THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 260, No. 28, Issue of December 5, pp. 15186-15193,1985 Printed in U.S.A.

0 1985 by The American Society of Biological Chemists, h e .

Human Lysosomal Acid Lipase/Cholesteryl Ester Hydrolase

(Received for publication, May 7, 1985, and in revised form, July 16, 1985)

Gloria N. Sandog and Louise M. Rosenbaum From the DeDartment of Internal Medicine and Arterwsclerosis/Specialized Center of Research, University of Zowa, Iowa City, Zowa 52242

Lysosomal acid lipase was purified to nearhomoge- The relationship of this effect to the possible requireneity in a yield of 25-30% from secretions of human ment for a natural activator substance has not been fibroblasts grownon microcarriers in spinner culture. determined. Thiols had no apparent effect on catalytic Ammonium chloride was added to the serum-freeme- activity when added to assays, but incombination with dium to stimulate production of extracellular enzyme detergents andglycerol, they were required for stabiand minimize modifications, including proteolytic lization of the purifiedacid lipase. Pretreatment of the processing and destruction of the mannose 6-phosphate enzyme with p-hydroxymercuribenzoate or N-ethylrecognition marker, that have been associated with maleimide destroyed activity, whereasphenylmethylpackaging and maturation of acid hydrolases inlyso- sulfonyl fluoride had no effect. Other inhibitors of somes. Chromatography of secretions by decyl-aga- hydrolysis of methylumbelliferyl oleate by purified rose, hydroxylapatite, phenylboronate-agarose, and acid lipase included phenyl- and dodecylboronic acid, gel filtration resulted in greater than 1500-fold puri- chlorpromazine, CaC12, MgSOa(millimolar concentrafication of the lipase, representing a 10,000-fold in- tions), chloride salts of Na, K, and ammonia (>0.1 M), crease above the specific activity of intracellular en- several purified proteins (>ZOO pg/ml), and heparin zyme. The apparent molecular weight of -49,000, es- (>1ng/ml) timated for the lipase by polyacrylamide gel electrophoresis inthe presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by Lysosomal acid lipase activity is essential for the hydrolysis gel filtration (Mr -47,000). By contrast, a smaller molecular weight (Mr -41,000) was estimated for the of cholesteryl esters that are taken upby cells in association intracellular enzyme. The purifiedenzyme was suscep- with low density lipoproteins (1).Cholesterol liberated by this tible to hydrolysis by endo-fl-N-acetylglucosaminidaiereaction has an important regulatory role in cellular sterol H, which resulted in at least two new forms, reduced metabolism. The activity has been attributed to an enzyme in apparent molecular weight by approximately termed lysosomal acid lipase, acid cholesteryl ester hydrolase, 4,000-6,000. Treatment with theendoglycosidase did or cholesterol esterase (EC 3.1.1.13) that acts on a variety of not alter the catalytic activityor heat stability of the substrates at acid pH, including cholesteryl esters, triglyceracid lipase. However, the treatedenzyme was no longer ides, and methylumbelliferyl and nitrophenyl fatty acyl esters. internalized by fibroblasts via the mannose 6-phos- Two human genetic lipid storage disorders, Wolman’s disease phate receptor and thereby had lost the capacity to and cholesteryl ester storage disease, are associated with a correct cholesteryl ester accumulation in cultured li- deficiency of this enzyme (2). The crucial role of the lipase in pase-deficient cells. Acid fatty acyl hydrolase activity cholesteryl ester metabolism prompted its consideration as for cholesteryloleate, triolein, and methylumbelliferyl an important factor inatherogenesis (3-6). oleate co-purified. All three esters were hydrolyzed To investigate the regulation of the acid lipase, its role in optimally at pH. 4.0, but the pH profile was alteredby lipid hydrolysis, and relationship to lipid storage disorders, addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty we set out to purify and study structural andcatalytic propacyl esters of 4-methylumbelliferone, a derivative with erties of the human enzyme. Purification of the lysosomal an intermediate chain length(9 carbons) was thebest acid lipase has been attempted from several sources (reviewed substrate and washydrolyzed at a rate comparable to in Ref. 7), including human liver (S), placenta (9, lo), aorta that of the oleate ester at pH 4. The optimal pH for (ll),and leukocytes (12). Yields were generally low due to instability of the preparations. The conflicting results rehydrolysis of the intermediate and shorter chain length esters washigher by about 2 pH units than that for theported for some properties of the enzyme emphasize the need longer chainesters (pH -4). The activityof the purified for highly purified, better characterized, and stable preparalipase was stimulated by several different proteins. tions. Studies of a number of lysosomal glycosidases, sulfatases, and cathepsin D showed that acid hydrolases have a * This research was supported by Arteriosclerosis/SpecializedCen- complicated life cycle (13) that involves extensive carbohyter of Research Grant HL 14230 from the National Heart, Lung, and drate modification (14) and limited proteolysis (15, 16). DemBlood Institute, National Institutes of Health. The costs of publica- onstrated roles for hydrolase-processing steps include mediation of this article were defrayed in part by the payment of page tion of selective transport in theendoplasmic reticulum (17), charges. This article must therefore be hereby marked “advertise- lysosomal packaging via the mannose 6-phosphate recogniment’’ in accordance with 18 U.S.C. Section 1734 solely to indicate tion marker (reviewed in Ref. 18), and, in the case of cathepsin this fact. $ T o whom correspondence should be addressed Department of D, activation of catalysis (19). To obtain a reproducible prepInternal Medicine, 528 MRC, University of Iowa, IowaCity, IA 52242. aration of the human acid lipase that is suitable for studies

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Purification of Secreted Human Fibroblast Acid Lipase of cellular recognition and structural properties and toavoid problems associated with the purification of various intracellular forms in different stages of processing, we isolated the acid lipase from secretions of cultured fibroblasts. The medium wassupplemented with ammonium chloride to stimulate production of enzyme that has been spared structural modifications associated with maturation of acid hydrolases in lysosomes (20, 21). Indeed, we have shown that a partially purified preparation of acid lipase from fibroblast secretions was efficiently internalized by the mannose 6-phosphate recognition system (22), whereas acid hydrolases isolated from tissue extracts are generally of the low uptake variety (23). An additional advantage of human fibroblasts as source material was the ability to confirm that the lipase under investigation corresponded to the affected enzyme in cells derived from patients with Wolman’s disease and cholesteryl ester storage disease (22). We present here procedures for the production and purification of the secreted form of acid lipase from human fibroblasts grown on microcarriers in spinner culture and describe physical, catalytic, and recognition properties of the enzyme. Preliminary results were reported (24). MATERIALS AND METHODS’ RESULTS

Purification Summary-The purification scheme for a typical preparation of acid lipaseis illustrated in Table I. A final yield of approximately 120 pg of protein represents a recovery of 25% of the acid lipase activity in secretions collected over a 5-week period from fibroblasts grown on 4 gof microcarriers. The specific activity of the purified enzyme was approximately 1500 times that of acid lipase in crude secretions and 10,000 times that of the intracellular fibroblast enzyme. Increases in specificactivity throughout the scheme were similar

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with respect to the hydrolysis of MUO: cholesteryl oleate, and triolein and were independent of the protein assay employed. By comparison of the lipase preparation in Coomassie Bluestained gels with standards at different concentrations (Fig. 5, lanes A-D), the major band in the preparation was estimated to comprise at least 90-95% of the total protein. Only one band was visible in the 1-pg lipase sample under conditions in which standard proteins with comparable molecular weights were detected in the 50-100-ng range. Whengels were silver stained, minor bands were detected (MI 27,51,60,81 x lo3).These varied in relative intensity among preparations and were separated from the major band associated with lipase

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FIG.5. SDS-polyacrylamide gel electrophoresis of acid lipase. Conditions were modified from those described by Laemmli (37)and Takacs (38).Slab gels (0.8 X 20 X 20 cm) were poured with glass plates (vertical gel electrophoresis system, Bethesda Research Laboratories). The stacking gel was formed with 5% acrylamide, 0.15% methylenebisacrylamide, 0.075% ammonium persulfate, and 0.06% tetramethylenediamine in 0.125% Tris-C1 buffer, pH 6.8. The TABLE I running gel consisted of 11% acrylamide and 0.33% bisacrylamide in 0.375 M Tris-CI, pH 8.8 (lanes A-D) or 10% monomer and 0.13% Summary of acid lipase purification Results are from a typical purification of acid lipase from secretions cross-linker in 0.75 M Tris-CI, pH 8.8 (E-K), with 0.05% of the catalysts. The SDS content of gels and of the running buffer (0.025 of normal foreskin fibroblasts. M Tris, 0.192 M glycine, pH 8.3) was 0.1% (A-D) or 1% plus 2 mM Specific activity‘ EDTA (E-K). Samples, which contained 0.063 M Tris-CI, pH 6.8,2% M U 0 Purification step Proteinb SDS, and 0.71 M 2-mercaptoethanol in a volume of 10-20 pl, were M U 0 c h ~ ~ Triolein ~ ~ ~ ’ yheated l a t 100 “C for 5 min, then adjusted to contain 5% sucrose and 0.04% bromphenol blue. Electrophoresis was at 250 V and 6 “C. Gels unitsx 10-3 mg milliunitslmg were stained with Coomassie Brilliant Blue G-250 (39),lanes A-D, (Fibroblasts) (0.0004)(0.0002) (0.015) or with silver reagents (40),lanes E-K. Lanes A, B, and C contained Secretions 0.1 720 72 1, 0.1, and 0.05 pg, respectively, of each of the standards, bovine Decyl-agarose 54 0.055 42 0.033 1.3 serum albumin (69,000),ovalbumin (44,500),carbonic anhydrase 12.0 4.2 50AcA 44 (29,000),/3-lactoglobulin(18,400),and ribonuclease A (13,600).Lanes Hydroxylapatite 10.0 3.2 32 H and K contained 0.15 pg of the first three standards and0.30 pg of Phenylboronate gel1.9 33.7 0.7726 3.0 myoglobin (17,200).Lane D contained 1 pg (-150 units) of the highly 5.54.4 94.2 0.21AcA 20 44 2nd purified acid lipase preparation. Lanes E-G contained 10-pl samples 3rd150 0.12 AcA 18 44 (with the indicated MU0 hydrolase activity) of fraction number 75 units), respectively, which were A unit of activity is defined as theamount of enzyme that results (