title: Purification and characterization of recombinant, human acid ceramidase. Catalytic reactions and interactions with acid sphingomyelinase.
abstract: Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(alpha) and 40 (beta)-kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly "high mannose" type oligosaccharides and that two distinct beta-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the approximately 2-4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.
authors.affiliation: Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA.
authors.forename: Xingxuan
authors.initials: X
authors.lastname: He
authors.forename: Nozomu
authors.initials: N
authors.lastname: Okino
authors.forename: Rajwinder
authors.initials: R
authors.lastname: Dhami
authors.forename: Arie
authors.initials: A
authors.lastname: Dagan
authors.forename: Shimon
authors.initials: S
authors.lastname: Gatt
authors.forename: Heike
authors.initials: H
authors.lastname: Schulze
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
authors.forename: Edward H
authors.initials: EH
authors.lastname: Schuchman
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002384:Catalysis; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000737:chemistry; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D002517:Galactosylgalactosylglucosylceramidase / Q000737:chemistry* / Q000302:isolation & purification*; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009439:Neuraminidase / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D012867:Skin / Q000166:cytology; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D008055:Lipids; D009844:Oligosaccharides; D011994:Recombinant Proteins; D003208:Concanavalin A; D012313:RNA; D012685:Sepharose; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase; D006596:Hexosaminidases; D009439:Neuraminidase; D002517:Galactosylgalactosylglucosylceramidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase