MODEL SV40-TRANSFORMED FIBROBLAST LINES FOR METABOLIC STUDIES OF HUMAN PROSAPOSIN AND ACID CERAMIDASE DEFICIENCIES

Skin fibroblasts from patients with Farber disease (acid ceramidase de ficiency) 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 transform ed cell lines conserved their original metabolic defects, and in parti cular they were free of detectable immunoreactivity when using anti-sa posin B and anti-saposin C antisera. Ultrastructurally, the cells cont ained heterogeneous lysosomal storage products. As found for their par ental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylcerami dase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radio active glucosylceramide or low density lipoprotein-associated radiolab elled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide stor age in prosaposin-deficient cells was also demonstrated by ceramide ma ss determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of aci d ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is r equired for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophy siological studies on human prosaposin deficiency. (C) 1997 Elsevier S cience B.V.