From the
In the present study, lecithin-cholesterol acyltransferase
(LCAT) catalyzed esterification of oxysterols was investigated by using
discoidal bilayer particles (DBP) containing various oxysterols,
phosphatidylcholines, and apolipoprotein A-I. The esterified oxysterols
were analyzed by high pressure liquid chromatography, gas
chromatography, and mass spectrometry. LCAT esterified all oxysterols
tested that are known to be present in human plasma. The esterification
yields in almost all cases were relatively high, often as high as the
yield of cholesterol esterification. When DBP preparations containing
27-hydroxycholesterol and various phosphatidylcholines were used for
the LCAT reaction, both monoesters and diesters were produced. The mass
spectrometry analysis showed that the monoester was produced by the
esterification of the 3
Lecithin-cholesterol acyltransferase (EC 2.3.1.43)
(LCAT)
In the human body, oxysterols are derived from the diet
primarily as products of cholesterol autoxidation
(14) or
produced endogenously (15). Some oxysterols are normal intermediates in
the metabolic pathways; for instance, 7
It has been
speculated that the LCAT reaction promotes the reverse transfer of
cholesterol
(1) . The removal of oxysterols from peripheral
tissues may also be promoted by the LCAT reaction. It is known that
oxysterols in human plasma are found not only in the free form but also
as fatty acyl esters
(13, 24, 26, 27) .
The esters of 27-OH-C were detected at more than five times higher
concentrations than the esters of other dihydroxysterols
(24) .
In the present study, we examined the esterification, by the LCAT
reaction, of oxysterols most frequently identified in human plasma. By
using 27-OH-C, we investigated the factors influencing the formation of
mono- and diesters.
Experiments were
performed, unless otherwise stated, in 39 mM phosphate buffer
(ionic strength 0.1, pH 7.4) containing 0.025% EDTA and 2 mM
sodium azide.
The mass spectra of palmitoyl diester of
27-OH-C obtained by the LCAT reaction (I) were identical
with those of the diester obtained synthetically. The palmitoyl diester
exhibited the highest abundance of
[M-2RCOOH]
Mass spectrometry analysis of oleoyl monoesters separated
by HPLC revealed that all oxysterols tested produced 3
The
LCAT reaction with various oxysterols and dioleoyl-phosphatidylcholine
was carried out primarily to characterize the esterification products
and to determine the possibility of diester formation. Therefore, the
incubation was performed at 37 °C for 1 h in the presence of excess
enzyme to obtain as much product as possible. Even under such
incubation conditions, the extent of esterification of oxysterol varied
widely, ranging from 30 to 90%. When cholesterol was substituted for
oxysterols, approximately 86% of cholesterol was esterified under
identical conditions. Among oxysterols tested, 7
In order to determine other factors
influencing the proportions of mono- and diesters in DBP preparations
containing 27-OH-C and dioleoyl-phosphatidylcholine, we studied the
effects of LCAT and DBP concentrations on the esterification
(Fig. 4). An increase in the LCAT concentration from 0.5 to 2.5
µg/ml not only increased the esterification yield but also changed
the relative amounts of mono- and diesters produced. At an LCAT
concentration as low as 0.5 µg/ml, 7.5 nmol of 27-OH-C was
esterified. A higher LCAT concentration, 5.38 µg/ml, gave an
esterification of about 13 nmol of 27-OH-C. While the esterification at
low concentrations yielded mainly monoesters, higher concentrations of
LCAT produced preferentially diesters. The plateau of the monoester
curve at LCAT concentrations higher than 2.5 µg/ml indicates that a
small part of monoester (approximately 15% of total esters) cannot be
converted into diesters. The effect of DBP concentrations on the
esterification of 27-OH-C was studied at a LCAT concentration of 5.2
µg/ml, the highest concentration used in the experiment for
Fig. 4
. Higher concentrations of DBP preparations resulted in
lower yields of diester and higher yields of monoester, thus giving
lower diester:monoester ratios (data not given). It was apparent that
the LCAT:DBP ratio governed the yields of mono- and diesters.
The ratio between 3
Significant amounts of
diesters were formed with the DBP preparations containing 27-OH-C and
phosphatidylcholine having saturated and unsaturated acyl groups at
sn-1 and sn-2 positions, respectively, such as
1-palmitoyl-2-oleoyl-phosphatidylcholine,
1-stearoyl-2-oleoyl-phosphatidylcholine, and
1-palmitoyl-2-linoleoyl-phosphatidylcholine (). We noted
that as much as 25% of diesters were produced with DBP containing
27-OH-C and 1-palmitoyl-2-oleoyl-phosphatidylcholine upon increasing
the enzyme concentration. It may be generalized that the
3
In the present study we have shown that all
oxysterols detected in human plasma are esterified by LCAT to form
3
DBPs containing
27-OH-C or cholesterol were incubated with LCAT as described under
``Experimental Procedures.'' The concentration of LCAT in the
reaction mixture was 5.2 µg/ml, and the incubation time was 1 h.
The distribution of the esters produced is given in mole percent with
respect to the initial amount, 40 nmol, of 27-OH-C or cholesterol used
for the LCAT reaction. Diesters of 27-OH-C obtained in detectable
amounts are listed. DBP containing 16:0/16:0-PC was prepared at 37
°C and dialyzed at room temperature. Duplicate experiments were
carried out. For each experiment, two HPLC assays were done, and four
values obtained were used to obtain the mean values and the standard
deviations.
We express our appreciation to J. Jerrell for mass
spectrum determination.
-hydroxyl group and not the 27-hydroxyl
group. The diesters were apparently produced by the esterification of
the 27-hydroxyl group only after the esterification of the
3
-hydroxyl group. Phosphatidylcholine containing a saturated acyl
group at sn-1 position and an unsaturated acyl group at sn-2 position
gave generally high esterification yield. The esterification of various
oxysterols was compared by using DBP containing
dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols
produced 3
-oleoyl monoesters. Unlike 27-hydroxycholesterol,
25-hydroxycholesterol, 7
-hydroxycholesterol,
7
-hydroxycholesterol, or cholestanetriol did not produce diesters.
Various factors influencing the formation of the monoesters and
diesters from 27-hydroxycholesterol were investigated. When
dioleoyl-phosphatidylcholine was used as the acyl donor, prolonged
dialysis of DBP preparations and increase in the ratio of the enzyme
concentration to substrate particle concentration increased the diester
formation. Significant amounts of diesters were also produced by using
1-palmitoyl-2-oleoyl-phosphatidylcholine and other phosphatidylcholines
as the acyl donors. By analyzing the conditions of monoester and
diester formation, a scheme for the LCAT reaction pathway was proposed.
(
)
catalyzes the transfer of the
phosphatidylcholine acyl group at sn-2 position to cholesterol, forming
lysophosphatidylcholine and cholesteryl
ester
(1, 2, 3, 4) . The cholesterol
present in discoidal high density lipoprotein particles containing
apolipoprotein (apo) A-I is preferentially esterified by the LCAT
reaction
(5) . Many studies have dealt with the specificity of
LCAT for acyl donor
phospholipids
(2, 6, 7, 8, 9) .
There are also studies relating LCAT specificity to the acyl acceptor.
It has been shown that not only cholesterol but also various other
sterols
(10) , saturated long-chain alcohols
(11) , and
water
(11, 12) can serve as the acyl acceptors. Although
the involvement of LCAT in the esterification of
7
-hydroxycholesterol (7
-OH-C) was speculated
(13) , no
study has been carried out concerning LCAT-catalyzed esterification of
oxysterols.
-OH-C is produced from
cholesterol by liver microsomal 7
-hydroxylase, the rate-limiting
enzyme in bile acid biosynthesis
(16) . The interest in
oxysterols is mainly due to their various biological effects:
cytotoxicity, inhibition of
-hydroxy-
-methylglutaryl CoA
reductase activity, activation of acyl-CoA acyltransferase, and
inhibition of low density lipoprotein binding, which have been
extensively reviewed
(17) . In addition, some oxysterols may be
involved in the atherosclerotic processes
(17) . Human aortic
tissue is known to contain 27-hydroxycholesterol (27-OH-C)
(
)
as a free sterol
(18, 19, 20, 21) and its monoesters
(22) and
diesters
(23, 24) . The aortic contents of 27-OH-C were
related to the severity of
atherosclerosis
(19, 21, 25) .
Materials
Cholesterol (>99%), cholesteryl oleate (CE 18:1),
cholesteryl linoleate (CE 18:2), cholesteryl palmitate (CE 16:0),
cholesteryl pentadecanoate (CE 15:0), cholesteryl arachidonate (CE
20:4), cholestanetriol (Triol-C), 7-OH-C, cholesterol
5
,6
-epoxide (
-epoxy-C), 20
-hydroxycholesterol,
apoA-I from human plasma (97%),
dipalmitoyl-sn-glycero-3-phosphorylcholine (16:0/16:0-PC),
dioleoyl-sn-glycero-3-phosphorylcholine (18:1/18:1-PC),
dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2/18:2-PC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine
(18:0/20:4-PC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphorylcholine
(16:0/18:2-PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine
(16:0/18:1-PC), 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine
(18:1/16:0-PC), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphorylcholine
(18:0/18:1-PC), sodium azide, sodium cholate, and 2-mercaptoethanol
were obtained from Sigma. Diarachidonoyl-sn-glycero-3-phosphorylcholine
(20:4/20:4-PC) and
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine
(16:0/20:4-PC) were purchased from Avanti Polar Lipids, Inc.
(Alabaster, AL). 27-Hydroxycholesterol (27-OH-C), cholesterol
5
,6
-epoxide (
-epoxy-C), and 7
-OH-C were obtained
from Research Plus, Inc. (Bayonne, NJ). Steraloids, Inc. (Wilton, NH)
provided 7-ketocholesterol (7-keto-C), 25-hydroxycholesterol (25-OH-C)
and 5-cholestene-25-hydroxy-3
-oleate
(25-OH-C[3
]18:1). Fatty acid-free bovine serum albumin
was purchased from Boehringer Mannheim. Bis(trimethylsilyl)
trifluroacetamide and boron trifluoride in methanol (12%, w/w) were
purchased from Supelco, Inc, (Bellefonte, PA). Ethyl alcohol, USP
absolute, was obtained from Midwest Grain Co. (Pekin, IL).
Acetonitrile, chloroform, hexane, and methanol of HPLC grade were
purchased from Fisher Scientific (Fair Lawn, NJ).
Synthesis of 27-Hydroxycholesterol Palmitate Esters
The monoesters
(25R)-R-cholestene-27-hydroxy-3-palmitate
(27-OH-C[3
]16:0) and 27-palmitate
(27-OH-C[27]16:0) and the diester 3
,27-dipalmitate
(27-OH-C[3
]16:0[27]16:0) were synthesized and
purified essentially according to the method of Teng et al.(28) to use as standards for HPLC, GC, and mass spectrometry.
The identity of the esters was confirmed by mass spectrometry.
Preparation of DBP
DBP preparations containing phosphatidylcholine and
cholesterol or oxysterol were prepared essentially according to the
method of Kato et al.(29) . An ethanol solution (17.5
µl) containing 0.1 µmol of cholesterol or oxysterol and 0.5
µmol of phosphatidylcholine was rapidly injected with a Hamilton
microsyringe into 0.4 ml of phosphate buffer while stirring under a
stream of N. After addition of 38 µl of 200 mM
sodium cholate and 40 µl of apoA-I solution (2.5
mg/ml)
(29) , the mixture (0.49 ml) was dialyzed at 4 °C
under N
against phosphate buffer with stirring for 2 days
with four changes of 2 liters each of buffer. After dialysis, the
solution was adjusted to 0.5 ml with phosphate buffer and used
immediately for experiments. For some preparations, indicated by
Dialysis-4 days (Fig. 3), the samples were kept in
dialysis bags with stirring for 2 additional days.
Figure 3:
Effect of dialysis period for DBP
preparations on the LCAT reaction. DBP preparations containing 27-OH-C
and dioleoyl-phosphatidylcholine were prepared according to the
procedure given under ``Experimental Procedures.'' Dialysis
periods of 2 and 4 days designate the samples used immediately after 2
days of dialysis and those kept in dialysis bags for 2 additional days,
respectively, before the LCAT reaction. Incubation of the reaction
mixtures was carried out as described in the legend to Fig. 1. Results
are mean ±S.D. of four experiments. Filled bar,
27-OH-C; diagonally striped bar,
27-OH-C[3]18:1; vertically striped bar,
27-OH-C[3
]18:1[27]18:1; openbar,total ester.
LCAT Reaction
LCAT was purified from human plasma according to the method
described previously
(29, 30) . The final purification of
the enzyme was approximately 20,000-fold over the starting plasma. The
LCAT reaction was carried out using 200 µl of DBP solution and
purified LCAT with the amounts given in the text in the presence of 4
mM 2-mercaptoethanol and 5 mg of bovine serum albumin in
phosphate buffer in the final volume of 500 µl as described
previously
(30) . The reaction was stopped by adding 2 ml of
methanol to the incubation mixture and heating at 55 °C under
nitrogen for 30 min. Deviation from this general procedure is given in
the text.
Purification and Analysis of the Reaction Products
Extraction of Unreacted Oxysterols and of Oxysterol
Esters
A known amount of the internal standard, cholesteryl
pentadecanoate (CE 15:0) or cholesteryl palmitate (CE 16:0) in
CHCl, was added to the incubation mixtures treated with
methanol after the LCAT reaction. Lipids were extracted with 6 ml of
hexane-chloroform (4:1, v/v) as described previously
(30) .
Reversed Phase HPLC
HPLC analyses of lipid
extracts obtained after the LCAT reaction were carried out in the
reversed phase mode using a Waters Liquid Chromatography system with
Adsorbshere C18 (5 µm) column, 150 4.6 mm (Alltech
Associates, Inc.). For HPLC separation of the lipid extracts, mobile
phase of acetonitrile:isopropyl alcohol (1:1, v/v) with a flow rate of
1.0 ml/min was used. For the analysis of free oxysterol and
purification of monoesters, they were rechromatographed on the same
HPLC column using a solvent mixture, acetonitrile:isopropyl alcohol
(90:10, v/v), with a flow rate of 1.8 ml/min. The separated compounds
were subjected to GC fatty acid analysis, GC, and mass spectrometry
analyses.
Derivatization and Gas Chromatography
For
quantitative analyses of cholesterol and oxycholesterol by GC, the
samples were derivatized to form trimethylsilyl ethers in a similar
manner to that described by Maerker and Unruh
(31) . GC was
performed on a Hewlett-Packard 5890 Series II GC equipped with a
programmable cool-on column inlet for the capillary column, flame
ionization detector, and a 30-m 0.25-mm inner diameter fused
silica capillary DB5 column (J & W Scientific, Folsom, CA). For
fatty acid analysis, oxysterol esters and cholesteryl esters were
subjected to boron trifluoride-catalyzed methanolysis according to the
method of Morrison and Smith
(32) . Fatty acid methyl esters were
analyzed with a Hewlett-Packard gas chromatograph (model 5790) equipped
with a flame ionization detector and Supelcowax-10, 30-m
0.32-mm inner diameter fused silica column (Supelco, Inc., Bellefonte,
PA).
Mass Spectroscopy
Mass spectra were recorded on an
HP 5985B GC/mass spectrometry system operated in the electron impact
mode (20 eV). Samples were introduced by the direct probe and distilled
from the probe at 60-350 °C (30 °C/min).
Analysis of the Products of LCAT Reaction
The
products of the esterification of cholesterol and 27-OH-C by the LCAT
reaction with 1-palmitoyl-2-oleoyl-phosphatidylcholine were separated
by HPLC (Fig. 1). DBP preparations containing the
phosphatidylcholine and cholesterol or 27-OH-C were used as the
substrate particles. The cholesterol esterification yielded cholesteryl
oleate (panel A, peak 2) and cholesteryl palmitate
(peak3). The esterification of 27-OH-C produced both
monoesters and diesters. The monoesters, 27-OH-C[3]18:1
((25R)-5-cholestene-27-hydroxy-3
-oleate) (panelB, peak5) and
27-OH-C[3
]16:0 (3
-palmitate) (peak6), showed considerably lower retention time than the
corresponding cholesteryl esters. The appearance of smaller peaks of
palmitoyl esters of 27-OH-C and cholesterol as compared with
corresponding oleoyl esters reflects considerably higher specificity of
LCAT for the acyl group of phosphatidylcholines at sn-2 position than
that at sn-1 position
(2, 33, 34) . Quantitative
data with respect to the positional specificities are presented in a
later section (). The diesters of 27-OH-C produced were
27-OH-C[3
]18:1[27]18:1
((25R)-5-cholestene-3
,27-dioleate) (panelB, peak7),
27-OH-C[3
]16:0[27]16:0 (3
,27-dipalmitate)
(panelB, peak9), and the mixtures
of 27-OH-C[3
]18:1[27]16:0 (3
-oleate,
27-palmitate) and 27-OH-C[3
]16:0[27]18:1
(3
-palmitate, 27-oleate) (panelB, peak8). These diesters gave considerably longer retention
time than the monoesters. The retention times of the monoesters and the
diesters that were obtained with a mobile phase of
acetonitrile:isopropyl alcohol (1:1, v/v) are included in .
In view of relatively close retention times of the monoesters, the
second mobile phase of acetonitrile:isopropyl alcohol (9:1, v/v), was
used to obtain better separation of monoesters (). The
monoesters in this solvent system were collected for GC and mass
spectrometry analyses. The diesters were collected in the first solvent
system for the analyses.
Figure 1:
HPLC separation of lipid extracts
obtained from the reaction mixtures for esterification of cholesterol
(A) and 27-OH-C (B) with
1-palmitoyl-2-oleoyl-phosphatidylcholine by LCAT. DBP preparations
containing 40 nmol of cholesterol or 27-OH-C and 200 nmol of
1-palmitoyl-2-oleoyl-phosphatidylcholine were incubated for 1 h with
2.6 µg of LCAT in the final volume of 0.5 ml as described under
``Experimental Procedures.'' The lipid extract was applied to
a column of Adsorbsphere C18 (150 4.6 mm). Isopropyl
alcohol:acetonitrile (1:1, v/v) was used as the mobile phase, at the
flow rate of 1.0 ml/min. The absorbance at 205 nm versus the
retention time was plotted. The numbers1-9 label the peaks of cholesterol (peak 1), CE18:1 (peak
2), CE16:0 (peak 3), 27-OH-C (peak 4),
27-OH-C[3
]18:1 (peak 5),
27-OH-C[3
]16:0 (peak 6),
27-OH-C[3
]18:1[27]18:1 (peak 7),
27-OH-C[3
]18:1[27]16:0 with
27-OH-C[3
]16:0[27]18:1 (peak8), 27-OH-C[3
]16:0[27]16:0
(peak9), and CE 15:0 (internal standard)
(IS).
The fatty acid composition of the mono- and
diesters produced from 27-OH-C were verified by GC analysis of the
fatty acid methyl esters. Oxysterol components of the esters were
analyzed by GC analysis after saponification and silylation. The mass
spectra data for mono- and diesters of 27-OH-C are included in Tables
II and III, respectively. All fragment ions listed could be ascribed to
relatively simple cleavage processes and were assigned on the basis of
their analogy with published spectral data. The mass spectra analysis
of monoesters showed that the hydroxyl group of 27-OH-C are esterified
at the position of C-3 and not at the position of C-27. This
conclusion is based on the following observations. The
3
-monoesters exhibited a high abundance of
[M-RCOOH]
(). The
synthetic 27-monoesters of palmitic acid exhibited a high abundance of
[M-Sc-H
O]
and of
[M-H
O]
(,
Footnote c). Moreover, the 27-monoester exhibited
[M-85]
and
[M-111]
representing
[M-H
O-C
H
]
and
[M-H
O-C
H
]
,
respectively. These two ions are known to be produced from
-ene-3
-alcohols but not from their
3
-esters
(28, 35) . These two ions were absent in
the spectra of the 3
-monoesters of 27-OH-C obtained by the LCAT
reaction ().
while the oleoyl and
linoleoyl diesters gave the highest abundance of
[M-RCOOH-Sc]
. Apparently the
side chain became more susceptible for cleavage with unsaturated
diesters. In the case of diesters of 27-OH-C with two different acyl
groups (Fig. 1B, peak8), mass spectra
data proved the presence of palmitoyl
([M-RCOOH]
, m/z 648,
19%) and oleoyl ([M-RCOO]
,
m/z 623, 42%) groups.
LCAT Esterification of Different Oxysterols Incorporated
into DBP
The esterification of various oxysterols by the LCAT
reaction was investigated by using DBP containing
dioleoyl-phosphatidylcholine and individual oxysterols.
Dioleoyl-phosphatidylcholine was used instead of
1-palmitoyl-2-oleoyl-phosphatidylcholine to simplify the analysis of
the esterification products. The HPLC analysis of lipid extracts from
all incubation mixtures revealed that only 27-OH-C gave, in addition to
one monoester peak, one diester peak. The retention times of the
monoester and diester were 7.9 and 33.6 min, respectively, when
analyzed with the mobile phase of acetonitrile:isopropyl alcohol (1:1,
v/v) (). All other oxysterols gave the single peaks of
monoesters with retention times as given in . No hydroxyl
groups, other than 3 hydroxyl groups, of 25-OH-C, 7
-OH-C,
7
-OH-C, and Triol-C were esterified by the LCAT reaction. Only
monoester formation can be expected from 7-keto-C,
-epoxy-C, and
-expoxy-C. The retention times of oleoyl monoesters of oxysterols
() generally agree with the polarity of the sterol
moieties.
-monoesters.
The lack of molecular ions in electron impact spectra, because of
facile elimination of fatty acids from
-ene-3
-esters, is well
documented
(36, 37) . The oleoyl monoesters of 27-OH-C,
25-OH-C, 7
-OH-C, and 7
-OH-C exhibited the highest abundance
of [M-RCOOH]
().
Furthermore, synthetically prepared 3
-oleoyl monoester of
25-hydroxycholesterol (25-OH-C[3
]18:1) gave identical
fragmentation as obtained for the oleoyl monoester of 25-OH-C produced
by the LCAT reaction. The monoester of 7-keto-C gave the highest
abundance of [M-RCOO]
, and
[M-RCOOH]
was absent. This ion
formation may be characteristic of 7-keto-C oleoyl monoester and
indicates the 3
-monoester formation. Oleoyl monoesters of both
-epoxy-C and
-epoxy-C gave the highest abundance of
[M-RCOO-H
O]
, which
may be the characteristic ion produced from the 3
oleoyl
monoesters of epoxy-C having an epoxy group between carbons 5 and 6.
The oleoyl monoester of Triol-C gave the highest abundance of
[M-RCOO-H
O]
and also a
very high abundance of
[M-RCOO-2H
O]
, which
indicated that the two hydroxyl groups at carbon 5 and 6 are easily
eliminated as the oleoyl group esterified at 3
-position.
-OH-C, 7-keto-C,
and
-epoxy-C gave the highest degree of esterification. 25-OH-C
and 7
-OH-C gave about 70% of esterification, and
-epoxy-C
yielded about 50% of esterification. Triol-C was not an effective
substrate for the esterification, possibly reflecting the pronounced
hydrophilicity of the sterol. 27-OH-C was unique among oxysterols as
described in later sections, by giving lower degrees of esterification
and yet substantial degrees of diester formation.
Time Dependence of the Esterification of 27-OH-C with
1-Palmitoyl-2-oleoyl-phosphatidylcholine by LCAT
The time course
of the LCAT reaction for 27-OH-C is presented in Fig. 2,
panelA, and that of the esterification of
cholesterol is given in panelB for comparison.
Almost half of the initial amount of 27-OH-C was esterified after 15
min as apparent from the decrease in free 27-OH-C and increase in total
esters produced. The total amount of 27-OH-C esters formed reached a
plateau at 1 h of reaction time. Although the initial rate of
cholesterol esterification was greater than that of 27-OH-C, the
overall esterification yields became similar for cholesterol and
27-OH-C after l h of reaction. For both 27-OH-C and cholesterol, the
oleoyl esters were preferentially formed as compared with palmitoyl
esters, reflecting the enzyme
specificity
(2, 33, 34) . The yield of palmitoyl
esters was about one-tenth that of the oleoyl monoesters (Fig. 2,
and ). Among the diesters produced, only the oleoyl
diester of 27-OH-C was plotted. The amounts of this diester and other
diesters produced are given in , and the formation of a
very small amount of palmitoyl diester was already indicated
(Fig. 1, peak9).
Figure 2:
Time course of the LCAT reaction for
27-OH-C and cholesterol. DBP preparations containing 20 nmol of 27-OH-C
(panelA) or cholesterol (panelB)
and 100 nmol of 1-palmitoyl-2-oleoyl-phosphatidylcholine were incubated
with 1.3 µg of LCAT in the final volume of 0.25 ml as described
under ``Experimental Procedures.'' The enzymatic reaction was
stopped by adding methanol to each incubation mixture at various time
periods. Results are means of four separate experiments. Typical
standard deviations for values of free and esterified forms greater
than 10 nmol were less than 0.5 nmol and for values less than 10 nmol
were a maximum of 1.2 nmol. PanelA shows 27-OH-C
(⊡), 27-OH-C[3]18:1 (
),
27-OH-C[3
]16:0 (
),
27-OH-C[3
]18:1[27]18:1 (
), and total
esters (
). PanelB shows cholesterol
(⊡), CE 18:1 (
), CE 16:0 (
), and total esters
(
).
Factors Influencing the Esterification of 27-OH-C with
Dioleoyl-phosphatidylcholine by LCAT
When DBP preparations
containing 27-OH-C and dioleoyl-phosphatidylcholine were used as
substrates for LCAT reaction, significant variations were noted in the
amounts of monoesters and diesters produced depending upon the length
of dialysis period (Fig. 3). When the DBP preparations dialyzed
for 2 days were used immediately for the LCAT reaction, it gave about
43% of unreacted 27-OH-C, 55% of monoesters, and 2% of diesters. In
contrast, the DBP kept in the dialysis bag for 2 additional days
reduced the extent of esterification, giving about 70% of unreacted
27-OH-C. Furthermore, the diester formation was considerably greater
than the monoester formation. It was observed that the increased
dialysis of the DBP preparations results in the formation of the
particles with considerably larger molecular weight. The DBP dialyzed
for 2 days gave the apparent particle mass of 200 kDa upon gel
permeation chromatography on Biogel A 1.5 m column. The 4-day dialysis
gave the particle masses ranging from 800 kDa to 1.2 10
Da. The size of DBP preparations might have influenced the extent
of esterification as well as the formation of mono- and diesters. When
27-OH-C and phosphatidylcholine containing both saturated and
unsaturated acyl groups, such as
1-palmitoyl-2-oleoyl-phosphatidylcholine, were used to prepare DBP, no
significant differences in the particle weight and in the
esterification were observed by the increased dialysis period. Both
preparations dialyzed for 2 and 4 days gave an apparent particle mass
of approximately 200 kDa.
Figure 4:
Influence of LCAT concentration on the
esterification of 27-OH-C in DBP. DBP preparations containing
dioleoyl-phosphatidylcholine and 27-OH-C with a dialysis period of 4
days were used for the LCAT reaction. The reaction mixtures contained
40 nmol of 27-OH-C and 200 nmol of dioleoyl-phosphatidylcholine in 200
µl of DBP preparations and various amounts of LCAT in a final
reaction volume of 0.5 ml. Reaction time was 1 h. Duplicate experiments
and duplicate HPLC assays for each sample were carried out, and the
mean values and the standard deviations refer to the four values thus
obtained. , 27-OH-C[3
]18:1;
,
27-OH-C[3
]18:1[27]18:1;
, total
esters.
LCAT Reaction with 27-OH-C and Various
Phosphatidylcholines in DBP
This experiment was carried out to
determine the effects of various phosphatidylcholines on the
esterification of 27-OH-C, especially the formation of the diesters, by
using the DBP preparations dialyzed for 2 days. The acyl donor
activities of various phosphatidylcholines for the esterification of
27-OH-C were compared with the donor activities for the cholesterol
esterification. All phosphatidylcholines tested were able to donate
acyl groups to 27-OH-C (). With the majority of
phosphatidylcholines tested, comparable degrees of esterification
yields were obtained for 27-OH-C and cholesterol.
Dipalmitoyl-phosphatidylcholine exhibited the lowest esterification of
both 27-OH-C and cholesterol among various phosphatidylcholines tested.
This reflects the limited fluidity of the bilayer structures containing
saturated phosphatidylcholine
(7) . The yield of esterification
of 27-OH-C was high with 1-palmitoyl-2-oleoyl-phosphatidylcholine,
1-stearoyl-2-oleoyl-phosphatidylcholine, and
1-palmitoyl-2-linoleoyl-phosphatidylcholine. With all
phosphatidylcholine containing saturated acyl group at sn-1 position
and unsaturated acyl group at sn-2 position, the acyl group at sn-2 was
transferred preferentially. The distributions of these sn-1 and sn-2
acyl groups in the monoesters of 27-OH-C were similar to their
distributions in cholesterol esters. The extent of the utilization of
sn-1 acyl group by LCAT for cholesterol esterification was in agreement
with previous results
(2, 33, 34) . The diesters
of 27-OH-C were also formed in variable proportions for each
phosphatidylcholine tested. The highest level of diester formation
occurred with dilinoleoyl-phosphatidylcholine.
1-Palmitoyl-2-oleoyl-phosphatidylcholine,
1-stearoyl-2-oleoyl-phosphatidylcholine, and
1-pal-mitoyl-2-linoleoyl-phosphatidylcholine gave considerably higher
degrees of diester formation than dioleoyl-phosphatidylcholine under
the conditions employed. Regardless of the types of phosphatidylcholine
used for the esterification, only 3-monoesters were formed as
indicated by the mass spectra data () already described.
DISCUSSION
In the present study, the esterification of a variety of
oxysterols by LCAT was investigated. The LCAT reaction of oxysterols
having more than one hydroxyl group, unlike that of cholesterol,
requires the determination of the location and number of the hydroxyl
groups esterified. The esterification of 3-hydroxyl group of all
oxysterols suggests that this hydroxyl group satisfies the steric
requirement for the transfer of an acyl group from the acyl-enzyme
intermediate
(3, 10) . The diester formation by the
esterification of the side chain 27-OH group proceeded only after the
esterification of the 3
-hydroxyl group. Apparently, the 27-OH
group that is located at the end opposite to the 3
ester oxygen
can be positioned at the enzyme active site for the esterification
after the monoester formation. Such an optimal positioning might have
not been obtained with other dihydroxysterols, such as 25-OH-C,
7
-OH-C, and 7
-OH-C, for diester formation with
dioleoyl-phosphatidylcholine.
-monoesters
and diesters produced from 27-OH-C as well as the yield of the esters
seems to be influenced by the physical characteristics of the DBP such
as the particle size. It is possible that the aggregation of DBP
containing 27-OH-C and dioleoyl-phosphatidylcholine may reduce the
surface flexibility of the substrate particles and interfere with the
removal of products from the active site of the bound enzyme. This
would increase the retention time of monoesters at the active site and
would enhance the formation of diesters especially when enzyme
concentration is high (Fig. 4). The highest LCAT concentration
(5.4 µg/ml) used in this study is roughly the physiological
concentration of LCAT in human plasma.
-monoester of 27-OH-C produced at the active site of LCAT has two
possibilities: 1) to leave the active site and return to the surface
layer of DBP or 2) to position itself into the hydrophobic cavity of
the enzyme, making the 27-hydroxyl group available for the diester
formation. The pathway proposed in Fig. 5could explain the
formation of both monoesters and diesters. The adsorption of the enzyme
to lipid-water interface leads to accommodation or binding of
phosphatidylcholine and 27-OH-C to the enzyme active site (step 1) and
to the formation of the acyl enzyme, E-Ac (step 2). The acyl group is
transferred to the bound 27-OH-C, thus producing the bound monoester
(step 3). The enzyme-3
-monoester complex could release the
monoester and the free enzyme (step 4) or could lead to the formation
of enzyme-3
-monoester-phosphatidylcholine complex (step 5), which
can be converted to an acyl-enzyme-3
-monoester complex (step 6).
The acylation of the bound 3
-monoester by the acyl group of the
enzyme complex could lead to the formation of the enzyme-bound diester
(step 8), which is subsequently released from the enzyme (step 9). The
diester formation (step 8) may require the flip-flop of the
3
-monoester for the correct positioning of the 27-hydroxyl group
near the active site of the acyl-enzyme. Alternatively,
3
-monoester can be released from the acyl-enzyme transition
complex upon displacement by 27-OH-C (step 7). Thus, 3
-monoester
formation could proceed through the cyclic reaction pathway (steps 3,
5, 6, and 7), by incorporating phosphatidylcholine (step 5) and 27-OH-C
(step 7) and by releasing lysophosphatidylcholine (step 6) and
3
-monoester (step 7).
Figure 5:
Proposed mechanism for the formation of
mono- and diesters of 27-OH-C by the LCAT reaction. E, enzyme;
E-Ac, acyl-enzyme.
The LCAT reaction usually occurs at the
lipid-water interface, and efficient transfer of the enzyme between
substrate particles is required
(38) . The transfer of the enzyme
is preceded by the enzyme desorption, which could occur, for example,
in step 4 (Fig. 5), leaving the product and also substrates at
the lipid-water interface
(38) . The cyclic reaction, therefore,
would occur only during the periods that the enzyme is associated with
substrate particles. The LCAT reaction may best be explained by an
interrupted cyclic or chain reaction mechanism. The number of cycles
occurring during the successful encounter between the enzyme and
substrate particle may be determined by various factors such as the
rate of removal of lysophosphatidylcholine from the interface and
apolipoprotein distribution at the interface
(38) . The chain
reaction pathways are supposed to be energetically more advantageous as
compared with the Michaelis-Menten type mechanism and are the sole
contributor to the catalytic process occurring with alcohol
dehydrogenase and other enzymes
(39) . It was previously observed
that LCAT exerts phospholipase A activity
(2, 10, 11) . This activity,
however, is relatively low and was thought to be due to the limited
penetration of water to the active site of the enzyme for the
hydrolysis of acyl-enzyme
(3, 10) . The low activity
could also be the result of the absence of the cyclic or chain
reaction, which may require the presence of an efficient acyl acceptor
such as cholesterol.
-acyl esters. Among various oxysterols tested, 27-OH-C formed
diesters by the LCAT reaction. This represents the first demonstration
of the enzymatic formation of sterol diesters. It was previously
reported that approximately 70% of 27-OH-C is present in the esterified
form in human plasma
(25) although the proportions of mono- and
diesters are not known. We are currently investigating the extent of
the formation of mono- and diesters of 27-OH-C under conditions similar
to those of human plasma. The presence of 50-84% of 7
-OH-C
in the esterified form in human plasma
(13) may reflect the
presence of the 3
-monoesters since no diester formation occurred
with this sterol. Approximately 50% of 7-ketocholesterol was reported
to exist in esterified form
(27) . It is possible that major
portions of the plasma oxysterol esters, like cholesteryl esters, are
derived by the LCAT reaction. There exists the possibility that
oxysterols may exert more pronounced atherogenic effects than
cholesterol
(17) . Then the LCAT reaction may represent an
important step in the reverse transport of oxysterols, alleviating
their toxic effects. The clarification of the processes of the reverse
transport awaits future investigations.
Table: 0p4in
Mixture of diesters, having two
different acyl groups at different positions, with identical retention
time.(119)
Table: 0p4in
367 (92.7),
[M-RCOO-2H
O].(119)
Table: The products of the LCAT
reaction obtained from DBP preparations containing 27-OH-C or
cholesterol and various phosphatidylcholines
,27-diol);
25-OH-C, 25-hydroxycholesterol (5-cholestene-3
,25-diol);
7
-OH-C, 7
-hydroxycholesterol
(5-cholestene-3
,7
-diol); 7
-OH-C,
7
-hydroxycholesterol (5-cholestene-3
,7
-diol); 7-keto-C,
7-ketocholesterol (5-cholestene-3
-ol-7-one);
-epoxy-C,
cholesterol 5
,6
-epoxide
(cholestan-5
,6
-epoxy-3
-ol);
-epoxy-C, cholesterol
5
,6
-epoxide (cholestan-5
,6
-epoxy-3
-ol);
Triol-C, cholestanetriol (cholestan-3
,5
,6
-triol); SE,
side chain.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.