title: Posttranslational regulation of acid sphingomyelinase in niemann-pick type C1 fibroblasts and free cholesterol-enriched chinese hamster ovary cells.
abstract: Niemann-Pick type C disease is characterized by the accumulation of cholesterol and other lipids within the lysosomal compartment, a process that is often accompanied by a reduction in acid sphingomyelinase activity. These studies demonstrate that a CHO cell mutant (CT-60), which accumulates lysosomal cholesterol because of a defective NP-C1 protein, has approximately 5-10% of the acid sphingomyelinase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. The cholesterol-induced reduction in acid sphingomyelinase activity can be reproduced in CHO-K1 cells by incubation in the presence of low density lipoprotein (LDL) and progesterone, which impairs the normal egress of LDL-derived cholesterol from the lysosomal compartment. Kinetic analysis of sphingomyelin hydrolysis in cell extracts suggests that the CT60 cells have a reduced amount of functional acid sphingomyelinase as indicated by a 10-fold reduction in the apparent V(max). Western blot analysis using antibodies generated to synthetic peptides corresponding to segments within the carboxyl-terminal region of acid sphingomyelinase demonstrate that both the CT60 and the LDL/progesterone-treated CHO-K1 cells possess near normal levels of acid sphingomyelinase protein. Likewise, Niemann-Pick type C fibroblasts also displayed normal acid sphingomyelinase protein but negligible levels of acid sphingomyelinase activity. These data suggest that cholesterol-induced inhibition is a posttranslational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in acid sphingomyelinase protein trafficking and maturation.
authors.affiliation: Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Reagan
authors.forename: M L
authors.initials: ML
authors.lastname: Hubbert
authors.forename: G S
authors.initials: GS
authors.lastname: Shelness
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000201:enzymology; D008969:Molecular Sequence Data; D009542:Niemann-Pick Diseases / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics / Q000378:metabolism*
chemical_list: D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase