Overexpression and mass spectrometry analysis of mature human acid ceramidase
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Heike Schulze
Abstract
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 GlcNAc2(Fuc)Man3 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.
©2007 by Walter de Gruyter Berlin New York
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- SARS-CoV accessory protein 7a directly interacts with human LFA-1
- Overexpression and mass spectrometry analysis of mature human acid ceramidase
- Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes
- Macrophage paraoxonase 2 (PON2) expression is upregulated by unesterified cholesterol through activation of the phosphatidylinositol 3-kinase (PI3K) pathway
- Acknowledgment
- Contents Biological Chemistry, Volume 388, 2007
- Author Index
- Subject Index
Articles in the same Issue
- FOXM1, a typical proliferation-associated transcription factor
- Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer
- A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences
- Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5)
- Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain
- SARS-CoV accessory protein 7a directly interacts with human LFA-1
- Overexpression and mass spectrometry analysis of mature human acid ceramidase
- Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes
- Macrophage paraoxonase 2 (PON2) expression is upregulated by unesterified cholesterol through activation of the phosphatidylinositol 3-kinase (PI3K) pathway
- Acknowledgment
- Contents Biological Chemistry, Volume 388, 2007
- Author Index
- Subject Index
