title: Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.
abstract: Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.
authors.affiliation: Laboratoire de Biochimie Maladies Métaboliques, INSERM U 466, Institut Louis Bugnard, Toulouse, France.
authors.forename: M
authors.initials: M
authors.lastname: Chatelut
authors.forename: K
authors.initials: K
authors.lastname: Harzer
authors.forename: H
authors.initials: H
authors.lastname: Christomanou
authors.forename: J
authors.initials: J
authors.lastname: Feunteun
authors.forename: M T
authors.initials: MT
authors.lastname: Pieraggi
authors.forename: B C
authors.initials: BC
authors.lastname: Paton
authors.forename: Y
authors.initials: Y
authors.lastname: Kishimoto
authors.forename: J S
authors.initials: JS
authors.lastname: O'Brien
authors.forename: J P
authors.initials: JP
authors.lastname: Basile
authors.forename: J C
authors.initials: JC
authors.lastname: Thiers
authors.forename: R
authors.initials: R
authors.lastname: Salvayre
authors.forename: T
authors.initials: T
authors.lastname: Levade
mesh_terms: D055573:Acid Ceramidase; D000581:Amidohydrolases / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics*; D055572:Ceramidases; D005333:Fetus; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D006023:Glycoproteins / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D016464:Lysosomal Storage Diseases / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D049231:Saposins; D013539:Simian virus 40 / Q000235:genetics*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D006023:Glycoproteins; C483345:PSAP protein, human; D011498:Protein Precursors; D049231:Saposins; D000581:Amidohydrolases; C526693:ASAH1 protein, human; D055573:Acid Ceramidase; D055572:Ceramidases