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Molecular and Enzymatic Analyses of Lysosomal Acid Lipase in Cholesteryl Ester Storage Disease
MOLECULAR GENETICS AND METABOLISM ARTICLE NO.
64, 126–134 (1998)
GM982707
Molecular and Enzymatic Analyses of Lysosomal Acid Lipase in Cholesteryl Ester Storage Disease Hong Du,*,1 Sulaiman Sheriff,*,2 Jorge Bezerra,† Tatyana Leonova,* and Gregory A. Grabowski* *Division of Human Genetics, and †Division of Gastroenterology and Nutrition, Children’s Hospital Research Foundation of Children’s Hospital Medical Center, and University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229-3039 Received March 15, 1998
Human lysosomal acid lipase (LAL; EC 3.1.1.13) plays a central role in the hydrolysis of cholesteryl esters and triglycerides that are delivered to lysosomes via the low-density lipoprotein (LDL) receptor pathway. The enzyme contributes to the homeostatic control of plasma lipoprotein levels and to the prevention of cellular lipid overload in liver, spleen, and macrophages (1). The hLAL-mediated release of free cholesterol within the cell has the following effects: (1) downregulation of endogenous cholesterol synthesis by negative feedback to the rate-limiting enzyme, 3-hydroxyl-3-methylglutaryl-CoA reductase (2), (2) downregulation of LDL uptake that is mediated by transcriptional regulation of the LDL-receptor gene via membrane-bound transcription factors (SREBP-1 and SREBP-2) released by sterol-regulated proteolysis (3–5), and (3) upregulation of cholesterol esterification by direct activation of fatty acyl CoA:cholesterol acyltransferase (6). Deficiency of hLAL results in one of two phenotypes: Wolman disease (WD) or cholesteryl ester storage disease (CESD). These diseases are inherited as autosomal recessive disorders. WD is a severe infantile-onset variant with death usually occurring before 1 year of age. Hepatosplenomegaly, steatorrhea, abdominal distention, adrenal calcification, and failure to thrive are observed in the first week of life. Massive intracellular storage of both cholesteryl esters and triglycerides is observed in the liver, adrenal gland, and intestine (1,7). CESD is a milder, later-onset disease. Lipid deposition is widespread although hepatomegaly may be the only clinical manifestation. Survival beyond middle age can occur with development of premature atherosclerosis. As deduced from the cDNA, the hLAL amino acid
Human lysosomal acid lipase (hLAL) is essential for the hydrolysis of cholesteryl esters and triglycerides in the lysosome. Defective hLAL activity leads to two autosomal recessive traits, Wolman disease (WD) or cholesteryl ester storage disease (CESD). Phenotypically, WD has accumulation of both triglycerides and cholesteryl esters, while CESD has mainly elevated cholesteryl esters. We characterized mutations in the hLAL gene from two CESD siblings. By reverse transcriptase-PCR (RTPCR) and cDNA cloning and sequencing, we identified homozygous deletion mutations of nucleotides 863 to 934, in the hLAL transcript. Normal levels of LAL mRNA were detected. The deletion in mRNA is due to a G to A transition in the last nucleotide of exon 8 of the hLAL gene, a splice junction mutation (E8SJM) that resulted in exon skipping, and a predicted in-frame deletion of the 24 amino acids. [35S]Met metabolic labeling studies in fibroblasts showed a low level of E8SJM LAL (Ç38%) that was highly unstable. Heterologous expression of E8SJM LAL in insect cells gave an LAL with low catalytic activity toward cholesteryl oleate and triolein. The effects of this mutation are complex with the production of decreased amounts of an unstable LAL that is catalytically defective. The results suggest that E8SJM leads to essentially a null allele and that the differences in WD and CESD phenotype involve other factors. q 1998 Academic Press Key Words: lysosomal acid lipase; Wolman’s disease; cholesterol ester storage disease; immunoprecipitation.
1
To whom correspondence should be addressed. Current Address: Division of Gastrointestinal Hormones, Department of Surgery, University of Cincinnati, College of Medicine, Cincinnati, OH 45267. 2
126 1096-7192/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Wolman Disease and CESD Mutations Allele
Exon
G66V H108P L179P P181L fs177 E7SJM Q277X H274Y E8SJM01 E8SJM/1 L273S E10DAG L336P
4 4 6 6 6 6 (intron) 8 8 8 8 (intron) 8 10 10
a b
cDNA No.a
Base substitution
Amino acid No.b
Amino acid substitution
Ref.
260 386 599 605 594
G–T A–C T–C C–T T–TT a–g C–T C–T G–A g–a T–C del AG T–C
66 108 179 181 177 205–253 277 274 254–277 254–277 273 303 336
Gly–Val His–Pro Leu–Pro Pro–Leu frame shift/stop del 48 a.a. Q–stop His–Tyr del 24 aa del 24 aa Leu–Ser frame shift/stop Leu–Pro
(24) (26) (13,17) (24) (13) (24) (25) (19) (17,20,21,22,23) (23) (24) (21) (18)
892 923 823–894 881 967–968 1070
WD
/
CESD / / / /
/ / / / / / / / /
The cDNA No. is started from the first ATG, since the cDNAs isolated from different groups have different 5* untranslated length. The amino acid No. is from mature N-terminus.
sequence has high homology with rat lingual and human gastric lipases (8). The mouse and rat LAL cDNAs predict highly similar proteins (9,10). The hLAL gene occupies 37 kb of chromosome 10q23.2– 23.3 (11) and has 10 exons (12,13). The reported hLAL cDNAs contain two polymorphic variants [C to A at 86, Thr(06) to Pro(06) and G to A at 107, Gly2 to Arg2] (14–16). The mutations in hLAL (Table 1) that have been identified in WD are missense (L179P) and nonsense mutations (fs177, Q277X, and E8SJM/1), and in CESD are missense (G66V, H108P, L179P, P181L, L336P, and H374Y) and nonsense mutations (E10DAG, E8SJM01). We report here the identification of homozygosity for a splice junction mutation that leads to the deletion of LAL exon 8 in two Brazilian siblings with CESD. Metabolic labeling with [35S]Met in normal and CESD fibroblasts showed the latter having a low level of E8SJM LAL that is unstable, t1/2 õ50% of wild types. Biochemical and heterologous expression studies indicate that the resultant protein has equally low activity toward cholesteryl esters and triglycerides although the patients’ phenotype is CESD rather than WD. These findings suggest that a factor(s) other than residual hLAL activity contributes to the differences in CESD and WD. MATERIALS AND METHODS Materials. The following were from commercial sources: [1-14C]cholesterol oleate (40–60 mCi/mmol), tri[1-14C]oleoylglycerol (80–120 mCi/mmol), [a-32P]-
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dCTP (3000 Ci/mmol), [35S]methionine (ú600 Ci/ mmol), and [a-35S]dATP (1500 Ci/mmol) (DuPont NEN); Triton X-100, 4-methylumbelliferyl oleate (4-MUO), protein A-sepharose CL4B beads, and S. aureus cells (Sigma); sodium taurocholate, sodium taurodeoxycholate, and L-a-phosphatidylcholine (CalBiochem); cholesteryl oleate and triolein (Nu Chek Prep, Elysian, MN); Geneclean kit (BIO 101, Inc.); reverse transcriptase (Promega, Madison WI); restriction endonuclease (New England Biolabs Inc.); Puregene D-5000 DNA isolation kit (Gentra); pCR-II cloning kit and Microfast track mRNA isolation kit (Invitrogen); oligonucleotides synthesizer (Pharmacia Gene Assembler); sequenase and sequenase kit (U.S. Biochemical Corp.); anti-rabbit IgG conjugated to alkaline phosphatase and alkaline phosphatase substrates (Bio-Rad); and high molecular weight protein kit and cell culture reagents (Life Technologies, Inc.). Human fibroblast cell cultures were established from explants after informed consent was obtained. All other chemicals were reagent grade. DNA, mRNA extraction, and oligonucleotide. Genomic DNA was isolated from cultured fibroblasts (107 cells) using Puregene D-5000 DNA isolation kit. mRNA was extracted from 107 cells using the Microfast track mRNA isolation kit. Oligonucleotides were synthesized (Pharmacia Gene Assemble) based on sequences of the hLAL gene (13). RT-PCR, PCR, and DNA sequencing. The cDNAs were synthesized using 0.5 mg fibroblast mRNA, 1 pmol of random primer pd(N)6, and 2 unit of AMV
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reverse transcriptase as described (27). After incubation for 90 min at 427C, 5 ml of the newly synthesized cDNA was used to amplify the entire hLAL coding region by PCR. The primers from 5* and 3* untranslated sequences were as follows: upstream primer 1 (UP1), 5*-GCCCGGCAGGACAGCTCCAGAATGAAA3*, and downstream primer 1 (DP1), 5*-CATAATCATTGACTTGGTGGTACACAGCTC-3*. The reaction (100 ml) contained 100 pmol of each oligonucleotide primers, 200 mM each dATP, dCTP, dGTP, and dTTP; 1X reaction buffer (50 mM KCl, 10 mM Tris-HCl at pH 8.4, 1.5 mM MgCl2) and 1 unit Taq DNA polymerase. Amplification was for 35 cycles (947C for 1 min, 557C for 1 min, and 727C for 2 min). The exon 8 and intronic junction sequences were amplified from genomic DNA using primers flanking exon 8 named hE8f and hE8r (hE8f, 5*-TCAATGCCACCTTAATGC-3*, and hE8r, 5*-GGAAAGGGTTTTGCATGCC-3*) (13). These PCR products were subjected to restriction enzyme digestion and cloned into pCR-II vector (Invitrogen). The inserts were sequenced by dideoxynucleotide termination with sequenase. Western blot analysis and acid lipase enzyme assay. Immunoblots were conducted with antihLAL antiserum as described (28,9). hLAL activities were estimated with the fluorogenic substrate, 4MUO, and natural substrates, 14C-labeled cholesteryl oleate and triolein, as described (27). All assays were conducted in duplicate and the results are the mean of three experiments. Assays were linear within the time frame used and less than 10% of substrates were cleaved. Metabolic labeling studies. Labeling and immunoprecipitation were as described (29). Briefly, confluent normal and CESD fibroblasts were rinsed once with methionine-free DMEM, containing 10% dialyzed FBS, and replaced with prewarmed methionine-free DMEM containing 10% dialyzed FBS and 100 mCi/ml [35S]methionine. After incubation for 0.5 h, the cells were rinsed three times with DMEM and nonradioactive chase medium was added and incubated for the indicated time. Then, cells were washed with 0.5% bovine serum albumin in phosphate-buffered saline (150 mM NaCl, 25 mM Na2HPO4 , 25 mM K2HPO4 , pH 7.5) and were lysed in 250–500 ml of lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride). After incubation for 30 min on ice with shaking, these mixtures were centrifuged at 10,000g (20 min) and an equal volume of radioimmune precipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5, 1% sodium
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deoxycholate, 0.1% SDS) was added to the supernatant. Nonspecific components were removed by treatment with S. aureus cells (1:10, v/v) for 1.5 h. After centrifugation (10,000g; 20 min), the supernatants were incubated with 1% preimmune serum overnight (47C). Nonspecific complexes were precipitated with protein A-Sepharose CL4B beads. The clarified supernatant was incubated (47C) overnight with anti hLAL antiserum (1:200 dilution). The immune complexes were precipitated with protein ASepharose CL4B beads and washed three times with radioimmune precipitation buffer by centrifugation and once with 0.1% Nonidet P-40 in 5 mM Tris-HCl, pH 7.5. The washed precipitates were heated (857C, 7 min) in fourfold-diluted (v/v) SDS-PAGE sample buffer. Aliquots were electrophoresed in 12.5% SDSPAGE. Autoradiography was performed by exposure of the dried gels to phosphor screens (Molecular Dynamics, Sunnyvale, CA) for 2–4 days. Expression of hLAL in insect cells. For direct comparison, the wild-type hLAL containing the polymorphic variant present in the CESD patients was constructed by site-directed mutagenesis. For the CESD mutant hLAL expression, the RT-PCR product was cloned into pCR-II vector and the coding region of hLAL (1.2 kb) was amplified by PCR with primer containing restriction sites (BglII site at upstream, F1: 5*-GAAGATCTCATGAAAATGCGGTTTCTTGG GG-3*; and NheI site at downstream, R1: 5*-CTAGCTAGCTCACTGATATTTCCTCATTAGATT-3*). The PCR products were digested with BglII and NheI, gel purified with Geneclean, and cloned into BamHI/ Nhe I-digested baculovirus vector pVT-Bac (28). The expression constructs of wild-type and the E8SJM mutant were sequenced to ensure the fidelity of the cDNA. Purification of recombinant baculoviruses, titering, and expression in insect cells were performed as described (29). hLAL expression was monitored in cell lysate by immunoblot analysis and enzyme assays at 72 h postinfection with pure recombinant virus. Case reports. A Brazilian girl had a history of abdominal distention, chronic diarrhea, and poor weight gain in the first year of life. At 1 month she was noted to have hepatomegaly and increased serum aminotransferase. Liver biopsy at 25 months of age showed a yellow, greasy core specimen. A skin biopsy was also performed and fibroblasts were shown to have decreased hLAL activity. Her parents were not consanguineous. Her asymptomatic 6-yearold brother was evaluated and found to have hepatomegaly. Liver biopsies from both patients at ages 2 and 6 showed mild portal inflammation, fibrosis, and
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TABLE 2 Clinical Data on CESD Siblings Patient
Age
Sex
HSM
AST/ALT
TC
TG
Adrenal calcification
P1 P2
5 year 9 year
Female Male
/ /
120/192 78/103
262 208
149 93
— —
Note. HSM, hepatosplenomegaly; AST/ALT, aspartate transaminase/alanine transaminase (mg/dl); TC, total cholesterol (normal range: 120–170 mg/dl); TG, total triglycerides (normal range: 41–138 mg/dl).
vacuolated hepatocytes and Kupffer cells. Both patients were started on the HMG-CoA reductase inhibitor, Lovastatin. The girl’s symptoms gradually improved and both patients continued to be asymptomatic. A mild increase of serum aminotransferase and hepatomegaly persisted. Clinical summaries of their current status are shown in Table 2. RESULTS hLAL Enzyme Activity and Protein Levels The deficiency of hLAL activity toward 4-MUO, cholesteryl oleate, and triolein in Cases 1 (P1) and 2 (P2) was confirmed in skin fibroblast cell cultures with the 4-MUO, cholesteryl oleate, and triolein acid lipase assays (Table 3). Compared to the wild-type fibroblast extract, the hLAL activity in Case 1 and 2 fibroblast extracts was 6.21 to 5.03% for [14C]cholesteryl oleate and 35.20 to 36.20% for [14C]triolein. Very low levels of hLAL protein were detectable with anti-hLAL antiserum on Western blots of
cultured fibroblasts from Cases 1 and 2 (Fig. 1). These results suggest that the mutant hLALs were unstable or that only a low level of hLAL was synthesized in the CESD cells. To distinguish between these mechanisms, we determined the amount of LAL mRNA and conducted metabolic labeling studies. The comparative levels of normal and CESD hLAL mRNA were determined by RT-PCR. A 500-bp PCR product from human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) was the internal control. Using several concentrations of cDNA template and cycle numbers for the PCR, no significant differences in the hLAL-specific RT-PCR product levels were observed with mRNA from control or CESD fibroblasts (Fig. 2). However, the size of the RT-PCR products from CESD patients is smaller than that from wild-type control. Metabolic labeling with [35S]Met was conducted in normal and patient fibroblasts. After a pulse of 0.5 h, a chase of 0 to 24 h, and specific immunoprecipitation, newly synthesized hLAL was analyzed on SDSPAGE and autoradiography. After 3 h of chase,
TABLE 3 Hydrolysis of 4-MUO, Cholesteryl Oleate, and Triolein by hLALs from Fibroblasts and Expressed in Insect Cells Enzyme activity 4-MUO (nmol/min/mg protein) Fibroblast lysates Normal WD (GM03111)a WD (GM01644)a WD (GM01606A)a Case 1 Case 2 Insect cell lysatesb HLAL wild type E8SJM a b
6.55 0.75 0.89 0.70 1.67 1.39
{ { { { { {
0.20 0.01 0.05 0.01 0.10 0.06
1086 { 75 0.249 { 0.03
Cholesteryl oleate Triolein (pmol/min/mg protein)
106.46 3.04 3.53 3.30 6.62 5.35
{ { { { { {
1.93 0.17 0.45 0.25 1.16 0.81
7230 { 110 250 { 8
206.69 51.59 46.35 43.53 72.84 74.23
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39.88 11.24 10.23 8.34 20.29 14.56
22100 { 190 595 { 34
Wolman disease fibroblasts, GM03111, GM01644, GM01606A, are from Human Genetic Mutant Cell Repository. The enzyme activity is shown as nmol/min/per mg CRIM.
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8. All eight clones from either patient contained this mutation. The homozygosity of this G to A mutation was further confirmed by restriction enzyme digestion of the genomic PCR product. Incubation of genomic PCR products from normals with BstNI gave complete cleavage of the 179-bp fragment into 103and 76-bp fragments. In comparison, the genomic PCR products from Case 1 and 2 samples could not be cleaved by BstNI (data not shown). This splice donor site mutation predicts a skipping of exon 8 and a 72-bp deletion in the hLAL cDNA in these two patients. FIG. 1. Western blots of hLAL from normal and CESD fibroblasts. Extracts from normal and CESD patient fibroblasts were subjected to 12.5% SDS-PAGE and Western blotting. The resulting immunoblots were visualized with alkaline phosphataseconjugated goat anti-rabbit IgG as secondary antibody. HF, normal human fibroblasts; P1 and P2 are CESD patient fibroblasts from Cases 1 and 2, respectively.
about 30–50% of the wild-type hLAL had been converted to a smaller molecular weight form and the total amount of LAL was present in smaller amounts than initially (Fig. 3A). After 24 h of chase, little of the wild-type hLAL remained, and the LAL t1/2 was about 6.6 h. In comparison, the hLAL from the CESD patient cells was present at low levels after the pulse and was nearly undetectable at 24 h of chase (Fig. 3B). The half-life of this mutant hLAL was estimated at 2.5 h or 2.6-fold less than that of wild-type hLAL (t1/2 Å 6.6 h). Mutation Identification in CESD Patients To identify the hLAL mutations, RT-PCR was used to amplify the complete coding region of hLAL. A smaller RT-PCR product was observed from Cases 1 and 2 than that from control fibroblasts (Fig. 4A). No normal-sized RT-PCR product was observed in any of several samples from these patients. The exon 8 region of hLAL contains a unique XbaI restriction site. Digestion of the RT-PCR product from normal fibroblast mRNA produced the expected 844- and 316-bp fragments. In comparison, the RT-PCR products from Case 1 and 2 mRNA were not cleaved by XbaI (Fig. 4B). Sequence analyses of these RT-PCR products showed a deletion from nucleotide 863 to 944, i.e., the entire exon 8 [the nucleotide numbers are from (8)]. Using forward and reverse primers flanking the exon 8 of hLAL gene (hE8f and hE8r), the genomic region flanking exon 8 was amplified, cloned, and sequenced. Figure 5 shows the presence of a G to A transition at the last nucleotide of exon
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Heterologous Expression Studies To directly evaluate the catalytic effect of the exon 8 deletion, recombinant baculovirus containing the normal or mutant hLAL cDNAs was produced and the proteins were expressed in Sf9 insect cells. Since the polymorphic variants of hLAL have different catalytic activities (15), the normal and exon 8-deleted hLAL were made to contain the same polymorphisms as in the patients [T(06)G2, (15)]. Expression of wild-type and mutant hLALs in insect cells was detected by Western blot (Fig. 6). The mutant hLAL was poorly expressed. This indicated that the mutant hLAL was unstable in this system. To directly compare the catalytic activities of the normal and mutant hLALs, enzyme activities were referenced to the amount of cross-reacting immunologic material (CRIM) on Western blot. These results are summarized in Table 3 with the [14C]triolein and [14C]cholesteryl ester substrates. Compared to the wild type, the expressed E8SJM form of hLAL had
FIG. 2. Semiquantitation of steady-state mRNA by RT-PCR. mRNA (0.5 mg) were used in each RT-PCR (35 cycles) with hLAL primers from 5* and 3* untranslated regions; hGAPDH-specific primers were used for 20 cycles. MW is the 1-kb DNA ladder; HF is human fibroblasts; P1 and P2 are the CESD patients’ fibroblasts.
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FIG. 3. Pulse-chase studies of hLAL from normal (A) and CESD (B) fibroblasts. The hLALs in fibroblasts were pulse labeled with [35S]methionine for 30 min and chased with nonradioactive media as indicated, immunoprecipitated with anti-hLAL antiserum, and analyzed by 12.5% SDS-PAGE.
3.5 and 2.7% of activities toward [14C]cholesteryl ester and [14C]triolein substrates, respectively, when equal amounts of CRIM were used. These results indicated that 30–40 times more mutant enzyme would be required to have equal activity toward these substrates as the normal enzyme. DISCUSSION Homozygosity for splice junction mutation of hLAL in two siblings was associated with CESD. The LAL cDNAs from these siblings showed absence of exon 8 with a predicted 24 amino acid deletion. Importantly, only shorter RT-PCR products were obtained from collected skin fibroblasts, and no normal length mRNAs, as RT-PCR products were detected. The level of mRNA in normal and CESD patients appeared similar although the amount of LAL detected in pulse studies was significantly less than in normals. These results were consistent with the
FIG. 4. RT-PCR analysis of hLAL mRNA from normal and CESD patients. The cDNAs were synthesized by random primers, and hLAL coding region was amplified by hLAL-specific primers as described under Methods and Materials. (A) The RT-PCR products were subjected to 1.0% agarose gel electrophoresis. (B) The RT-PCR products were subjected to XbaI digestion and electrophoresis in 1.2% agarose gel. M, DNA molecular weight, 1-kb ladder; P1 and P2, CESD patients; C, normal human fibroblasts control.
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production of only the deleted variant of mRNA without alternative splicing in fibroblasts. The exon 8 skipping is caused by the point mutation in the last nucleotide of exon 8, a G to A transition at splice donor site, allowing the synthesis of a low amount of unstable, catalytically defective LAL. No other point mutations were detected in the coding region except for the polymorphic variants at 86A (Pro-6) and 107G (Gly2) (15). The E8SJM mutation has been reported in six independent cases of CESD: heterozygously with L179P (17), DAG302 (21), a null mutation (20), and G66V (24). Two homozygotes are also known (16,21). In addition, two WD patients were homozygous for a point mutation, G to A at /1 of the exon 8 splice donor site, that resulted in an exon 8 deletion (23). Thus, our patients with CESD and those of Aslanidis et al. with WD produce enzyme without the 24 amino acids encoded by exon 8. Heterologous expression showed that hLAL missing exon 8 had very low activity toward 4MUO (0.023%), cholesteryl ester (3.5%), and triolein (2.7%). These results indicated the 24 amino acid deletion produced an enzyme with very low, but not absent, activities. This differs with the studies of Aslanidis et al, based on expression of E8JSM in insect cells that indicated a completely inactive enzyme (23). The present results indicate a low steady-state level of the deleted hLAL in CESD fibroblasts that is coupled with the low intrinsic activity. These results are consistent with the very low hLAL activity in these cells. Although the reason for the differences in the two studies is not readily apparent, both studies support the conclusion that WD and CESD with homozygous exon 8 deletions should have the same degree of enzyme deficiency. Aslanidis et al. provided data that the E8SJM mutation, G to A01, led to Ç3% correctly spliced hLAL mRNA (23). In comparison, the G to
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A/1 in WD did not allow any correct splicing (23). Thus, the milder CESD phenotype would have greater residual activity due to the production of a small amount of normal protein. WD would have no normal hLAL and lower (to absent) hLAL activity. Our RT-PCR and cDNA cloning studies did not detect any full-length hLAL mRNA nor did our restriction digestion studies of RT-PCR products in Case 1 or 2 show the presence of a transcript with intact exon 8. Since our PCR primers for RTPCR were in the 5* and 3 * untranslated region, we should have amplified any full-length mRNA that was present. In addition, the metabolic labeling studies showed the rapid degradation of the mutant hLAL and little if any steady-state hLAL in the cells. Thus, our data do not support the presence of normal hLAL in our CESD patient fibroblasts. However, this does not exclude the possibility of alternatively spliced full-length transcripts in other tissues. From our results, we conclude that other factors, in addition to the residual activity, might contribute to the pathogenesis of CESD and WD. Comparing the catalytic activity in CESD fibroblasts and that in heterologous expression system, the trioleylglycerol lipase activity in CESD fibroblasts is relatively high compared to that obtained from insect cell expression. Since we used the equal amount CRIM from mutant and wild-type LAL in the enzyme assays, we directly compared the catalytic rate constants and have avoided the issues of different levels of enzyme protein. Also, it is unlikely that the instability of the E8SJM LAL protein in the insect overexpression system would have differentially affected the cholesteryl esterase vs triglyceri-
FIG. 6. Immunoblots of normal and mutant hLAL expressed in Sf9 cells. Extracts from Sf9 cells expressing normal or E8SJM hLALs were subjected to 12.5% SDS-PAGE and Western blot as described in the legend of Fig. 1.
dase activity. It has been reported in several cases that the triglyceridase activity in CESD patient fibroblasts is only 20–30% of normal control activity (30–32). The heterologous expression in Sf9 cells of LAL mutants (28) has shown dramatic decreases of catalytic activity toward both cholesteryl oleate and triolein substrates. Expression of six other LAL mutants in HeLa cells, using the vaccinia T7 expression system (33), has also revealed catalytic activity defects toward both substrates. However, the endogenous activities obscure the low level of mutant LAL activity. Thus, the explanation for the preferential substrate storage in CESD is not clear from the enzymology alone. To elucidate such a basis, site-directed mutagenesis analyses have suggested a preferential effect on cholesteryl esterase activity when LAL with altered Cys227 or Cys236 were expressed in HeLa cells (33,34). These data and ours suggested that residual LAL activity and other factor(s) are involved in causing the two distinctive phenotypes of WD and CESD. The correlation of in vitro catalytic activity toward two substrates and the pathophysiology of two distinctive phenotypes of WD and CESD requires further investigation. ACKNOWLEDGMENTS
FIG. 5. DNA sequencing of the PCR-amplified exon/intron boundary of exon 8 of the hLAL gene from wild-type control and a CESD patient. The G to A transition at the last nucleotide of Exon 8 is marked (*). Patient 2 has the same G to A transition as Patient 1 (data not shown).
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This work was supported in part by grants to G.A.G from the NIH (DK36729 and NS34071) and to H.D. from the AHA National (95013240) and Ohio Affiliates (SW-94-06-I). Additional support was from the Lucille P. Markey Charitable Trust and the Children’s Hospital Research Foundation.
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REFERENCES 1. Assmann G, Seedorf U. Acid lipase deficiency: Wolman disease and cholesteryl ester storage disease. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds.), 6th ed. New York: McGraw Hill, 1995, pp 2563–2587. 2. Brown MS, Goldstein JL. Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J Lipid Res 21:505– 517, 1980. 3. Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP1, a membrane bound transcription factor released by sterolregulated proteolysis. Cell 77:53–62, 1994. 4. Hua X, Yokoyama C, Wu J, Briggs R, Brown MS, Goldstein JL, Wang X. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element. Proc Natl Acad Sci USA 90:11603–11607, 1993. 5. Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL. Nuclear protein that binds sterol regulatory element of lowdensity lipoprotein receptor promoter. J Biol Chem 268:14490–14496, 1993. 6. Goldstein JL, Dana SE, Faust JR, Beaudet AL, Brown MS. Role of lysosomal acid lipase in the metabolism of plasma low-density lipoprotein. J Biol Chem 250:8487–8495, 1975. 7. Hoeg JM, Demosky SJ Jr., Pescovitz OH, Brewer HB Jr. Cholesteryl ester storage disease and Wolman’s disease: Phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency. Am J Hum Genet 36:1190–1203, 1984. 8. Anderson RA, Sando GN. Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. J Biol Chem 266:22479–22484, 1991. 9. Du H, Witte DP, Grabowski GA. Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein. J Lipid Res 36:937–949, 1996. 10. Nakagawa H, Matsubara S, Kuriyama M, Yoshidome H, Fujiyama J, Yoshida H, Osame M. Cloning of rat lysosomal acid lipase cDNA and identification of the mutation in the rat model of Wolman’s disease. J Lipid Res 36:2212–2218, 1995. 11. Anderson RA, Rao RA, Byrum RS, Rothschild CB, Bowden DW, Hayworth R, Pettentai M. In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in Wolman disease to chromosome 10q23.2-q23.3 Genomics 1:245–247, 1993. 12. Aslanidis C, Klima H, Lackner KJ, Schmitz G. Genomic organization of the human lysosomal acid lipase gene (LIPA). Genomics 20:329–331, 1994. 13. Anderson RA, Byrum RS, Coates PM, Sando GN. Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman disease. Proc Natl Acad Sci USA 91:2718–2722, 1994. 14. Ameis DM, Merkel M, Eckerskorn C, Greten H. Purification, characterization and molecular cloning of human hepatic lysosomal acid lipase. Eur J Biochem 219:905–914, 1994. 15. Du H, Sheriff S. Two polymorphic forms of human lysosomal acid lipase have different levels of activity. Am J Hum Genet 57:1017A, 1995. 16. Muntoni S, Wiebusch H, Funke H, Seedorf U, Roskos M,
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Schulte H, Saku K, Arakawa K, Balestrieri A, Assmann G. A missense mutation (Thr-6Pro) in the lysosomal acid lipase (LAL) gene is present with a high frequency in three different ethnic populations impact on serum lipoprotein concentrations. Hum Genet 97:265–267, 1996. 17. Maslen CL, Babcock D, Illingworth DR. Occurrence of a mutation associated with Wolman disease in a family with cholesteryl ester storage disease. J Inher Metab Dis 18:620– 623, 1995. 18. Seedorf U, Wiebusch H, Muntoni S, Skovby F, Christensen NC, Nickel V, Roskos M, Funke H, Ose L, Assmann G. A novel variant of lysosomal acid lipase (Leu336 r Pro) associated with acid lipase deficiency and cholesteryl ester storage disease. Arther Thromb Vasc Biol 15:773–778, 1995. 19. Pagani F, Zagato L, Merati G, Paone G, Gridelli B, Maier JA. Histidine to tyrosine replacement in lysosomal acid lipase causes cholesteryl ester storage disease. Hum Mol Genet 3:1605–1609, 1995. 20. Klima H, Ullrich K, Aslanidis C, Fehringer P, Lackner KJ, Schmitz G. A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease. J Clin Invest 92:2713–2718, 1993. 21. Ameis D, Brockmann G, Knoblich R, Merkel M, Ostland RE, Jr, Yang JW, Coates PM, Cortner JA, Feiman SV, Greten H. A 5* splice-region mutation and a dinucleotide deletion in the lysosomal acid lipase gene in two patients with cholesteryl ester storage disease. J Lipid Res 36:241–250, 1995. 22. Muntoni S, Wiebusch H, Funke H, Ros E, Seedorf U, Assmann G. Homozygosity for a splice junction mutation in exon 8 of the gene encoding lysosomal acid lipase in a Spanish kindred with cholesteryl ester storage disease (CESD). Hum Genet 95:491–494, 1995. 23. Aslanidis C, Ries S, Fehringer P, Buchler C, Klima H, Schmitz G. Genetic and biochemical evidence that CESD and Wolman disease are distinguished by residual lysosomal acid lipase activity. Genomics 33:85–93, 1996. 24. Pagani F, Garcia R, Pariyarath R, Stuani C, Gridelli B, Paone G, Baralle F. Expression of lysosomal acid lipase mutants detected in three patients with cholesteryl ester storage disease. Hum Mol Genet 5:1611–1617, 1996. 25. Ries S, Aslanidis C, Fehringer P, Carel J, Gendrel D, Schmitz G. A new mutation in the gene for lysosomal acid lipase leads to Wolman disease in an African kindred. J Lipid Res 37:1761–1765, 1996. 26. Gasche C, Aslanidis C, Kain R, Exner M, Helbich T, Dejaco C, Schmitz G, Ferenci P. A novel varant of lysosomal acid lipase in cholesteryl ester storage disease associated with phenotype and improvement on lovastatin. J Hepatol 27:744–750, 1997. 27. Du H, Roy AL, Roeder RG. Human transcription factor USF stimulates transcription through the initial elements of the HIV-1 and Ad-ML promoters. EMBO J 12:501–511, 1993. 28. Sheriff S, Du H, Grabowski GA. Lysosomal acid lipase: characterization by site directed mutagenesis and heterologous expression. J Biol Chem 270:27766–27772, 1995. 29. Leonova T, Qi X, Bencosme A, Ponce E, Sun Y, Grabowski GA. Proteolytic processing patterns of prosaposin in insect and mammalian cells. J Biol Chem 271:17312–17320, 1996.
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30. Hoeg JM, Demosky SJ, Brewer B. Characterization of neutral and acid hydrolase in Wolman’s disease. Biochem Biophys Acta 711:59–65, 1982. 31. Burton BK, Emery D, Mueller HW. Lysosomal acid lipase in cultivated fibroblasts: Characterization of enzyme activity in normal and enzymatically defected cell lines. Clin Chim Acta 101:25–32, 1980. 32. Pagani F, Garcia R, Pariyarath R, Stuani C, Gridelli B, Paone G, Baralle FE. Expression of lysosomal acid lipase mutants detected in three patients with cholesteryl
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ester storage disease. Hum Mol Genet 5:1611 – 1617, 1996. 33. Pagani F, Pariyarath R, Stuani C, Garcia R, Baralle FE. Cysteine rsidues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity. Biochem J 326:265–269, 1997. 34. Lohse P, Loshe P, Chahrokh-Zadeh S, Seidel D. Human lysosomal acid lipase/cholesteryl ester hydrolase and human gastric lipase: site-directed mutagenesis of Cys227 and Cys236 results in substrate-dependent reduction of enzymatic activity. J Lipid Res 38:1896–1905, 1997.
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Molecular and Enzymatic Analyses of Lysosomal Acid Lipase in Cholesteryl Ester Storage Disease Hong Du,*,1 Sulaiman Sheriff,*,2 Jorge Bezerra,† Tatyana Leonova,* and Gregory A. Grabowski* *Division of Human Genetics, and †Division of Gastroenterology and Nutrition, Children’s Hospital Research Foundation of Children’s Hospital Medical Center, and University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229-3039 Received March 15, 1998
Human lysosomal acid lipase (LAL; EC 3.1.1.13) plays a central role in the hydrolysis of cholesteryl esters and triglycerides that are delivered to lysosomes via the low-density lipoprotein (LDL) receptor pathway. The enzyme contributes to the homeostatic control of plasma lipoprotein levels and to the prevention of cellular lipid overload in liver, spleen, and macrophages (1). The hLAL-mediated release of free cholesterol within the cell has the following effects: (1) downregulation of endogenous cholesterol synthesis by negative feedback to the rate-limiting enzyme, 3-hydroxyl-3-methylglutaryl-CoA reductase (2), (2) downregulation of LDL uptake that is mediated by transcriptional regulation of the LDL-receptor gene via membrane-bound transcription factors (SREBP-1 and SREBP-2) released by sterol-regulated proteolysis (3–5), and (3) upregulation of cholesterol esterification by direct activation of fatty acyl CoA:cholesterol acyltransferase (6). Deficiency of hLAL results in one of two phenotypes: Wolman disease (WD) or cholesteryl ester storage disease (CESD). These diseases are inherited as autosomal recessive disorders. WD is a severe infantile-onset variant with death usually occurring before 1 year of age. Hepatosplenomegaly, steatorrhea, abdominal distention, adrenal calcification, and failure to thrive are observed in the first week of life. Massive intracellular storage of both cholesteryl esters and triglycerides is observed in the liver, adrenal gland, and intestine (1,7). CESD is a milder, later-onset disease. Lipid deposition is widespread although hepatomegaly may be the only clinical manifestation. Survival beyond middle age can occur with development of premature atherosclerosis. As deduced from the cDNA, the hLAL amino acid
Human lysosomal acid lipase (hLAL) is essential for the hydrolysis of cholesteryl esters and triglycerides in the lysosome. Defective hLAL activity leads to two autosomal recessive traits, Wolman disease (WD) or cholesteryl ester storage disease (CESD). Phenotypically, WD has accumulation of both triglycerides and cholesteryl esters, while CESD has mainly elevated cholesteryl esters. We characterized mutations in the hLAL gene from two CESD siblings. By reverse transcriptase-PCR (RTPCR) and cDNA cloning and sequencing, we identified homozygous deletion mutations of nucleotides 863 to 934, in the hLAL transcript. Normal levels of LAL mRNA were detected. The deletion in mRNA is due to a G to A transition in the last nucleotide of exon 8 of the hLAL gene, a splice junction mutation (E8SJM) that resulted in exon skipping, and a predicted in-frame deletion of the 24 amino acids. [35S]Met metabolic labeling studies in fibroblasts showed a low level of E8SJM LAL (Ç38%) that was highly unstable. Heterologous expression of E8SJM LAL in insect cells gave an LAL with low catalytic activity toward cholesteryl oleate and triolein. The effects of this mutation are complex with the production of decreased amounts of an unstable LAL that is catalytically defective. The results suggest that E8SJM leads to essentially a null allele and that the differences in WD and CESD phenotype involve other factors. q 1998 Academic Press Key Words: lysosomal acid lipase; Wolman’s disease; cholesterol ester storage disease; immunoprecipitation.
1
To whom correspondence should be addressed. Current Address: Division of Gastrointestinal Hormones, Department of Surgery, University of Cincinnati, College of Medicine, Cincinnati, OH 45267. 2
126 1096-7192/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Wolman Disease and CESD Mutations Allele
Exon
G66V H108P L179P P181L fs177 E7SJM Q277X H274Y E8SJM01 E8SJM/1 L273S E10DAG L336P
4 4 6 6 6 6 (intron) 8 8 8 8 (intron) 8 10 10
a b
cDNA No.a
Base substitution
Amino acid No.b
Amino acid substitution
Ref.
260 386 599 605 594
G–T A–C T–C C–T T–TT a–g C–T C–T G–A g–a T–C del AG T–C
66 108 179 181 177 205–253 277 274 254–277 254–277 273 303 336
Gly–Val His–Pro Leu–Pro Pro–Leu frame shift/stop del 48 a.a. Q–stop His–Tyr del 24 aa del 24 aa Leu–Ser frame shift/stop Leu–Pro
(24) (26) (13,17) (24) (13) (24) (25) (19) (17,20,21,22,23) (23) (24) (21) (18)
892 923 823–894 881 967–968 1070
WD
/
CESD / / / /
/ / / / / / / / /
The cDNA No. is started from the first ATG, since the cDNAs isolated from different groups have different 5* untranslated length. The amino acid No. is from mature N-terminus.
sequence has high homology with rat lingual and human gastric lipases (8). The mouse and rat LAL cDNAs predict highly similar proteins (9,10). The hLAL gene occupies 37 kb of chromosome 10q23.2– 23.3 (11) and has 10 exons (12,13). The reported hLAL cDNAs contain two polymorphic variants [C to A at 86, Thr(06) to Pro(06) and G to A at 107, Gly2 to Arg2] (14–16). The mutations in hLAL (Table 1) that have been identified in WD are missense (L179P) and nonsense mutations (fs177, Q277X, and E8SJM/1), and in CESD are missense (G66V, H108P, L179P, P181L, L336P, and H374Y) and nonsense mutations (E10DAG, E8SJM01). We report here the identification of homozygosity for a splice junction mutation that leads to the deletion of LAL exon 8 in two Brazilian siblings with CESD. Metabolic labeling with [35S]Met in normal and CESD fibroblasts showed the latter having a low level of E8SJM LAL that is unstable, t1/2 õ50% of wild types. Biochemical and heterologous expression studies indicate that the resultant protein has equally low activity toward cholesteryl esters and triglycerides although the patients’ phenotype is CESD rather than WD. These findings suggest that a factor(s) other than residual hLAL activity contributes to the differences in CESD and WD. MATERIALS AND METHODS Materials. The following were from commercial sources: [1-14C]cholesterol oleate (40–60 mCi/mmol), tri[1-14C]oleoylglycerol (80–120 mCi/mmol), [a-32P]-
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dCTP (3000 Ci/mmol), [35S]methionine (ú600 Ci/ mmol), and [a-35S]dATP (1500 Ci/mmol) (DuPont NEN); Triton X-100, 4-methylumbelliferyl oleate (4-MUO), protein A-sepharose CL4B beads, and S. aureus cells (Sigma); sodium taurocholate, sodium taurodeoxycholate, and L-a-phosphatidylcholine (CalBiochem); cholesteryl oleate and triolein (Nu Chek Prep, Elysian, MN); Geneclean kit (BIO 101, Inc.); reverse transcriptase (Promega, Madison WI); restriction endonuclease (New England Biolabs Inc.); Puregene D-5000 DNA isolation kit (Gentra); pCR-II cloning kit and Microfast track mRNA isolation kit (Invitrogen); oligonucleotides synthesizer (Pharmacia Gene Assembler); sequenase and sequenase kit (U.S. Biochemical Corp.); anti-rabbit IgG conjugated to alkaline phosphatase and alkaline phosphatase substrates (Bio-Rad); and high molecular weight protein kit and cell culture reagents (Life Technologies, Inc.). Human fibroblast cell cultures were established from explants after informed consent was obtained. All other chemicals were reagent grade. DNA, mRNA extraction, and oligonucleotide. Genomic DNA was isolated from cultured fibroblasts (107 cells) using Puregene D-5000 DNA isolation kit. mRNA was extracted from 107 cells using the Microfast track mRNA isolation kit. Oligonucleotides were synthesized (Pharmacia Gene Assemble) based on sequences of the hLAL gene (13). RT-PCR, PCR, and DNA sequencing. The cDNAs were synthesized using 0.5 mg fibroblast mRNA, 1 pmol of random primer pd(N)6, and 2 unit of AMV
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reverse transcriptase as described (27). After incubation for 90 min at 427C, 5 ml of the newly synthesized cDNA was used to amplify the entire hLAL coding region by PCR. The primers from 5* and 3* untranslated sequences were as follows: upstream primer 1 (UP1), 5*-GCCCGGCAGGACAGCTCCAGAATGAAA3*, and downstream primer 1 (DP1), 5*-CATAATCATTGACTTGGTGGTACACAGCTC-3*. The reaction (100 ml) contained 100 pmol of each oligonucleotide primers, 200 mM each dATP, dCTP, dGTP, and dTTP; 1X reaction buffer (50 mM KCl, 10 mM Tris-HCl at pH 8.4, 1.5 mM MgCl2) and 1 unit Taq DNA polymerase. Amplification was for 35 cycles (947C for 1 min, 557C for 1 min, and 727C for 2 min). The exon 8 and intronic junction sequences were amplified from genomic DNA using primers flanking exon 8 named hE8f and hE8r (hE8f, 5*-TCAATGCCACCTTAATGC-3*, and hE8r, 5*-GGAAAGGGTTTTGCATGCC-3*) (13). These PCR products were subjected to restriction enzyme digestion and cloned into pCR-II vector (Invitrogen). The inserts were sequenced by dideoxynucleotide termination with sequenase. Western blot analysis and acid lipase enzyme assay. Immunoblots were conducted with antihLAL antiserum as described (28,9). hLAL activities were estimated with the fluorogenic substrate, 4MUO, and natural substrates, 14C-labeled cholesteryl oleate and triolein, as described (27). All assays were conducted in duplicate and the results are the mean of three experiments. Assays were linear within the time frame used and less than 10% of substrates were cleaved. Metabolic labeling studies. Labeling and immunoprecipitation were as described (29). Briefly, confluent normal and CESD fibroblasts were rinsed once with methionine-free DMEM, containing 10% dialyzed FBS, and replaced with prewarmed methionine-free DMEM containing 10% dialyzed FBS and 100 mCi/ml [35S]methionine. After incubation for 0.5 h, the cells were rinsed three times with DMEM and nonradioactive chase medium was added and incubated for the indicated time. Then, cells were washed with 0.5% bovine serum albumin in phosphate-buffered saline (150 mM NaCl, 25 mM Na2HPO4 , 25 mM K2HPO4 , pH 7.5) and were lysed in 250–500 ml of lysis buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride). After incubation for 30 min on ice with shaking, these mixtures were centrifuged at 10,000g (20 min) and an equal volume of radioimmune precipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5, 1% sodium
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deoxycholate, 0.1% SDS) was added to the supernatant. Nonspecific components were removed by treatment with S. aureus cells (1:10, v/v) for 1.5 h. After centrifugation (10,000g; 20 min), the supernatants were incubated with 1% preimmune serum overnight (47C). Nonspecific complexes were precipitated with protein A-Sepharose CL4B beads. The clarified supernatant was incubated (47C) overnight with anti hLAL antiserum (1:200 dilution). The immune complexes were precipitated with protein ASepharose CL4B beads and washed three times with radioimmune precipitation buffer by centrifugation and once with 0.1% Nonidet P-40 in 5 mM Tris-HCl, pH 7.5. The washed precipitates were heated (857C, 7 min) in fourfold-diluted (v/v) SDS-PAGE sample buffer. Aliquots were electrophoresed in 12.5% SDSPAGE. Autoradiography was performed by exposure of the dried gels to phosphor screens (Molecular Dynamics, Sunnyvale, CA) for 2–4 days. Expression of hLAL in insect cells. For direct comparison, the wild-type hLAL containing the polymorphic variant present in the CESD patients was constructed by site-directed mutagenesis. For the CESD mutant hLAL expression, the RT-PCR product was cloned into pCR-II vector and the coding region of hLAL (1.2 kb) was amplified by PCR with primer containing restriction sites (BglII site at upstream, F1: 5*-GAAGATCTCATGAAAATGCGGTTTCTTGG GG-3*; and NheI site at downstream, R1: 5*-CTAGCTAGCTCACTGATATTTCCTCATTAGATT-3*). The PCR products were digested with BglII and NheI, gel purified with Geneclean, and cloned into BamHI/ Nhe I-digested baculovirus vector pVT-Bac (28). The expression constructs of wild-type and the E8SJM mutant were sequenced to ensure the fidelity of the cDNA. Purification of recombinant baculoviruses, titering, and expression in insect cells were performed as described (29). hLAL expression was monitored in cell lysate by immunoblot analysis and enzyme assays at 72 h postinfection with pure recombinant virus. Case reports. A Brazilian girl had a history of abdominal distention, chronic diarrhea, and poor weight gain in the first year of life. At 1 month she was noted to have hepatomegaly and increased serum aminotransferase. Liver biopsy at 25 months of age showed a yellow, greasy core specimen. A skin biopsy was also performed and fibroblasts were shown to have decreased hLAL activity. Her parents were not consanguineous. Her asymptomatic 6-yearold brother was evaluated and found to have hepatomegaly. Liver biopsies from both patients at ages 2 and 6 showed mild portal inflammation, fibrosis, and
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TABLE 2 Clinical Data on CESD Siblings Patient
Age
Sex
HSM
AST/ALT
TC
TG
Adrenal calcification
P1 P2
5 year 9 year
Female Male
/ /
120/192 78/103
262 208
149 93
— —
Note. HSM, hepatosplenomegaly; AST/ALT, aspartate transaminase/alanine transaminase (mg/dl); TC, total cholesterol (normal range: 120–170 mg/dl); TG, total triglycerides (normal range: 41–138 mg/dl).
vacuolated hepatocytes and Kupffer cells. Both patients were started on the HMG-CoA reductase inhibitor, Lovastatin. The girl’s symptoms gradually improved and both patients continued to be asymptomatic. A mild increase of serum aminotransferase and hepatomegaly persisted. Clinical summaries of their current status are shown in Table 2. RESULTS hLAL Enzyme Activity and Protein Levels The deficiency of hLAL activity toward 4-MUO, cholesteryl oleate, and triolein in Cases 1 (P1) and 2 (P2) was confirmed in skin fibroblast cell cultures with the 4-MUO, cholesteryl oleate, and triolein acid lipase assays (Table 3). Compared to the wild-type fibroblast extract, the hLAL activity in Case 1 and 2 fibroblast extracts was 6.21 to 5.03% for [14C]cholesteryl oleate and 35.20 to 36.20% for [14C]triolein. Very low levels of hLAL protein were detectable with anti-hLAL antiserum on Western blots of
cultured fibroblasts from Cases 1 and 2 (Fig. 1). These results suggest that the mutant hLALs were unstable or that only a low level of hLAL was synthesized in the CESD cells. To distinguish between these mechanisms, we determined the amount of LAL mRNA and conducted metabolic labeling studies. The comparative levels of normal and CESD hLAL mRNA were determined by RT-PCR. A 500-bp PCR product from human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) was the internal control. Using several concentrations of cDNA template and cycle numbers for the PCR, no significant differences in the hLAL-specific RT-PCR product levels were observed with mRNA from control or CESD fibroblasts (Fig. 2). However, the size of the RT-PCR products from CESD patients is smaller than that from wild-type control. Metabolic labeling with [35S]Met was conducted in normal and patient fibroblasts. After a pulse of 0.5 h, a chase of 0 to 24 h, and specific immunoprecipitation, newly synthesized hLAL was analyzed on SDSPAGE and autoradiography. After 3 h of chase,
TABLE 3 Hydrolysis of 4-MUO, Cholesteryl Oleate, and Triolein by hLALs from Fibroblasts and Expressed in Insect Cells Enzyme activity 4-MUO (nmol/min/mg protein) Fibroblast lysates Normal WD (GM03111)a WD (GM01644)a WD (GM01606A)a Case 1 Case 2 Insect cell lysatesb HLAL wild type E8SJM a b
6.55 0.75 0.89 0.70 1.67 1.39
{ { { { { {
0.20 0.01 0.05 0.01 0.10 0.06
1086 { 75 0.249 { 0.03
Cholesteryl oleate Triolein (pmol/min/mg protein)
106.46 3.04 3.53 3.30 6.62 5.35
{ { { { { {
1.93 0.17 0.45 0.25 1.16 0.81
7230 { 110 250 { 8
206.69 51.59 46.35 43.53 72.84 74.23
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22100 { 190 595 { 34
Wolman disease fibroblasts, GM03111, GM01644, GM01606A, are from Human Genetic Mutant Cell Repository. The enzyme activity is shown as nmol/min/per mg CRIM.
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8. All eight clones from either patient contained this mutation. The homozygosity of this G to A mutation was further confirmed by restriction enzyme digestion of the genomic PCR product. Incubation of genomic PCR products from normals with BstNI gave complete cleavage of the 179-bp fragment into 103and 76-bp fragments. In comparison, the genomic PCR products from Case 1 and 2 samples could not be cleaved by BstNI (data not shown). This splice donor site mutation predicts a skipping of exon 8 and a 72-bp deletion in the hLAL cDNA in these two patients. FIG. 1. Western blots of hLAL from normal and CESD fibroblasts. Extracts from normal and CESD patient fibroblasts were subjected to 12.5% SDS-PAGE and Western blotting. The resulting immunoblots were visualized with alkaline phosphataseconjugated goat anti-rabbit IgG as secondary antibody. HF, normal human fibroblasts; P1 and P2 are CESD patient fibroblasts from Cases 1 and 2, respectively.
about 30–50% of the wild-type hLAL had been converted to a smaller molecular weight form and the total amount of LAL was present in smaller amounts than initially (Fig. 3A). After 24 h of chase, little of the wild-type hLAL remained, and the LAL t1/2 was about 6.6 h. In comparison, the hLAL from the CESD patient cells was present at low levels after the pulse and was nearly undetectable at 24 h of chase (Fig. 3B). The half-life of this mutant hLAL was estimated at 2.5 h or 2.6-fold less than that of wild-type hLAL (t1/2 Å 6.6 h). Mutation Identification in CESD Patients To identify the hLAL mutations, RT-PCR was used to amplify the complete coding region of hLAL. A smaller RT-PCR product was observed from Cases 1 and 2 than that from control fibroblasts (Fig. 4A). No normal-sized RT-PCR product was observed in any of several samples from these patients. The exon 8 region of hLAL contains a unique XbaI restriction site. Digestion of the RT-PCR product from normal fibroblast mRNA produced the expected 844- and 316-bp fragments. In comparison, the RT-PCR products from Case 1 and 2 mRNA were not cleaved by XbaI (Fig. 4B). Sequence analyses of these RT-PCR products showed a deletion from nucleotide 863 to 944, i.e., the entire exon 8 [the nucleotide numbers are from (8)]. Using forward and reverse primers flanking the exon 8 of hLAL gene (hE8f and hE8r), the genomic region flanking exon 8 was amplified, cloned, and sequenced. Figure 5 shows the presence of a G to A transition at the last nucleotide of exon
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Heterologous Expression Studies To directly evaluate the catalytic effect of the exon 8 deletion, recombinant baculovirus containing the normal or mutant hLAL cDNAs was produced and the proteins were expressed in Sf9 insect cells. Since the polymorphic variants of hLAL have different catalytic activities (15), the normal and exon 8-deleted hLAL were made to contain the same polymorphisms as in the patients [T(06)G2, (15)]. Expression of wild-type and mutant hLALs in insect cells was detected by Western blot (Fig. 6). The mutant hLAL was poorly expressed. This indicated that the mutant hLAL was unstable in this system. To directly compare the catalytic activities of the normal and mutant hLALs, enzyme activities were referenced to the amount of cross-reacting immunologic material (CRIM) on Western blot. These results are summarized in Table 3 with the [14C]triolein and [14C]cholesteryl ester substrates. Compared to the wild type, the expressed E8SJM form of hLAL had
FIG. 2. Semiquantitation of steady-state mRNA by RT-PCR. mRNA (0.5 mg) were used in each RT-PCR (35 cycles) with hLAL primers from 5* and 3* untranslated regions; hGAPDH-specific primers were used for 20 cycles. MW is the 1-kb DNA ladder; HF is human fibroblasts; P1 and P2 are the CESD patients’ fibroblasts.
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FIG. 3. Pulse-chase studies of hLAL from normal (A) and CESD (B) fibroblasts. The hLALs in fibroblasts were pulse labeled with [35S]methionine for 30 min and chased with nonradioactive media as indicated, immunoprecipitated with anti-hLAL antiserum, and analyzed by 12.5% SDS-PAGE.
3.5 and 2.7% of activities toward [14C]cholesteryl ester and [14C]triolein substrates, respectively, when equal amounts of CRIM were used. These results indicated that 30–40 times more mutant enzyme would be required to have equal activity toward these substrates as the normal enzyme. DISCUSSION Homozygosity for splice junction mutation of hLAL in two siblings was associated with CESD. The LAL cDNAs from these siblings showed absence of exon 8 with a predicted 24 amino acid deletion. Importantly, only shorter RT-PCR products were obtained from collected skin fibroblasts, and no normal length mRNAs, as RT-PCR products were detected. The level of mRNA in normal and CESD patients appeared similar although the amount of LAL detected in pulse studies was significantly less than in normals. These results were consistent with the
FIG. 4. RT-PCR analysis of hLAL mRNA from normal and CESD patients. The cDNAs were synthesized by random primers, and hLAL coding region was amplified by hLAL-specific primers as described under Methods and Materials. (A) The RT-PCR products were subjected to 1.0% agarose gel electrophoresis. (B) The RT-PCR products were subjected to XbaI digestion and electrophoresis in 1.2% agarose gel. M, DNA molecular weight, 1-kb ladder; P1 and P2, CESD patients; C, normal human fibroblasts control.
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production of only the deleted variant of mRNA without alternative splicing in fibroblasts. The exon 8 skipping is caused by the point mutation in the last nucleotide of exon 8, a G to A transition at splice donor site, allowing the synthesis of a low amount of unstable, catalytically defective LAL. No other point mutations were detected in the coding region except for the polymorphic variants at 86A (Pro-6) and 107G (Gly2) (15). The E8SJM mutation has been reported in six independent cases of CESD: heterozygously with L179P (17), DAG302 (21), a null mutation (20), and G66V (24). Two homozygotes are also known (16,21). In addition, two WD patients were homozygous for a point mutation, G to A at /1 of the exon 8 splice donor site, that resulted in an exon 8 deletion (23). Thus, our patients with CESD and those of Aslanidis et al. with WD produce enzyme without the 24 amino acids encoded by exon 8. Heterologous expression showed that hLAL missing exon 8 had very low activity toward 4MUO (0.023%), cholesteryl ester (3.5%), and triolein (2.7%). These results indicated the 24 amino acid deletion produced an enzyme with very low, but not absent, activities. This differs with the studies of Aslanidis et al, based on expression of E8JSM in insect cells that indicated a completely inactive enzyme (23). The present results indicate a low steady-state level of the deleted hLAL in CESD fibroblasts that is coupled with the low intrinsic activity. These results are consistent with the very low hLAL activity in these cells. Although the reason for the differences in the two studies is not readily apparent, both studies support the conclusion that WD and CESD with homozygous exon 8 deletions should have the same degree of enzyme deficiency. Aslanidis et al. provided data that the E8SJM mutation, G to A01, led to Ç3% correctly spliced hLAL mRNA (23). In comparison, the G to
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A/1 in WD did not allow any correct splicing (23). Thus, the milder CESD phenotype would have greater residual activity due to the production of a small amount of normal protein. WD would have no normal hLAL and lower (to absent) hLAL activity. Our RT-PCR and cDNA cloning studies did not detect any full-length hLAL mRNA nor did our restriction digestion studies of RT-PCR products in Case 1 or 2 show the presence of a transcript with intact exon 8. Since our PCR primers for RTPCR were in the 5* and 3 * untranslated region, we should have amplified any full-length mRNA that was present. In addition, the metabolic labeling studies showed the rapid degradation of the mutant hLAL and little if any steady-state hLAL in the cells. Thus, our data do not support the presence of normal hLAL in our CESD patient fibroblasts. However, this does not exclude the possibility of alternatively spliced full-length transcripts in other tissues. From our results, we conclude that other factors, in addition to the residual activity, might contribute to the pathogenesis of CESD and WD. Comparing the catalytic activity in CESD fibroblasts and that in heterologous expression system, the trioleylglycerol lipase activity in CESD fibroblasts is relatively high compared to that obtained from insect cell expression. Since we used the equal amount CRIM from mutant and wild-type LAL in the enzyme assays, we directly compared the catalytic rate constants and have avoided the issues of different levels of enzyme protein. Also, it is unlikely that the instability of the E8SJM LAL protein in the insect overexpression system would have differentially affected the cholesteryl esterase vs triglyceri-
FIG. 6. Immunoblots of normal and mutant hLAL expressed in Sf9 cells. Extracts from Sf9 cells expressing normal or E8SJM hLALs were subjected to 12.5% SDS-PAGE and Western blot as described in the legend of Fig. 1.
dase activity. It has been reported in several cases that the triglyceridase activity in CESD patient fibroblasts is only 20–30% of normal control activity (30–32). The heterologous expression in Sf9 cells of LAL mutants (28) has shown dramatic decreases of catalytic activity toward both cholesteryl oleate and triolein substrates. Expression of six other LAL mutants in HeLa cells, using the vaccinia T7 expression system (33), has also revealed catalytic activity defects toward both substrates. However, the endogenous activities obscure the low level of mutant LAL activity. Thus, the explanation for the preferential substrate storage in CESD is not clear from the enzymology alone. To elucidate such a basis, site-directed mutagenesis analyses have suggested a preferential effect on cholesteryl esterase activity when LAL with altered Cys227 or Cys236 were expressed in HeLa cells (33,34). These data and ours suggested that residual LAL activity and other factor(s) are involved in causing the two distinctive phenotypes of WD and CESD. The correlation of in vitro catalytic activity toward two substrates and the pathophysiology of two distinctive phenotypes of WD and CESD requires further investigation. ACKNOWLEDGMENTS
FIG. 5. DNA sequencing of the PCR-amplified exon/intron boundary of exon 8 of the hLAL gene from wild-type control and a CESD patient. The G to A transition at the last nucleotide of Exon 8 is marked (*). Patient 2 has the same G to A transition as Patient 1 (data not shown).
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This work was supported in part by grants to G.A.G from the NIH (DK36729 and NS34071) and to H.D. from the AHA National (95013240) and Ohio Affiliates (SW-94-06-I). Additional support was from the Lucille P. Markey Charitable Trust and the Children’s Hospital Research Foundation.
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LYSOSOMAL ACID LIPASE DEFICIENCY
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