Document Detail


Purification and characterization of recombinant, human acid ceramidase. Catalytic reactions and interactions with acid sphingomyelinase.
MedLine Citation:
PMID:  12815059     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Xingxuan He; Nozomu Okino; Rajwinder Dhami; Arie Dagan; Shimon Gatt; Heike Schulze; Konrad Sandhoff; Edward H Schuchman
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.     Date:  2003-06-18
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  278     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2003 Aug 
Date Detail:
Created Date:  2003-08-25     Completed Date:  2003-10-02     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  32978-86     Citation Subset:  IM    
Affiliation:
Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA.
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MeSH Terms
Descriptor/Qualifier:
Adenoviridae / genetics
Amidohydrolases / pharmacology
Animals
Blotting, Northern
CHO Cells
Catalysis
Chromatography, Gel
Concanavalin A / chemistry
Cricetinae
DNA, Complementary / metabolism
Electrophoresis, Polyacrylamide Gel
Fibroblasts / metabolism
Galactosylgalactosylglucosylceramidase / chemistry*,  isolation & purification*
Glycosylation
Hexosaminidases / pharmacology
Humans
Hydrogen-Ion Concentration
Kinetics
Lipids
Macrophages / metabolism
Mice
Mice, Knockout
Neuraminidase / pharmacology
Oligosaccharides / chemistry
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Precipitin Tests
Protein Structure, Tertiary
RNA / metabolism
Recombinant Proteins / chemistry,  isolation & purification,  metabolism
Sepharose / chemistry
Skin / cytology
Sphingomyelin Phosphodiesterase / chemistry*
Grant Support
ID/Acronym/Agency:
5R03 TW 01372/TW/FIC NIH HHS; R01 DK54830/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Complementary; 0/Lipids; 0/Oligosaccharides; 0/Recombinant Proteins; 11028-71-0/Concanavalin A; 63231-63-0/RNA; 9012-36-6/Sepharose; EC 3.1.4.-/acid sphingomyelinase-1; EC 3.1.4.12/Sphingomyelin Phosphodiesterase; EC 3.2.1.-/Hexosaminidases; EC 3.2.1.18/Neuraminidase; EC 3.2.1.47/Galactosylgalactosylglucosylceramidase; EC 3.5.-/Amidohydrolases; EC 3.5.1.52/Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

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