Purification and characterization of human intestinal neutral ceramidase.
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-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa 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 Cu(2+) and Zn(2+) 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.
Ohlsson
Lena
L
Gastroenterology and Nutrition Laboratory, Biomedical Centre B11, Lund University, Tornavagen 10, S-22184 Lund, Sweden. lena.ohlsson@med.lu.se
Palmberg
Carina
C
Duan
Rui-Dong
RD
Olsson
Maria
M
Bergman
Tomas
T
Nilsson
Ake
A
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004386:Duodenum; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007422:Intestines / Q000201:enzymology*; D008969:Molecular Sequence Data; D055576:Neutral Ceramidase; D014407:Tumor Cells, Cultured
D002518:Ceramides; D002784:Cholesterol; D000581:Amidohydrolases; C526744:ASAH2 protein, human; C526747:Asah2 protein, mouse; D055572:Ceramidases; D055576:Neutral Ceramidase