(Received for publication, May 22, 1995; and in revised form, October 24, 1995)
From the
Phospholipase D from Streptomyces chromofuscus hydrolyzes lysophosphatidylcholine or lysophosphatidylethanolamine
in aqueous 1% Triton X-100 solution. In situ monitoring of
this reaction by P NMR revealed the formation of cyclic
lysophosphatidic acid (1-acyl 2,3-cyclic glycerophosphate) as an
intermediate which was hydrolyzed further by the enzyme at a
functionally distinct active site to lysophosphatidic acid (lyso-PA).
Synthetic cyclic lyso-PA (1-octanoyl 2,3-cyclic glycerophosphate) was
found to be stable in aqueous neutral solutions at room temperature. It
was hydrolyzed by the bacterial phospholipase D to lyso-PA at a rate
which was approximately 4-fold slower than the rate of formation of
cyclic lyso-PA. The addition of 5-10 mM sodium vanadate
could partially inhibit the ring opening reaction and thus increase
substantially the cyclic lyso-PA accumulation. Cyclic lyso-PA may act
as a dormant configuration of the physiologically active lyso-PA or may
even possess specific activities which await verification.
Activation of phospholipases has been implicated in a wide range
of signal transduction pathways(1) . With regard to
phospholipase D (PLase D), ()the current information as to
the molecules generated by PLase D activation and their mode of action
is still scarce (reviewed in (2) ).
The first step in the lytic activity of phospholipase D is the formation of a phosphoryl enzyme intermediate which is analogous to the acyl enzyme in the action of common esterases(3, 4) . This intermediate is generally cleaved by the ambient water molecules with the net hydrolysis of one phosphoester bond. However, alcoholic hydroxyl residues can compete for the cleavage of the phosphoryl enzyme intermediate to yield a phosphodiester product(5, 6) . If a hydroxyl group is positioned appropriately in the substrate it can also compete for the phosphoryl enzyme yielding a cyclic product. Cyclic products of phospholipase action have been detected in the action of phosphatidylinositol-specific phospholipase C (PLase C) on phosphatidylinositol yielding 1,2-cyclic inositol phosphate as the initial product(7, 8) , and in the PLase C hydrolysis of phosphatidylglycerol to 1,3-cyclic glycerophosphate(9) . Similar cyclization takes place in the hydrolysis of glycerophosphorylcholine or glycerophosphorylethanolamine by glycerophosphinicocholine diesterase, where 1,2-cyclic glycerophosphate is formed(10, 11) . All of these cyclic phosphates are relatively stable at neutral pH but can be hydrolyzed by specific phosphodiesterases to form the respective phosphate monoesters(12, 13) . It seems, therefore, that when the phosphoryl residue attached to the enzyme includes a free hydroxyl group, the formation of a cyclic phosphodiester by intramolecular transphosphorylation can take place. In analogy to the above reactions with a hydroxyl group, it has been suggested (9) that phospholipids with a free primary amine group (i.e. phosphatidylethanolamine and phosphatidylserine) may yield a cyclic phosphoramidate intermediate upon the action of PLase C. These putative five-membered cyclic phosphoramidates are expected to be hydrolyzed spontaneously(9) .
The action of PLase D on phospholipids forms a phosphatidyl enzyme intermediate which can either react with water to yield phosphatidic acid (PA) or with an alcohol (5, 6) to yield a new phospholipid (e.g. phosphatidylethanol if ethanol is added as the alcohol). This transphosphatidylation reaction raises the possibility that the action of PLase D on lysophospholipids may yield cyclic lyso-PA by intramolecular transphosphorylation with the hydroxyl on carbon 2 of the glycerol backbone as schematically presented in Fig. 1. The present work provides evidence that cyclic lyso-PA is formed upon the interaction of bacterial PLase D with lysophosphatidylcholine (lyso-PC) or lysophosphatidylethanolamine (lyso-PE).
Figure 1:
1-Octanoyl 2,3-cyclic glycerophosphate was prepared
by reacting -octanoyl glycerol (Avanti Chemicals) with phosphorus
oxychloride as described(15) . It was assumed that the
transacylation in this reaction did not exceed 10%(16) , and
therefore the prepared lysophospholipid was essentially of the
configuration.
The in situ enzymic experiments were
carried out in DO. Samples in 10-mm NMR tubes contained
30-40 mg of synthetic lyso-PC dispersed in
1.5 ml of 100
mM borate buffer, pD = 8, with 10 mM calcium
chloride. An appropriate amount of PLase D was added, depending on the
overall reaction time desired.
Preliminary screening has indicated that bacterial PLase D,
unlike cabbage PLase D, can cleave the headgroup of lyso-PC and lyso-PE
in the presence of ether or 1% Triton X-100. Fig. 2presents the
comparative degradation profiles of PC and lyso-PC solubilized in
Triton X-100 mixed micelles, pH 8.0, as assayed by TLC and
determination of the phosphorus content of each lipid spot. Under these
conditions lyso-PC is a substrate for bacterial PLase D, but it is not
as rapidly hydrolyzed as PC, in qualitative agreement with the findings
of Imamura and Horiuti(18) . In an analogous assay with
dipalmitoyl-PE and 1-palmitoyl-PE (lyso-PE), similar profiles and rates
of degradation were obtained. It should be noted that the enzyme
``lysophospholipase D'' (19, 20) acts
selectively on L--ether-linked lysophospholipids and not
on the common ester-linked L-
-lysophospholipids which
were studied here.
Figure 2:
Degradation profile of egg PC and egg
lyso-PC by bacterial PLase D. Egg PC (,
) or lyso-PC
(
,
), each at an initial concentration of 5 mg/ml, was
incubated with bacterial PLase D at pH 5.5 (100 mM acetate
buffer containing 10 mM calcium chloride, open
symbols) or at pH 8.0 (100 mM borate buffer containing 10
mM calcium chloride, filled symbols) in the presence
of 1% Triton X-100 at 25 °C. At each time point an aliquot was
removed from the reaction mixture and phospholipids were separated by
TLC and determined by phosphorus analysis (see ``Materials and
Methods'').
The aqueous medium consisting of phospholipid (5
mg/ml) dispersed in 1% Triton X-100, 10 mM borate buffer, pH
8.0, with 2 mM Ca (see Fig. 2and
``Materials and Methods'') was found suitable for in situ monitoring of the PLase D cleavage products of lysophospholipids
by
P NMR. However, short chain lyso-PC substrates (e.g. 1-octanoyl-3-glycerolphosphorylcholine) were found to be
hydrolyzed by bacterial PLase D without the use of Triton X-100 or
ether. Furthermore, for these compounds the lyso-PC concentration used
in the assay was below its critical micellar concentration, ensuring
that the substrate is essentially monomeric. During the course of the
lyso-PC hydrolysis, a new resonance was observed at 18.5 ppm as
presented in Fig. 3. Ordinary phosphate esters (e.g. lyso-PA or lyso-PC) are characterized by resonances in the range
of +5 to -6 ppm(21) . The chemical shift of the new
resonance, quite downfield from that of the substrate (0.5 ppm) and
product (5.1 or 0.8 ppm, with or without vanadate, respectively), was
consistent with the formation of a five-membered cyclic phosphodiester.
Five-membered cyclic phosphates have a downfield shift due to
-electron shielding effect(22) . Experimental conditions
were selected for the complete hydrolysis of the lysophospholipid
substrate by PLase D to occur over several hours. This allowed the
production of the five-membered cyclic phosphodiester and the more
gradual appearance of lyso-PA to be monitored for different substrate
systems as a function of time. In all of the incubations of lyso-PC and
lyso-PE with PLase D, the formation of a
P resonance at
18.5 ppm (i.e. a cyclic diester product) was observed
concomitant with the appearance of the lyso-PA resonance. After rapidly
reaching a certain intensity which was maintained for a while
(consistent with reaching a steady-state concentration), the resonance
at 18.5 ppm declined, while that of the phosphate monoester (i.e. lyso-PA) continued to increase in intensity (Fig. 3). The
most likely explanation for the unusual downfield shifted resonance was
the formation of a cyclic lyso-PA as an intermediate in the PLase D
reaction.
Figure 3:
In vitro monitoring by P(
H) NMR of lyso-PC degradation by bacterial
PLase D. Synthetic lyso-PC (1-octanoyl 3-glycerol phosphorylcholine) at
the initial concentration of 100 mM in borate buffer, pD 8.0
(containing 10 mM Ca
) was incubated with
bacterial PLase D in the NMR tube at 28 °C for 14 h. The recorded
P(
H) NMR spectra indicate the formation and
the decline of a peak of a five-membered phosphodiestering at
= 18.5 ppm (cyclic lyso-PA, c-LPA), decay of the
lyso-PC (LPC) resonance at
= 0.5 ppm, and the
subsequent emergence of a lyso-PA (LPA) resonance at
= 0.8 ppm. Inset,
P spectra of synthetic
cyclic lyso-PA. Scanning conditions: 70° pulses using 4-s
relaxation delay and composite pulse proton decoupling (see
``Materials and Methods'').
The formation of cyclic lyso-PA by the action of PLase D
on lyso-PC or lyso-PE was verified with P NMR spectra of
synthetic 1-octanoyl 2,3-cyclic glycerophosphate (i.e. cyclic
lyso-PA). The phosphorus chemical shift of this material corresponded
to 18.5 ppm, essentially the same shift as the resonance observed by
P NMR in assays of the action of PLase D on lyso-PC or
lyso-PE (Fig. 3). The synthetic 1-octanoyl 2,3-cyclic
glycerophosphate was examined for its stability under assay conditions
and as a substrate for bacterial PLase D, since in the NMR assays
eventually all the cyclic product was converted to lyso-PA. The
synthetic cyclic lyso-PA was found to be stable at pH 6-8 for at
least 10 h as was indicated by invariant intensity at +18.5 ppm (Fig. 4). However, in the presence of PLase D, the intensity of
this resonance decreased while that of lyso-PA increased, indicating
phosphatase activity. The addition of egg PC in 1% Triton X-100 or in a
1:1 mixture with lyso-PC did not affect the rate of this reaction which
excluded the possibility that the cyclization and the ring opening take
place at the same catalytic site.
Figure 4:
Hydrolysis profile of synthetic cyclic
lyso-PA. 1-Octanoyl 2,3-cyclic glycerophosphate in the presence
() and absence (
) of bacterial PLase D. The emerging
lyso-PA is also indicated (
). The experimental conditions are as
described in the legend to Fig. 3.
The cyclization and the ring opening activities could, in principle, be related to separate entities which copurified in the partial enzyme purification used to generate the PLase D. All the PLase D preparations used in the NMR experiments also exhibited a strong phosphatase activity with the conventional substrate p-nitrophenyl phosphate (data not shown). To explore this further, we have chromatographed PLase D preparations by gel filtration using Sephadex G-75, G-100, G-150, and S-300, DEAE-cellulose-52, and red Sepharose, which is an affinity binder of alkaline phosphatase, in an attempt to dissociate these activities. In all of these attempts the phosphatase activity, as monitored by hydrolysis of p-nitrophenyl phosphate, coincided with the PLase D activity. Therefore, we tentatively conclude that the phosphatase activity is inherent in the bacterial PLase D.
The reaction profiles presented above and the fact that cyclization and phosphatase activities always coincided suggest that PLase D carries out two sequential enzymatic reactions with lysophospholipids: (i) intramolecular cyclization to form cyclic lyso-PA, followed by (ii) hydrolysis to lyso-PA, namely a phosphodiesterase type cleavage.
An
alternative approach for dissecting the cyclization activity from that
of the ring opening was the use of phosphatase competitive inhibitors.
In a series of P NMR experiments which were carried out
under the same conditions as in Fig. 3, we have added
-glycerophosphate (5 mM), p-nitrophenyl
phosphate (5 mM), and sodium vanadate (2-100
mM). Sodium vanadate at concentrations of 5-10 mM could partially inhibit the phosphatase activity of the PLase D.
This was measured by a substantial increase in relative content of
cyclic lyso-PA compared with lyso-PA. An example of a reaction profile
in the presence of 5 mM sodium vanadate is shown in Fig. 5. Under these reaction conditions there was a clear
relative increase in the intensity of the resonance at +18.5 ppm.
The presence of sodium vanadate caused a downfield shift and splitting
in the resonance of the lyso-PA. One possible explanation for the
marked effect of vanadate on the lyso-PA resonances could be the
formation of pyrophosphovanadate which is stabilized by the presence of
Ca
.
V spectra recorded in the above
system (not shown) displayed an upfield shift similar to that recorded
in a pyrophosphovanadate formed between adenosine monophosphate (AMP)
and sodium vanadate(23) . The two resonances at the region of
lyso-PA (Fig. 5) might be related to
and
isomers.
Figure 5: Degradation profile of lyso-PC by PLase D in the presence of 5 mM sodium vanadate. The conditions and experimental setting were as in Fig. 3. Inset, a spectrum taken for the same reaction carried out with a 10-fold higher enzyme concentration (t = 5 min). The proposed lyso-PA-vanadate structure is as follows:
Table 1represents a summary of the P chemical
shifts of the compounds presented in this communication.
Assuming
that each of the above PLase D reactions follows a Michaelis-Menten
type kinetic scheme with two independent enzyme sites, E and E
then the reaction steps may be
presented as
follows,
where A is the starting lysophospholipid (e.g. lyso-PC), B is the putative cyclic lyso-PA, and C is the lyso-PA
(in principle in either or
form). V
and V
are the V
values,
while K
and K
are the corresponding K
values.
Accordingly(24) ,
The solution for the reduction of A with time is
or
To a first approximation one can assume that along the whole
reaction profile A < K, which upon integration
of -1c leads to a simple first order decline in
A.
Assuming further that the same approximation also holds for B (i.e. B < K)
then
The rapid cleavage of the putative cyclic lyso-PA implied, from
the leveling off and decline in the 18.5 ppm band (see Fig. 3),
hampered our attempts to apply the kinetic analysis described above. In
practice, however, this could be carried out only in the presence of
sodium vanadate which acted as an inhibitor of the phosphadiesterase
component (see Fig. 5). A typical experimental presentation of
the change in A, B, and C with time in the presence of 5 mM sodium vanadate is shown in Fig. 6. The presented values
were obtained by pick integration under instrumental setup for
quantitative evaluation (see ``Materials and Methods''). The
profiles comply with , , and and with the implied correspondence C = A - A - B. Approximate analysis of the kinetics
described above was carried out by first evaluating V
/K
according to and . For the experimental conditions used in Fig. 6, the corresponding rate of cyclization was around V
/K
= 0.04
min
. V
/K
could be evaluated in an independent experiment (as the one
presented in Fig. 5) measuring the hydrolysis of synthetic B,
1-octanoyl 2,3-cyclic glycerophosphate. The value obtained was V
/K
= 0.01
min
. Insertion of these V
/K
and V
/K
values into yielded the change in the cyclic lyso-PA concentration as a
function of reaction time. As seen in Fig. 6, the correspondence
between the observed and calculated presence of cyclic lyso-PA is
reasonable, considering the series of approximations that were applied.
Figure 6:
The time profiles of the reduction in
lyso-PC and increase in lyso-PA and cyclic lyso-PA by the action of
bacterial PLase D in the presence of 0.01 M sodium vanadate.
The reaction was carried out as described in the legend to Fig. 5. The experimental parameters of the P NMR
setup for quantitative evaluation are described under ``Materials
and Methods.'' The relative content of lyso-PC (
) lyso-PA
(
) and cyclic lyso-PA (
) were obtained by area integration
of the resonances at 0.5, 5.1, and 18.5 ppm, respectively. The
calculated profile of the expected change in cyclic lyso-PA according
to is also presented(- - -
-).
Lyso-PA has been recently cited as an important modulator of cell
functions(25, 26) . It is synthesized either by the
action of PLase A on PA or by phosphorylation of
-monoglyceride by a specific kinase. Cyclic lyso-PA can in
principle be formed in cells by the action of PLase A
on
membrane phospholipids (27) followed by the action of PLase D
which is described here. These consecutive enzyme reactions may impose
a serious stringency on the formation of cyclic lyso-PA. Furthermore,
the conversion of cyclic lyso-PA to lyso-PA will demand a third
enzymatic action. Detection of these yet unexplored reactions will
clarify whether cyclic lyso-PA is actually formed in cells and whether
it serves as a dormant form of lyso-PA or acts as a modulator of cell
functions on its own right.