(Received for publication, July 24, 1995; and in revised form, November 2, 1995)
From the
We have previously identified a novel CoA-independent
transacetylase in the membrane fraction of HL-60 cells that transfers
the acetate group from platelet activating factor (PAF) to a variety of
lysophospholipid acceptors (Lee, T.-c., Uemura, Y., and Snyder,
F.(1992) J. Biol. Chem. 267, 19992-20001). In the
present study, we demonstrate that a similar transacetylase can
transfer the acetate group from PAF to sphingosine forming N-acetylsphingosine (C-ceramide). The chemical
structure of the reaction product, C
-ceramide, was
established by its identical R
value with
authentic C
-ceramide standard on thin-layer plate,
sensitivity to acid treatment, resistance to alkaline hydrolysis, and
ability to form the C
-ceramide dibenzoate derivative.
Nonspecific transfer of the acetate from PAF to sphingosine in the
absence of enzyme and nonlinearity of the reaction rates were rectified
by complexing sphingosine to bovine serum albumin in a 1:1 molar ratio.
Under these conditions, the apparent K
for PAF is 5.4 µM, which is in the same range
as the K
(12.0 µM) when
lysoplasmalogen is the acetate acceptor. PAF:sphingosine transacetylase
has a narrow substrate specificity and strict stereochemical
configuration requirements. Ceramide, sphingosylphosphocholine,
stearylamine, sphingosine 1-phosphate, or sphingomyelin are not
substrates, whereas sphinganine has a limited capacity to accept the
acetate from PAF. Also, only the naturally synthesized D-erythroisomer but not the synthetic L-erythro-, D-threo-, or L-threoisomers of sphingosine can serve
as a substrate. PAF transacetylase activity is widely distributed among
several tissues and may involve histidine and cysteine for its
catalytic activity due to inhibitory effects to the enzyme by diethyl
pyrocarbonate and N-ethylmaleimide, respectively.
C
-ceramide is produced via PAF:sphingosine transacetylase,
and physiological levels of C
-ceramide are detected in both
undifferentiated and differentiated intact HL-60 cells. Collectively,
because C
-ceramide has many biological activities that
differ from that of PAF and sphingosine, the CoA-independent,
PAF-dependent transacetylase serves as a modifier of PAF, and
sphingosine functions by generating a variant lipid mediator,
C
-ceramide.
Platelet-activating factor (PAF) ()is a potent lipid
mediator involved in diverse pathophysiological processes, e.g. inflammation, allergic reactions, and many aspects of reproduction
(see recent reviews by Hanahan(1992), Shukla(1992), Venable et
al.(1993), and Snyder (1995a, 1995b)). The biosynthetic routes for
PAF via either de novo (Lee et al., 1986, 1988;
Renooij and Snyder, 1981; Woodard et al., 1987) or remodeling
(Wykle et al., 1980; Lee, 1985) pathways are well established.
In addition to the putative conversion of
1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(alkylarachidonoyl-GPC) to alkyllyso-GPC by phospholipase A
in the remodeling pathway, alkyllyso-GPC can also be generated by
a CoA-independent transacylase that transfers the long-chain acyl
groups (primarily 20:4) from alkylacyl-GPC to other lysophospholipid
acceptors containing either ethanolamine or choline, e.g. alk-1-enyllyso-sn-glycero-3-phosphoethanolamine (Uemura et al., 1991; Venable et al., 1991). The
alkyllyso-GPC formed by the transacylase can subsequently be acetylated
by the alkyllyso-GPC:acetyl-CoA acetyltransferase to produce PAF
(Uemura et al., 1991; Nieto et al., 1991).
PAF is degraded by the acetylhydrolase to form acetate and alkyllyso-GPC (Blank et al., 1981, 1983b; Farr et al., 1980, 1983; Stafforini et al., 1987); the latter is rapidly transacylated to enter a membrane pool of alkylacyl-GPC that is highly enriched with arachidonic acid (MacDonald and Sprecher, 1991; and Snyder et al., 1992). Recently, we identified a unique membrane-associated CoA-independent transacetylase that can transfer the acetate group from PAF to a wide variety of lysophospholipids (radyllyso-GPC, radyllyso-sn-glycero-3-phosphoethanolamine, acyllyso-sn-glycero-3-phosphoserine, and acyllyso-sn-glycero-3-phosphoinositol), radyllyso-sn-glycero-3-phosphate, and long-chain fatty alcohols (Lee et al., 1992). This enzyme appears to be the preferential in vivo route for the biosynthesis of the ethanolamine plasmalogen and acyl analogs of PAF. It has been proposed (Lee et al., 1992) that the transacetylase plays a role in the fine tuning of PAF biological responses and cross-talk between de novo and remodeling pathways of PAF biosynthesis via acetylation of intermediates involved in the generation of bioactive lipid molecules.
Sphingosine (see reviews by Hannun and Bell(1987, 1989), Merrill and Stevens(1989), Merrill(1991), Hannun and Linardic(1993), and Hannun and Bell(1993)) and its related metabolites such as ceramides (Kolesnick, 1991), sphingosine 1-phosphate (Zhang et al., 1991), and sphingosylphosphorylcholine (Desai et al., 1993) have also emerged as important signaling molecules involved in many cellular processes. The fact that sphingosine 1-phosphate and radyllyso-glycerophosphate, as well as sphingosylphosphorylcholine and radyllyso-GPC, are structurally similar to each other and acetylated sphingomyelin has been reported to possess PAF-like activity (Berdyshev and Getmanova, 1991) prompted us to investigate the possibility that the transacetylase may be able to donate the acetate group from PAF to sphingolipids, thus modifying the biological activities of both PAF and sphingolipid derivatives.
Acetyl-CoA-dependent acylation of sphingosine, ceramide, and
alkyllyso-GPC was assessed by incubating 50 µM [H]acetyl-CoA (0.4 µCi), 100 mM Tris-HCl (pH 7.4), the test lipid substrate (20 µM each), and 100 µg of membrane protein from undifferentiated
HL-60 cells in a final volume of 0.5 ml. The protein content of the
enzyme preparations was determined by the method of Lowry et
al.(1951).
A similar protocol was used to
quantitate the mass of C-ceramides in HL-60 cells. Briefly,
lipids from differentiated and undifferentiated HL-60 cells
(10
-10
cells) were extracted and separated on
TLC to isolate the C
-ceramide. The C
-ceramide
dibenzoates were prepared as described above except a known amount of
C
-ceramide as an internal standard was added during the
isolation of C
-ceramide from the thin-layer plates. The
dibenzoates were analyzed by HPLC. The HPLC retention time for
C
-ceramide dibenzoates was 16.59 min. Amounts of
C
-ceramide were quantitated based on a molar extinction
coefficient of 2.6
10
liter/mol
cm at 230 nm.
A more detailed description of the method on the quantitative analysis
of C
-ceramide in the biological samples will be submitted
for publication elsewhere.
Figure 1:
Thin-layer
chromatographic scan of reaction products formed by the PAF:sphingosine
CoA-independent transacetylase associated with the membrane fraction
(100,000 g 60 min pellet) of undifferentiated HL-60
cells. Hexadecyl-[
H]acetyl-GPC (1
µM, 0.3 µCi in 50 µl 0.1% BSA-saline) was
incubated with 50 µM sphingosine (in 50 µl of 0.1%
BSA-saline), 1 mM sodium acetate, 5 mM EGTA, and 100
mM Tris-HCl (pH 7.4), with/without or boiled (100 °C for
10 min) membrane proteins (100 µg) in a final volume of 0.5 ml for
60 min at 37 °C. Reaction products were analyzed after lipid
extraction by TLC and liquid scintillation counting as described under
``Experimental Procedures.'' Authentic standards, PAF,
N-acetylsphingosine (N-Ac-Sph), and N,O
-triacetylsphingosine (N,O
-Ac-Sph) migrated to the areas
indicated by the brackets below the fraction
numbers.
Figure 2:
Time course for the formation of N-acetylated sphingosine (N-Ac-Sph) by the
PAF:sphingosine CoA-independent transacetylase associated with the
membrane fraction (100,000 g 60 min pellet) of
undifferentiated HL-60 cells. The assay system was the same as
described in the legend of Fig. 1except the incubation times
were varied as indicated. Results, the average of duplicate
determinations with variations < 10%, are representative of five
similar experiments.
Figure 3:
Effect of BSA:sphingosine molar ratio on
the formation of N-acetylsphingosine by the PAF:sphingosine
CoA-independent transacetylase associated with the membrane fraction
(100,000 g 60 min pellet) of undifferentiated HL-60
cells. The assay system was the same as described in the legend of Fig. 1except 50 µM sphingosine was added with
various amounts of BSA. Molar ratios of BSA/sphingosine (BSA/Sph) are indicated in the inset. The results are
the averages of duplicate determinations with variations < 10%.
Linearity of the reaction rates with a molar ratio of BSA/sphingosine
as 1 has been confirmed in numerous other
experiments.
The effect of
[H]PAF concentrations on the formation of N-acetylsphingosine was determined at a molar ratio of
BSA:sphingosine of 1 (Fig. 4). Maximal enzyme activity occurred
between 10 and 20 µM of [
H]PAF, with
an apparent K
of 5.4 µM for PAF. The
effect of sphingosine concentrations on the generation of N-acetylsphingosine by the PAF:sphingosine CoA-independent
transacetylase can be seen in Fig. 5. A Lineweaver-Burk plot
gave a curved reciprocal line with an upward divergence (Fig. 5B). This type of kinetic behavior makes the
determination of the Michaelis constant difficult and also suggests
that sphingosine not only serves as a substrate but may act as an
activator (Dixon and Webb, 1964) for the transacetylase.
Figure 4:
Effect of
hexadecyl-[H]acetyl-GPC concentrations on the
PAF:sphingosine CoA-independent transacetylase activity associated with
the membrane fraction (100,000
g 60 min pellet) of
undifferentiated HL-60 cells. The assay system was the same as
described in the legend of Fig. 1except the concentrations of
[
H]PAF were varied, the 50 µM sphingosine was suspended with equal molar amounts of BSA, and the
incubation times were 30 and 60 min. Results are expressed as means
± S.E. (n = 4). Some of the error bars do not show up on the graph due to the small ranges of
S.E.
Figure 5:
Effect
of sphingosine concentrations on the PAF:sphingosine CoA-independent
transacetylase activity associated with the membrane fraction (100,000
g 60 min pellet) of undifferentiated HL-60 cells. The
assay system was the same as described in the legend of Fig. 4except 15 µM
hexadecyl-[
H]acetyl-GPC was used and the
concentrations of sphingosine were varied as indicated. The molar ratio
of BSA/sphingosine was 1. The results are expressed as means ±
S.E. (n = 12). One of the error bars did not
show up on the graph due to the small range of
S.E.
Figure 6:
Substrate specificities of PAF:sphingosine
CoA-independent transacetylase activity associated with the membrane
fraction (100,000 g 60 min pellet) of undifferentiated
HL-60 cells. The assay system was the same as described in the legend
of Fig. 1except 15 µM
hexadecyl-[
H]acetyl-GPC and 50 µM various acceptors (suspended in equal molar concentration of BSA)
were incubated in the presence and the absence of membrane proteins for
30 and 60 min. The products were identified by their co-migration on
TLC plates with authentic standards. The amount of products formed
nonenzymatically has been subtracted from the total amount of product
formed to obtain the values depicted for the enzymatic conversions. A
duplicate experiment showed similar results. spc,
sphingosylphosphorycholine; spp, sphingosine
1-phosphate.
The possibility of sphingosine accepting a
long-chain acyl group, instead of the short-chain acetyl grouping, was
examined by incubating 1 µM (0.3 µCi)
[H]-dipalmitoyl-GPC or
hexadecyl[
H]arachidonoyl-GPC instead of
[
H]PAF with 50 µM sphingosine
(complex with equal molar of BSA) using the same conditions under which
PAF:sphingosine CoA-independent transacetylase was assayed. Under these
conditions, no measurable amount of ceramide could be detected.
Furthermore, when sphingosine, ceramide, or alkyllyso-GPC was incubated
with [
H]acetyl-CoA in the presence of membrane
proteins from undifferentiated HL-60 cells, only alkyllyso-GPC (75
pmol/min
mg protein) but neither sphingosine nor ceramide was
acetylated by the lyso-PAF:acetyl-CoA acetyltransferase. These results
are consistent with our finding that the amounts of acetate transferred
from PAF to sphingosine remained the same when
[
H]PAF and sphingosine were incubated either in
the presence or absence of sodium acetate (1 mM) with membrane
proteins of HL-60 cells (data not shown). Moreover, no acetate was
incorporated into sphingosine when 1 mM sodium
[
H]acetate (0.3 µCi) was incubated under the
same conditions with 50 µM sphingosine and membrane
proteins of undifferentiated HL-60 cells.
As one means of addressing the question as to whether a single enzyme or two different enzymes catalyze the acetylation of lysophospholipids and sphingosine by PAF, mixed substrate experiments were conducted. Results indicate that sphingosine, acyllyso-GPC, and sphingosylphosphorylcholine at equal molar concentration with that of lysoplasmalogen (50 µM) inhibited the production of acetylated plasmalogen by 20, 55, and 27%, respectively. On the other hand, lysoplasmalogen, acyllyso-GPC, and sphingosylphosphorylcholine at 50 µM had no effect on the acetylation of 50 µM sphingosine. When the concentration of sphingosine was reduced to 25 µM, 50 µM lysoplasmalogen (molar ratio of sphingosine to lysoplasmalogen, 1:2) was still ineffective in inhibiting N-acetylsphingosine formation. With 10 µM sphingosine, a mere 13% decrease in N-acetylsphingosine generation was caused by 50 µM lysoplasmalogen. In addition, sphingosine 1-phosphate, not a substrate for the transacetylase, exerts no influence on the transacetylation of either lysoplasmalogens or sphingosine. Thus, either the CoA-independent transacetylase has a higher substrate affinity for sphingosine than any of these other substrate analogs or the possibility of two isoforms of the transacetylase might be involved in the transfer of the acetate from PAF to sphingosine and lysophospholipids.
Figure 7:
Tissue distribution of PAF:lysoplasmalogen (A) and PAF:sphingosine CoA-independent transacetylases (B). Postnuclear membrane fractions (100,000 g 60 min pellet) isolated from homogenates of various rat tissues
and homogenates of undifferentiated HL-60 cells were used to assay
PAF:lysoplasmalogen and PAF:sphingosine transacetylase activities. The
assay system was the same as described in the legend of Fig. 1except 50 µM [
H]PAF and
300 µM lysoplasmalogen in a final volume of 0.25 ml were
used for assaying PAF:lysoplasmalogen transacetylase and 15
µM [
H]PAF and 50 µM
sphingosine (complexed with equal molar concentration of BSA) were used
for assaying PAF:sphingosine transacetylase. The results are expressed
as the fold increase in specific activity of a tissue or the cells over
that of the brain (151 pmol/min
mg protein for PAF:lysoplasmalogen
transacetylase and 1 pmol/min
mg protein for PAF:sphingosine
transacetylase). Values are the averages of two separate experiments
± ranges; data from each experiment represent the means of four
determinations for two incubation time periods (15 and 30
min.).
We have demonstrated that an enzyme from the membrane
fraction of undifferentiated HL-60 cells can transfer the acetate group
of PAF to sphingosine with the formation of C-ceramide as
depicted by Fig. R1. The product was identified as N-acetylsphingosine by its co-migration with an authentic
standard on thin-layer plates, its sensitivity to acid hydrolysis, its
resistance to alkaline treatment, which indicated the presence of an
amide linkage, and its ability to form dibenzoate derivatives. Several
other lines of evidence also support the notion that this reaction is
catalyzed by a CoA-independent transacetylase. The reactions were
normally carried out in the presence of excess unlabeled sodium acetate
(1 mM) in order to prevent or minimize any radioactive acetate
released from the PAF being directly incorporated into sphingosine. In
addition, the presence of CoA up to 0.5 mM in the incubation
mixture had no effect on the generation of N-acetylsphingosine
(data not shown). Under the assay conditions where lysoplasmalogens are
acetylated by the acetyl-CoA acetyltransferase, sphingosine does not
serve as a substrate. Furthermore, transacylation of the amino group on
sphingosine apparently is highly specific for short-chain acyl groups (i.e. acetate), because dipalmitoyl-GPC and
hexadecylarachidonoyl-GPC cannot transfer either palmitate or
arachidonate, respectively, to sphingosine under similar experimental
conditions. However, the possibility could also exist that
dipalmitoyl-GPC and hexadecylarachidonoyl-GPC can not serve as a
substrate due to their inability to insert into membrane.
Figure R1: Reaction 1.
A small
but significant amount of N-acetylsphingosine is produced from
[H]PAF and sphingosine nonenzymatically
(chemical) ( Fig. 2and 3B), and the reaction rate is
not linear with incubation time (Fig. 2). However, both of these
problems can be circumvented by complexing sphingosine with equal molar
amounts of BSA (Fig. 3). Reasons for why complexing the
sphingosine with BSA would reduce the nonenzymatic formation of N-acetylsphingosine between sphingosine and PAF and improve
the linearity of the reaction are not clear at present. It is possible
that sphingosine may exist in both monomeric and aggregate forms in the
absence of BSA, which could favor the chemical formation of
N-acetylsphingosine. Furthermore, Merrill(1991) has reported the
formation of 1:1 molar complex of sphingosine with BSA stabilizes the
amphoteric sphingosine molecule. Nevertheless, even though the
sphingosine-BSA complex reduces the nonenzymatic formation of N-acetylsphingosine and provides satisfactory linear kinetics
for the reaction, it also diminishes the reaction rate significantly (Fig. 3). When kinetic parameters were measured using the
sphingosine-BSA complex as the substrate, the calculated apparent K
for [
H]PAF (5.4
µM, Fig. 4) of the PAF:sphingosine transacetylase
is in the same range as we previously reported for the
PAF:lysoplasmalogen transacetylase (12.0 µM) (Lee et
al., 1992). The apparent K
for
lysoplasmalogens was 106.4 µM (Lee et al., 1992),
whereas the apparent K
for sphingosine in this
study could not be assessed due to its kinetic behavior (Fig. 5).
Among the variety of sphingosine-related compounds (i.e. stearylamine, sphinganine, sphingosine 1-phosphate,
sphingosylphosphorylcholine, ceramide, and sphingomyelin) tested, only
sphinganine, sphingosylphosphorylcholine, and ceramide showed limited
ability to accept the acetate group from PAF (Fig. 6). These
results indicate the transacetylase has a strict substrate structural
requirement for sphingolipids in that it prefers to transfer the
acetate group to the -NH instead of -OH
grouping; moreover, the presence of -OH and trans double bond
within the structure appears to potentiate the transacetylase activity.
For example, stearylamine is inactive, whereas sphinganine (a
metabolite of the de novo biosynthetic pathway (Merrill,
1991)) is less active than sphingosine (a turnover product of
sphingolipids) as a substrate for the transacetylase. In addition,
neither lysoplasmalogen and related substrate analogs (i.e. acyllyso-GPC) nor sphingosine analogs (i.e. sphingosylphosphorylcholine and sphingosine 1-phosphate) affected
the transfer of acetate from PAF to sphingosine, whereas the transfer
of acetate from PAF to lysoplasmalogen was easily inhibited by the
presence of these substrate analogs (with the exception of sphingosine
1-phosphate). These findings suggest that the transacetylase has a
different affinity for sphingosine and lysoplasmalogens. Consistent
with these findings are the observations that concentration-dependent
responses to the modification of cysteine and histidine residues of the
transacetylases by the inhibitors are different for the acetylation of
sphingosine and lysoplasmalogens (Table 1).
It is difficult at present to assess whether two different enzyme activities carry out the transfer of the acetate from PAF to lysophospholipids and sphingosine. Even though PAF:lysoplasmalogen transacetylase differs from PAF:sphingosine transacetylase in its substrate specificities, tissue distribution (Fig. 7), and enzyme inhibitor responses (Table 1), both enzymatic activities respond to temperature inactivation in a similar manner when either preincubating the membrane fractions at 60 °C for various times or when assayed at different incubation temperatures (data not shown). We are currently attempting to purify the transacetylase protein(s) and clone the cDNA(s) for the transacetylases in order to address this important issue.
Recently,
Liu and Subbaiah(1994) reported that purified lecithin-cholesterol
acyltransferase from human plasma can catalyze the transfer of the
acetate group from PAF to lysophosphatidylcholine (acyllyso-GPC)
forming acyl analog of PAF. Lecithin-cholesterol acyltransferase with a
molecular mass of 65,000-69,000 Da (Marcel, 1982; Chung et
al., 1979) is synthesized in the liver and secreted into plasma
where it associates with high density lipoproteins (Marcel et
al., 1980; Glomset, 1972). It requires serine, histidine, and
cysteine for catalytic activity (Jauhiainen and Dolphin, 1986). On the
other hand, the PAF-dependent transacetylase we investigated is a
membrane-associated enzyme. In addition, the PAF:lysophospholipid
transacetylase (solubilized from rat kidney membranes with 0.02% Tween
20) has an estimated molecular mass of 47 kDa (determined by Sephacryl
S-200 column), ()and both histidine and cysteine (Table 1), but not serine (not inhibited by phenylmethylsulfonyl
fluoride) (Lee et al., 1992) are required for expression of
its activity. Furthermore, the PAF-dependent transacetylases do not
transfer long-chain acyl groups to acceptor molecules. These properties
indicate that lecithin-cholesterol acyltransferase and transacetylase
are two distinct enzymes.
C-ceramide, which has been
extensively used by many investigators as an unnatural, cell-permeable
analog of long-chain acyl-ceramides, possesses many of the biological
activities associated with the naturally occurring ceramides containing
long-chain acyl moieties. At micromolar concentrations, it stimulates
the activities of a mitogen-activated protein kinase (Raines et
al., 1993), a cytosolic protein phosphatase (Dobrowsky and Hannun,
1992), and protein phosphatase 2A (Dobrowsky et al., 1993) and
induces programmed cell death (Obeid et al., 1993), cell cycle
arrest (Jayadev et al., 1995), cellular differentiation, and
c-myc down-regulation (Kim et al., 1991). In
addition, C
-ceramide inhibits the stimulation of DNA
synthesis and phospholipase D activity by phosphatidic acid and
lysophosphatidic acid (Gomez-Muñoz et
al., 1994). It also inhibits fMet-Leu-Phe- and phorbol
12-myristate 13-acetate-induced superoxide formation in neutrophils
(Wong et al., 1995). However, unlike sphingosine and
sphingosylphosphorylcholine, the C
-ceramide does not
inhibit protein kinase C (Hannun and Bell, 1987) or cause
Ca
release (Ghosh et al., 1990).
Furthermore, C
-dihydroceramide (N-acetylsphinganine) acts instead as an inhibitor of protein
phosphatase 2A (Dobrowsky et al., 1993) and fails to induce
apoptosis (Obeid et al., 1993) or block fMet-Leu-Phe-activated
superoxide generation (Wong et al., 1995). Our results on the
identification and characterization of PAF:sphingosine transacetylase,
the demonstration of the formation of C
-ceramide by this
enzyme in intact HL-60 cells, and that this enzyme only uses the
naturally occurring stereoisomer, D-erythro-sphingosine, as a
substrate, suggest that one function of the PAF:sphingosine
transacetylase is to modulate the biological responses of PAF and
sphingosine by producing a different signaling molecule,
C
-ceramide. The detection of physiological levels of
C
-ceramide in HL-60 cells further supports the notion that
C
ceramide is a naturally occurring lipid mediator.