Overexpression and mass spectrometry analysis of mature human acid ceramidase.
Human acid ceramidase catalyzes the last step of lysosomal sphingolipid degradation, the hydrolysis of ceramide to sphingosine and free fatty acid. Inherited deficiency of acid ceramidase activity leads to Farber disease (Farber lipogranulomatosis). In this study, we describe the overexpression and processing of recombinant human acid ceramidase in Sf21 insect cells, its purification and characterization. Infection of Sf21 cells with a recombinant baculovirus encoding acid ceramidase precursor led to a mixture of human acid ceramidase precursor and mature enzyme secreted into the medium. Acidification of the cell culture supernatant to pH 4.2-4.3 triggered the processing of the precursor and resulted in a homogeneous sample of mature human acid ceramidase. The enzyme was purified by chromatography on Concanavalin A Sepharose and Octyl Sepharose yielding 1 mg purified protein per liter of supernatant. The recombinant enzyme was deglycosylated with peptide N-glycosidase F and the main component of the released oligosaccharides was identified as GlcNAc(2)(Fuc)Man(3) by electrospray mass spectrometry. Apparently, five of the six potential N-glycosylation sites were used. Tryptic digestion of the functional recombinant enzyme and matrix-assisted laser desorption/ionization time-of-flight- and electrospray ionization-mass spectrometry analysis of the resulting peptides indicated disulfide bridges between C10-C319, C122-C271 and C367-C371.
Schulze
Heike
H
LIMES Membrane Biology and Lipid Biochemistry Unit, University Bonn, c/o Kekulé-Institute, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.
Schepers
Ute
U
Sandhoff
Konrad
K
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003470:Culture Media; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D002517:Galactosylgalactosylglucosylceramidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000493:pharmacokinetics; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D016464:Lysosomal Storage Diseases / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D003470:Culture Media; D004220:Disulfides; D009844:Oligosaccharides; D010455:Peptides; D011994:Recombinant Proteins; D002517:Galactosylgalactosylglucosylceramidase; D014357:Trypsin; D003545:Cysteine