},
  series       = {Biochimie},
  title        = {Purification and characterization of human intestinal neutral ceramidase.},
  url          = {http://dx.doi.org/10.1016/j.biochi.2007.03.009},
  volume       = {89},
  year         = {2007},
}