THE JOURNAL OF BIOLOGKX CHEMWIW Vol. 250, No. 21, Issue of November 10, pp. 8487-8795,1975 Printed in U.S.A. Role of Lysosomal Acid Lipase in the Metabolism of Plasma Low Density Lipoprotein OBSERVATIONS IN CULTURED FIBROBLASTS FROM A PATIENT WITH CHOLESTERYL ESTER STORAGE DISEASE* (Received for publication, May 22, 1975) JOSEPH L. GOLDSTEIN,+ SUZANNA E. DANA, JERRY R. FAUST, ARTHUR L. BEAUDET,~ AND MICHAEL S. BROWN lI From the Department of Internal Medicine, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 and the Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77025 The hydrolysis of cholesteryl esters contained in plasma low density lipoprotein was reduced in cultured fibroblasts derived from a patient with cholesteryl ester storage disease, an inborn error of metabolism in which lysosomal acid lipase activity is deficient. While these mutant cells showed a normal ability to bind low density lipoprotein at its high affinity cell surface receptor site, to take up the bound lipoprotein through endocytosis, and to hydrolyze the protein component of the lipoprotein in lysosomes, their defective lysosomal hydrolysis of the cholesteryl ester component of the lipoprotein led to the accumulation within the cell of unhydrolyzed cholesteryl esters, the fatty acid distribution of which resembled that of plasma lipoprotein. When the cholesteryl ester storage disease cells were incubated with low density lipoprotein, the reduced r&e of liberation of free cholesterol by these mutant cells was associated with a delay in the occurrence of two lipoprotein-mediated regulatory events, suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and activation of endogenous cholesteryl ester formation. In contrast to their defective hydrolysis of exogenously derived lipoprotein-bound cholesteryl esters, the cholesteryl ester storage disease cells showed a normal rate of hydrolysis of cholesteryl esters that had been synthesized within the cell. These data lend support to the concept that in cultured human fibroblasts cholesteryl esters entering the cell bound to low density lipoprotein are hydrolyzed within the lysosome and that one of the functions of this intracellular organelle is to supply the cell with free cholesterol. Cultured human fibroblasts possess a specific mechanism for the net uptake of cholesterol derived from plasma low density lipoprotein (LDL),’ the major cholesterol-carrying protein in human blood (l-6). Previous data suggest that this mechanism involves the following sequences of events: (a) LDL binds to a specific receptor on the cell surface (1, 2); (b) the surface- bound LDL becomes incorporated into endocytotic vesicles; (c) the internal endocytotic vesicles containing bound LDL fuse with lysosomes (4, 5); (d) the cholesteryl ester and protein components of LDL are hydrolyzed by lysosomal enzymes to products, including free cholesterol and amino acids (2, 4, 5); *This research was supported by grants from the American Heart Association (74983), the National Foundation-March of Dimes, and the National Institutes of Health (GM 19258 and HL 16024). $ Recipient of a Research Career Development Award (GM 70,277) from the United States Public Health Service. 5 Investigator of the Howard Hughes Medical Institute. IT Established Investigator of the American Heart Association. ‘The abbreviations used are: LDL, low density lipoprotein; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reduc- tase; LPDS, lipoprotein-deficient serum; [sH]CL-LDL, [3H]choles- teryl linoleate bound to low density lipoprotein. and (e) the liberated free cholesterol is transferred from lysosomes to cellular membranes (6). The resultant accumula- tion of cholesterol within the cell regulates two events in cellular cholesterol metabolism: (a) cholesteryl ester formation is stimulated through an activation of a membrane-bound fatty acyl-CoA:cholesterol acyltransferase (7, 8) and (b) cholesterol synthesis is reduced through a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (9). Formulation of this model of LDL metabolism in human fibroblasts has been facilitated by a comparison of the behavior of normal fibroblasts with that of mutant cells from subjects with the homozygous form of familial hypercholesterolemia, which lack the cell surface LDL receptor and thus fail to manifest all of the metabolic conse- quences of high affinity LDL binding (l-9). A critical aspect of the above model involves the role of the lysosome in hydrolyzing the cholesteryl ester and protein components of LDL so as to make free cholesterol available to the cell. This requirement for lysosomes has been inferred from studies in which lysosomal hydrolytic activity has been blocked in intact cells through the use of inhibitors such as 84878488 chloroquine (4, 5). Monolayers of fibroblasts treated with chloroquine are unable to hydrolyze the LDL taken up from the medium and as a consequence their cellular cholesteryl ester formation is not activated and HMG-CoA reductase activity is not suppressed (4). To investigate further the role of the lysosome in cellular LDL metabolism, the present studies were conducted utilizing mutant fibroblasts that are deficient in lysosomal acid lipase activity (10-12). Extracts of these mutant cells, which were derived from a subject with cholesteryl ester storage disease (12), manifest a 95% reduction in their ability to hydrolyze the cholesteryl esters contained in LDL. Comparison of LDL metabolism in monolayers of the mutant cells and normal cells has confirmed that: (a) lysosomal acid lipase plays a major role in hydrolyzing cholesteryl esters that enter the cell bound to LDL and (b) the