(Received for publication, July 14, 1995)
From the
Lysosomal acid lipase (LAL) is essential for the hydrolysis of
cholesterol esters and triglycerides that are delivered to the
lysosomes via the low density lipoprotein receptor system. The
deficiency of LAL is associated with cholesteryl ester storage disease
(CESD) and Wolman's disease (WD). We cloned the human LAL cDNA
and expressed the active enzyme in the baculovirus system. Two
molecular forms (M
41,000 and
46,000)
with different glycosylation were found intracellularly, and
24%
of the M
46,000 form was secreted into the
medium. Tunicamycin treatment produced only an inactive M
41,000 form. This result implicates
glycosylation occupancy in the proper folding for active-site function.
Catalytic activity was greater toward cis- than trans-unsaturated fatty acid esters of 4-methylumbelliferone
and toward esters with 7-carbon length acyl chains. LAL cleaved
cholesterol esters and mono-, tri-, and diglycerides. Heparin had a
biphasic effect on enzymatic activity with initial activation followed
by inhibition. Inhibition of LAL activity by tetrahydrolipstatin and
diethyl p-nitrophenyl phosphate suggested the presence of
active serines in binding/catalytic domain(s) of the protein.
Site-directed mutagenesis at two putative active centers,
GXSXG, showed that Ser
was important to
catalytic activity, whereas Ser
was not and neither was
the catalytic nucleophile. Three reported mutations (L179P, L336P, and
AG302 deletion) from CESD patients were created and expressed in
the Sf9 cell system. None cleaved cholesterol esters, and L179P and
L336P cleaved only triolein at
4% of wild-type levels. These
results suggest that mechanisms, in addition to LAL defects, may
operate in the selective accumulation of cholesterol esters or
triglycerides in CESD and WD patients.
Lysosomal acid lipase (LAL) ()hydrolyzes cholesteryl
esters and triglycerides that are delivered to the lysosomes by low
density lipoprotein receptor-mediated endocytosis. LAL is important to
the regulation of cholesterol synthesis and homeostasis since it
liberates free cholesterol for negative feedback of
hydroxymethylglutaryl-CoA reductase(1) . Defective human LAL
(hLAL) activity has been associated with two rare autosomal recessive
traits, Wolman's disease (WD) and cholesteryl ester storage
disease (CESD). WD is lethal within the first year of life due to
hepatosplenomegaly, adrenal calcification, and massive accumulation of
triglycerides and cholesterol esters in these organs as well as
macrophages and blood vessels. CESD is a less severe disorder with
longer survival, hepatomegaly, premature atherosclerosis, and
dyslipoproteinemias. Residual LAL activity has been detected in CESD,
but not in WD(2) .
The hLAL cDNA and chromosomal gene have
been characterized, and the locus maps to human chromosome
10q23.2-23.3(3, 4) . The gene has 10 exons spread over
36 kilobase pairs(5, 6) . Several hLAL mutations
have been detected in cDNAs derived from mRNAs of CESD and WD patient
cells. In CESD, a splice donor site G to A transition leads to aberrant
splicing of exon 8 and a 72-base pair (24-amino acid) deletion (7) . An AG deletion leads to frameshift at amino acid 302
(
AG302) and a truncated lipase with a 34-amino acid C-terminal
deletion(8) . Two proline to leucine substitutions (L179P and
L336P) have been detected in different CESD
patients(5, 9) . In WD, a T insertion at nucleotide
635 results in a frameshift (fs177) and premature translation
termination at amino acid 189(5) .
hLAL is a member of a
highly conserved lipase family that includes gastric and lingual
lipases(3) . The hLAL cDNA encodes a 372-amino acid mature
protein and a 27-amino acid signal sequence(10) . The predicted
mature polypeptide contains six cysteines. Three of these cysteines are
conserved among members of the lipase family at positions 227, 236, and
244. This glycoprotein has six glycosylation consensus sequences
(Asn-X-Ser/Thr), and three at Asn,
Asn
, and Asn
are conserved among members of
the lipase gene family(3) . The enzyme is trafficked to the
lysosome via the mannose 6-phosphate receptor
system(11, 12) . However, the dependence of catalytic
activity on glycosylation is unknown. All the members of the lipase
gene family have conserved GXSXG pentapeptide
sequences that contain the active-site serine nucleophiles (13, 14, 15) . hLAL has two such sequences at
residues 97-101 and 151-155 with potential serine
nucleophiles at residues 99 and 153(3) . These are conserved in
mouse LAL, (
)but their participation in the activity of LAL
is unknown.
The very low amount of LAL within mammalian cells (12, 16, 17) has limited its structure/function characterization. Kinetic studies have been limited to highly purified preparations of rabbit and human LALs. Rabbit liver LAL cleaves cholesteryl esters and tri-, di-, and monoglycerides with apparent specificity for the 1- and 3-acyl groups and no activity for the 2-monoglycerides(18) . Hepatic hLAL will cleave 2-monoglycerides(19) . hLALs from liver (10) or expressed in COS cells have activity toward triglyceride and cholesteryl esters(3) . The rabbit enzyme also cleaves 4-methylumbelliferyl fatty acid esters of various chain lengths with preference for medium (7-carbon) size chain esters(20, 21) . Sulfonic acid and boronic acid derivatives, diethyl pyrocarbonates, and heparin inhibit hLAL(22) .
We have cloned and expressed normal and mutant hLAL cDNAs to develop a more detailed understanding of the properties of hLAL. Specifically, we explored the roles of GXSXG sequences and glycosylation in catalysis. Expression of identified CESD and WD alleles suggests a more complex molecular pathogenesis than an isolated hLAL deficiency.
For tunicamycin treatment, Sf9 cells were infected with recombinant virus; 1 h later, the virus-containing medium was aspirated, and the infected cells were washed once and changed to serum-free and protein-free Grace's medium containing tunicamycin (6 µg/ml). The cells and medium were harvested 3 days later for enzyme assays and immunoblot analysis.
Purified His tag-containing hLAL was used to
raise polyclonal anti-hLAL antibody. New Zealand White rabbits (2.3 kg)
were injected intradermally (10 areas) with 300 µg (total) of
purified antigen emulsified in Freund's adjuvant. Intramuscular
boosts were given every 2-3 weeks with 60 µg of antigen.
Serum was collected 10-15 days after each boost. The antibody
titers from each bleeding were analyzed by enzyme-linked immunosorbent
assay using affinity column-purified recombinant as an antigen and
horseradish peroxidase-conjugated goat anti-rabbit IgG as secondary
antibody. The antibody used in this study had a titer of 1:10 using 2 ng of purified protein. The specificity of the antibody
was evaluated by immunoblotting using recombinant hLAL and lysates from
human fibroblasts and HepG2 cells. A single band with M
41,000 in lysates of bacteria expressing hLAL and M
41,000 and
46,000 proteins in human
fibroblasts were detected by this antibody.
Immunoblotting of insect
and bacterial cell-expressed recombinant enzymes was performed using
polyclonal anti-hLAL antibody as described for anti-human
-glucosidase (27) with slight modifications. Briefly, Sf9
cells were infected with wild-type or pure recombinant AcNPV; 1 h
later, the virus-containing medium was aspirated, and cells were washed
and changed to serum-free and protein-free Grace's medium. Three
days after infection, the medium and cells were collected separately by
centrifugation (525
g, 10 min). The cell pellet was
further washed three times in phosphate-buffered saline and solubilized
in 0.1 M phosphate, pH 6.8, containing 10 mM
-mercaptoethanol, 0.25% Triton X-100, 1 mM EDTA, and
0.02% sodium azide (22) by ultrasonic irradiation at 4 °C
using a cup sonicator (30, 20, and 20 s; 4 °C). The sonicates were
centrifuged (875
g, 20 min), and the supernatant was
used for immunoblot analysis. Immunoblotting was done with the
polyclonal anti-hLAL antibody (1:2000 dilution) and alkaline
phosphatase-conjugated goat anti-rabbit IgG as secondary antibody.
Extracts of uninfected and wild-type virus-infected Sf9 cells did not
show any signal. The density of individual visualized bands was
quantified by transmission densitometry using purified bacterially
expressed hLAL as a reference on each gel.
Briefly, 25
µmol of 4-MU fatty acid in hexane and 40 µmol of L--phosphatidylcholine in chloroform were evaporated
under N
. The residue was resuspended in 2.4 mM sodium taurodeoxycholate and sonicated at 4 °C (using a cup
sonicator for 3-5 min at 50 watts). The resultant translucent and
homogeneous dispersions were used as the substrates. Under the standard
assay conditions, 50 µl of
4-MUO/L-
-phosphatidylcholine liposomes were added to the
reaction mixture. Enzyme assays were in 0.2 M sodium acetate,
pH 5.5 (400 µl). Assays were stopped with 1.6 ml of 0.1 M Tris-HCl, pH 7.6, and the fluorescence intensity was quantified
(Aminco Bowman spectrofluorometer).
For the lipid substrates,
cholesteryl ester or tri-, di-, or monoglycerides were emulsified in
individual mixtures of 0.05% Triton X-100, 1.6% serum albumin (fatty
acid-free), 3.13% sodium taurocholate, and 0.15 M acetate, pH
5.5, by sonication on ice at 50 watts for 1 min. The reactions (1 ml)
with enzyme were stopped with 4 ml of isopropyl alcohol/sulfuric acid
(40:1, v/v). To extract the lipids, 2 ml of HO and 5 ml of
hexane were added to the mixture. After vigorous shaking, the hexane
phase was collected and evaporated to dryness under N
, and
the residues were solubilized in chloroform (500 µl). The various
lipids in 25-µl aliquots were resolved by TLC in
1,2-dichloroethane/methanol (98:2, v/v). Autoradiograms were developed
from TLC plates, and the bands were visualized by fluorography and/or
quantitated in a PhosphorImager (Molecular Dynamics). For activity,
hexane were fractions shaken for 10 min with 0.1 M KOH (1 ml),
and the lipids in the aqueous (alkali) phase were quantitated in a
scintillation spectrometer. All assays were conducted in duplicate, and
the results are the means of three experiments. Assays were linear in
the time frame used, and <10% of the substrates were cleaved during
these reactions.
To evaluate the effect of enzyme modifiers on hLAL
activity, the required amounts of DNP, THL, heparin sulfate, and
tunicamycin were added to the enzyme solution and incubated for 15 min
at ambient temperature prior to the addition of substrate. DNP and THL
were added in acetate/EtOH, and heparin sulfate and tunicamycin in
sodium acetate, pH 5.5. A blank solution of an acetate/EtOH mixture had
no effect on enzymatic activity. Enzyme kinetic data were analyzed
using Kaleidagraph version 3.0 (Abelbeck Software).
Active hLAL was present in cell lysates and spent medium of
Sf9 cells infected with recombinant baculovirus containing the hLAL
cDNA. Using the 4-MUO substrate, the apparent K values for LAL from lysates and medium were 20.3 ± 3.9 and
37.7 ± 5.2 µM, respectively. The respective V
values for these cell lysates were 501.4
± 20.2 nmol/min/mg of protein and 386.0 ± 14.53
nmol/min/10 ml of medium (Fig. 1). On immunoblotting, equal
amounts of enzymatic activity from cell lysates and medium gave about
equal densitometric signals (data not shown), indicating similar k
values from these sources. The pH activity
profile was bell-shaped, with a pH optimum for 4-MUO of 5.5 and
7-10-fold decreases in activity at pH 4.5 and 6.5 (data not
shown). The K
values were unchanged in this pH
range. Using hLAL from spent medium, the substrate specificity was
evaluated with 4-MU fatty acid esters (250 µM) of varying
alkyl chain lengths (Fig. 2). Substrates with acyl chains less
than C
were poorly hydrolyzed (
17% of C
rates). The maximal hydrolytic rates were obtained with the
C
alkyl esters. The C
-cis-4-MU (4-MUO) had 56% of the rate
achieved with the heptonate derivative. The C
-trans-elaidate ester was hydrolyzed at 21%
of the rate observed with 4-MUO. The stearate (C
) and
palmitate (C
) derivatives were very poor substrates.
Figure 1:
Effect of substrate concentration on
recombinant hLAL activity from Sf9 cell lysates () or spent
medium (&cjs2024;). The molar ratio of L-
-phosphatidylcholine to 4-MUO was the same for all
experiments. Data points represent the means ± S.E. of three
independent experiments performed in duplicate. The apparent K
and V
values
were calculated using the Kaleidagraph
program. Assays
were at 37 °C for 30 min.
Figure 2:
Substrate specificity of recombinant hLAL
from spent medium of Sf9 cells. The 4-MU fatty acid esters (250
µM) of varying acyl chain lengths were incubated at 37
°C. Values represent the means of three experiments performed in
duplicate. For clarity, error bars are not included, but were 10%.
, 4-MU oleate; ◊, 4-MU heptonate;
, 4-MU elaidate;
, 4-MU palmitate; ⊞, 4-MU stearate;
, 4-MU
propionate and 4-MU acetate were the same.
hLAL also hydrolyzed cholesteryl esters and tri-, di-, and
monooleins, but with 30-100-fold lower rates than obtained
with 4-MUO ( Table 1and Table 2). By using sn-1 or sn-3 randomly radiolabeled trioleins, we monitored the rates
of di- to monoolein conversion and oleic acid appearance. These
analyses showed that the accumulation of diolein was in excess of that
for monoolein, whereas the appearance of free oleic acid was rapid (Fig. 3). The ratio for production of diolein versus monoolein was
35. The diolein production rate was
2.5-fold greater than that for oleic acid. Similar results were
obtained in lysates from normal cultured skin fibroblasts at 60 min of
incubation. These results indicate substantial preference of hLAL for
the tri- and monooleins as compared with the dioleins when triolein was
the initial substrate. However, the enzyme did cleave triolein
completely. The apparent preference for tri- and monooleins was not
observed when individual tri-, di-, or monooleins were used as
substrates (Table 2), i.e. the rates of cleavage for
these pure substrates were very similar. Since sn-1 and sn-3 were randomly labeled, no preferential cleavage of these
substrates could be determined.
Figure 3: Cleavage of triolein by hLAL from Sf9 lysates (lanes 2, 5, and 8) and normal cultured skin fibroblasts (lanes 1, 4, and 7). Lanes 3, 4, and 9 are controls from Sf9 cells infected with nonrecombinant baculovirus. The reactions were conducted for 15 min (lanes 1-3), 30 min (lanes 4-6), and 60 min (lanes 7-9). The various lipids were resolved on polyester-supported silica gel plates with dichloroethane/methanol (50:1, v/v) as the running solvent.
To evaluate the similarities of
active-site function between hLAL and other lipases, the active
center-directed inhibitors DNP and THL were
used(30, 31) . DNP inhibition of hLAL activity from
cell lysates or medium fit a single exponential curve with an IC of
4.1 ± 0.14 µM at 250 µM 4-MUO substrate (Fig. 4A). Dilution experiments of
the fully DNP-inhibited enzyme did not result in recovery of enzymatic
activity, i.e. DNP is an irreversible inhibitor of hLAL.
THL's inhibition of hLAL was more complex and potent. The
inhibition curves did not fit a single exponential (Fig. 4B), suggesting that THL could be turned over by
hLAL. An IC
of
40 nM was estimated for this
inhibitor. Dilution experiments showed that THL is a reversible
inhibitor of hLAL. Heparin had a biphasic effect on hLAL: at low
concentrations (0-2.5 µg/ml), enzymatic activity was enhanced
2-fold (Fig. 5). In comparison, at higher concentrations
(>2.5 µg/ml), the activity was inhibited with an IC
of
25 µg/ml.
Figure 4:
Inhibition of recombinant hLAL activity by
DNP (A) and THL (B). Different concentrations of DNP
or THL were incubated with 5 µl of spent medium from Sf9 cells at
room temperature for 30 min. Substrate (250 µM 4-MUO)-containing liposomes were added, and the enzymatic activity
was determined. Control () and S99A (
) refer to wild-type
hLAL and mutant LAL with a Ser
Ala substitution,
respectively.
Figure 5:
Effect of heparin on recombinant hLAL
activity. Cell protein (; 0.5 µg) or spent medium (
;
5 µl) from Sf9 cells was incubated with different amounts of
heparin for 30 min at ambient temperature, and enzyme assays were done
at 37 °C for 30 min.
THL and DNP were shown to bind to putative
catalytic nucleophilic serines of other lipases in the conserved
sequence (GXSXG). hLAL has two such putative
active-site sequence surrounding Ser and
Ser
. To determine if either of these serines participates
in the catalytic process, each was individually substituted with
alanines that cannot participate as nucleophiles. The S99A (but not
S153A) enzyme was active to a similar degree as the wild-type enzyme (Table 1). The S153A enzyme was completely inactive to the 4-MUO
substrate, even though similar amounts of hLAL protein were detected by
immunoblotting of Sf9 cells expressing the normal, S99A, or S153A cDNA (Fig. 6A). The S99A enzyme also was inhibited by DNP
and THL with respective IC
values of 2.0 ± 0.1
µM and 34.0 ± 4.7 nM. S99A also had
essentially normal enzymatic activity toward cholesteryl ester and
tri-, di-, and monooleins ( Table 1and Table 2).
Figure 6:
Immunoblots of normal and mutant hLALs
expressed in Sf9 cells. Extracts from Sf9 cells expressing normal hLAL
cDNA or mutant hLAL were subjected to 12.5% polyacylamide gel
electrophoresis. The resulting immunoblots were visualized with rabbit
anti-hLAL antibody. Sf9 refers to lysates from wild-type
virus-infected Sf9 cells. C is a high density gel showing
faster migration of truncated AG302 mutant hLAL. MW refers to prestained molecular weight standards
(
10
).
Three
additional mutant hLALs were expressed in the Sf9 system, including
L179P, L336P, and AG302. These mutations in hLALs had been
identified homozygously or as compound heterozygotes in patients with
CESD and WD(5, 8, 9) . In lysates from Sf9
cells infected with the various mutant recombinant viruses, no
enzymatic activity toward 4-MUO and cholesterol esters and only very
low activity toward triolein could be detected (L179P and L336P) ( Table 1and Table 2). Immunoblots demonstrated the presence
of normal size hLAL in Sf9 cells infected with the L179P and L336P
recombinant viruses (Fig. 6). In comparison, the
AG302
protein had a smaller molecular weight, indicating the premature
termination of translation.
Although hLAL is targeted to the
lysosomes via the mannose 6-phosphate receptor system, it is unknown if
glycosylation is needed to preserve or to develop catalytically
competent conformers. The single molecular species of normal or mutant
hLALs could not be obtained in an active form in E. coli. In
Sf9 cells, two molecular weight forms, M
41,000 and
46,000, were detected at a ratio of
5:1 (Fig. 7). The M
46,000 form was the
predominant form in spent medium. To determine the relationships of
these forms, hLALs from cellular lysates and spent medium were
deglycosylated with N-glycanase. Only the M
41,000 form was detected following
deglycosylation. This indicates that the larger form was glycosylated
and the smaller form was either unglycosylated or severely
underglycosylated. Similarly, when Sf9 cells were treated with
tunicamycin prior to infection with the hLAL recombinant virus, only a
single band at M
41,000 was detected (Fig. 7). No enzyme was detected in the medium. By
immunofluoresence, the generalized intracellular distribution of hLAL
in Sf9 cells was unaltered by tunicamycin treatment (data not shown).
hLAL for tunicamycin-treated cells was catalytically inactive (Table 3).
Figure 7:
Effect of tunicamycin on hLAL expression
in Sf9 cells. Extracts of Sf9 cells (lane 2) and spent medium (lanes 3 and 4) expressing hLAL from normal cDNA were
subjected to 12.5% polyacrylamide gel electrophoresis. The spent medium
was concentrated 20-fold by trichloroacetic acid precipitation. MW refers to the prestained high molecular weight markers
(10
). Lane 1 contains hLAL expressed
in E. coli.
Although hLAL plays an essential role in the lysosomal
catabolism of cholesterol esters and triglycerides, its low level of
expression in most tissues has limited biochemical studies of this
enzyme. We and others (3) have recently cloned the cDNA for
hLAL, and except for minor polymorphic variants(10) , the coding sequences are identical. This facilitated the
heterologous expression of hLAL in mammalian and insect cell systems at
high levels for further characterization. The pVT plasmid vector was
used in these studies since it contained an insect-derived leader
sequence from honeybee that has been shown in other systems to
facilitate the expression of some mammalian proteins in insect
cells(23) . Our results show that this expression system
provides sufficient amounts of normal and mutant hLALs for biochemical
analyses of mutations associated with WD and CESD and to examine the
role of particular residues in the catalytic function. These results
provide insights into the roles of glycosylation and two putative
nucleophilic serines (Ser
and Ser
) in the
development of catalytically active hLAL as well as an appreciation for
the role of hLAL in WD and CESD.
N-Glycosylation,
particularly the presence of a mannose 6-phosphate residue, is
important to the lysosomal localization of many
hydrolases(32) . However, the potential role of glycosylation
in the formation or maintenance of catalytically active conformers of
lysosomal enzymes has not been fully explored. Acid -glucosidase,
the enzyme deficient in Gaucher's disease, is known to require
glycosylation at a site near the N terminus for the development of a
catalytically active conformer(33) . Based on deglycosylation
of hLAL with either endoglycosidase H or F, investigators concluded
that glycosylation was not important for the catalytic function of
hLAL(10, 22) . Indeed, a preliminary report suggested
that active hLAL could be expressed in bacterial systems that
completely lack the glycosylation apparatus(9) . We could not
achieve expression of active hLAL in E. coli using our pET
vector system. In addition, tunicamycin treatment of Sf9 cells infected
with the recombinant virus containing LAL resulted in the production of
a catalytically inactive hLAL. By immunofluorescence, no difference in
the distribution of hLAL between untreated and tunicamycin-treated Sf9
cells was observed; all unglycosylated hLAL was retained
intracellularly. In the absence of tunicamycin, two major molecular
weight forms of hLAL were detected by immunoblotting of cell lysates.
The lower molecular weight form (M
41,000)
appeared to be unglycosylated or severely underglycosylated, i.e. retained only the core N-acetylglucosamine residues or
was totally unglycosylated. The higher molecular weight species in
cells or in spent medium were glycosylated as demonstrated by mobility
shifts following N-glycanase treatment or treatment of cells
with tunicamycin. Also, equal amounts of high molecular weight (M
46,000) cross-reacting immunologic
material from hLAL within cells or in spent medium provided about the
same level of enzymatic activity. These results suggest that the lower
molecular weight form was unglycosylated and inactive. We conclude from
these above studies that cotranslational glycosylation of hLAL is
required for the formation of a catalytically active conformer, similar
to the results with acid
-glucosidase (33) . The
preservation of enzymatic activity following deglycosylation indicates
that occupancy of glycosylation sites is unnecessary for the
maintenance of a catalytically active conformer. These results suggest
that glycosylation may be involved in the global folding of hLAL and/or
in directing disulfide bond formation. We have no explanation for the
presence of large amounts of unglycosylated LAL in insect cells. This
has been noted with other proteins, herpes simplex virus-1 and -2
glycoprotein, and tissue plasminogen
activator(34, 35) , expressed in this system, but the
basis for this is not known.
Recombinant hLAL had similar catalytic
properties to those reported for lysosomal acid lipases from human
fibroblasts and liver and rabbit aorta and
liver(10, 20, 21, 22) . These
properties include a pH optimum of 5.3, a broad substrate
specificity, a preference for intermediate fatty acid acyl chain length
substrates and cis-unsaturated fatty acid acyl esters, K
values of 38 µM for 4-MUO, and
stabilization by bovine serum albumin and
-mercaptoethanol.
Despite the broad substrate hydrolysis, our studies show a clear
selectivity and structural requirements for recognition of substrates
by the enzyme. The kink at the ninth carbon in the oleate chain, due to
its
-cis-unsaturation, creates a linear
9-carbon chain for potential interaction with hLAL. The similar kinetic
properties for the 4-MU oleate and heptonate substrates raise the
interesting possibility that 7-9-carbon fatty acid acyl chain
lengths may be the ideal size for enzyme binding and catalysis by
accommodation in the active site. The absence of the double bond and
the rigidity of the long fatty acid chain in the stearate and palmitate
substrates may impair substrate interaction. Similarly, the 4-MU
elaidate, which has a
-trans-unsaturation,
was catalyzed 80% less efficiently by LAL than the corresponding
oleate. These preferences were further indicated by the cleavage of
triglyceride (triolein) to its di- and monoacylglycerols and fatty acid
derivatives. The accumulation of diolein during cleavage of the
triolein shows that diacylglycerols are poorer substrates for hLAL than
either triacylglycerols or monoacylglycerols. The studies with tri-,
di-, and monoacylglycerols as pure substrates also indicated a
potentially different binding mechanism, i.e. no differences
were found in the level of enzymatic activity or preference since the
same catalytic activities were obtained with each of the substrates.
This is in contrast to the clear preference for triolein or monoolein
substrates when triolein was used as the initial substrate. One
explanation for these results would be the inability of LAL to release
the diolein rapidly after removal of the first fatty acid acyl chain.
This would then require a rebinding and possibly reorientation so that
a preferential (sn-1 or sn-3) bond could be cleaved.
In contrast, when the diolein is supplied as a pure substrate, its
orientation for preferential bond cleavage in the active site would be
determined during the initial binding step. An equally plausible
explanation for this effect would be a slow reconformation of LAL that
was required for the cleavage of fatty acid from diolein compared with
the triolein or monoolein without release of the substrate. In either
case, there appears to be a preference for the sn-1- or sn-3-primary ester group compared with the secondary ester on
carbon 2. This is consistent with the lack of cleavage of
2-monooleylglycerol by rabbit LAL(18) . Although additional
studies will be required to elucidate the absolute substrate preference
using more highly purified enzymes, these results are consistent with
previous data of rabbit and human LAL substrates preferences. These
results are similar to those on other mammalian lipases that show
preference for primary ester bonds in substrates such as lipoprotein,
pancreatic, hepatic, and lingual
lipases(36, 37, 38) . In comparison, bile
salt-activable lipase and monoglycerol lipase lack positional
specificities(39, 40) .
The presence of two
putative active-site sequences (GXSXG) in hLAL
suggested the possibility that either one of these could determine the
substrate specificity for cholesteryl ester and/or triacylglycerols.
Since serine has been identified as an active-site nucleophile in
several lipases and we demonstrated DNP and THL inhibition of hLAL, a
serine was likely involved as the catalytic nucleophile for hLAL. By
mutagenizing serine 99 or 153 to alanine, we obliterated the
nucleophilic capacity of these residues. The results demonstrate that
serine 99 is uninvolved in the catalytic activity of hLAL, whereas
serine 153 is important, but not necessary, for the activity. Our
results do not support the complete separability of cholesteryl ester
and triglyceride activities as determined by serine 99 or 153. In
addition, serine 153 appears to be very important for the cleavage of
4-MUO, cholesteryl oleate, and triolein as well as mono- and
diacylglycerols. However, the S153A mutant retains some activity toward
triolein (4% of the wild type). This very low level of activity is
reproducible, whereas cleavage of diolein and monoolein as well as
cholesterol ester could not be demonstrated by this mutant enzyme. We
conclude that serine 153 is very important to the catalytic mechanism
of LAL, but is not necessarily the catalytic nucleophile. In
comparison, S99A mutant LAL had normal activity toward all substrates,
except the monoacylglycerol, with which activity was increased by
2-fold. This suggests that although Ser
is not
important as a catalytic nucleophile, conformational changes near
Ser
or its direct participation may influence catalytic
activity or entrance to the active site. In either event, it is not
essential to catalysis.
To explore the pathogenesis of WD or CESD,
we created the three identified mutations. These two autosomal
recessively inherited disorders result in the accumulation
preferentially of cholesteryl esters in CESD or cholesteryl esters and
triglycerides in Wolman's disease. Wolman's disease is a
much more severe disorder, leading to death in the first year of life,
whereas CESD may be compatible with long-term survival(2) .
Several investigators have suggested that the primary differences in
phenotype in WD and CESD relate to the level of residual LAL activity
present in various tissues(29, 41, 42) .
Expression of each of the mutant enzymes in Sf9 cells indicated
exceedingly low to absent cholesterol ester cleavage activity for any
of the enzymes. In comparison, the L179P and L336P enzymes showed
reproducible cleavage (4% level) of triglyceride, whereas the
AG302 enzyme was inactive. In addition, the L336P mutant had very
low levels of activity toward the diacylglycerol substrate. The overall
conclusion is that cholesterol esterase activity is absent in all three
mutant enzymes, whereas low level triglyceride activity is present in
L179P and L336P. It is instructive to examine the genotypes and
phenotypes of the patients who had these mutant alleles. For example, a
L336P homozygote had CESD, whereas L179P was found in CESD and WD in
the presence of null alleles, i.e. an exon 8 splice junction
and frameshift mutations, respectively. Thus, from the heteroallelic
CESD and WD variants, no enzymatic basis is evident for distinguishing
between CESD and WD. Although it is possible that our in vitro studies do not adequately reflect the in situ residual
enzymatic activity, these findings support the hypothesis of Burke and
Schubert (42) that other factors or mechanisms besides LAL must
be operative to account for the selective accumulation of cholesteryl
esters and triglycerides in CESD and WD.