RESUMO
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.
Assuntos
Galactosilgalactosilglucosilceramidase/biossíntese , Galactosilgalactosilglucosilceramidase/química , Animais , Baculoviridae/química , Baculoviridae/genética , Western Blotting , Linhagem Celular , Meios de Cultura , Cisteína/química , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Galactosilgalactosilglucosilceramidase/farmacocinética , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Insetos , Cinética , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Espectrometria de Massas , Oligossacarídeos/química , Oxirredução , Peptídeos/química , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/químicaRESUMO
Among the many processes regulating cell death, ceramide signaling is a vital component. We previously determined that acid ceramidase (AC) is upregulated in 60% of primary prostate cancer (PCa) tissues, suggesting that AC may play a role in tumor development. In order to determine the significance of AC elevation, stable clones of DU145 cells with AC overexpression (AC-EGFP) were generated. Compared to controls (EGFP), AC-EGFP cells exhibited enhanced cell proliferation and migration. Subcutaneous injection of AC-EGFP cells into Nu/Nu mice resulted in larger tumor volumes compared to EGFP controls. Moreover, using the MTS viability assay, AC-EGFP cells were more resistant to cell death induced by doxorubicin, cisplatin, etoposide, gemcitabine or C6-ceramide. Conversely, knock down of AC using siRNA, sensitized AC-EGFP cells to these drugs. In addition, mass spectroscopic analysis of sphingolipids indicated that long chain ceramide levels were decreased in AC-EGFP cells treated with either doxorubicin or etoposide. In conclusion, this study implicates AC as a critical regulator of PCa progression by affecting not only tumor cell proliferation and migration but also responses to drug therapy, suggesting AC as a potential therapeutic target in advanced PCa.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Galactosilgalactosilglucosilceramidase/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Ceramidas/metabolismo , Progressão da Doença , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
Protein glycosylation modifies the processing of several key proteins involved in the molecular pathogenesis of Alzheimer's disease (AD). Aberrant glycosylation of tau and down-regulation of sialyltransferase in AD brain suggest a possible dysregulation of protein glycosylation that may play a role in AD. We therefore isolated major glycoproteins from AD brain by using lectin-affinity chromatographies and ion-exchange chromatography and further separated them using SDS-polyacylamide gel electrophoresis. Mass spectrometry analysis of 11 isolated glycoproteins led to their identification as: neuronal cell adhesion molecule, beta-globin, IgM heavy chain VH1 region precursor, contactin precursor, dipeptidylpeptidase VI, CD81 partner 3, prenylcysteine lyase, adipocyte plasma-associated protein, acid ceramidase and two novel proteins. We found that the level and activity of acid ceramidase (AC), one of the major identified human brain glycoproteins, were significantly elevated in AD brain. Immunohistochemical staining indicated that AC was located mainly in the cell bodies of neurons and colocalized with neurofibrillary tangles. Our findings suggest that AC might play a role in controlling neuronal apoptosis and that AC-mediated signalling pathways might be involved in the molecular mechanism of AD.
