| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 35, 32733-32743, August 29, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
||
From the
Department of Biochemistry and Molecular
Biology, Medical University of South Carolina, Charleston, South Carolina
29425 and the ¶Department of Microbiology,
University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, January 28, 2003 , and in revised form, June 2, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
75 kDa and one of
30–35 kDa. Interestingly, the highly purified SM synthase preparation
also showed neutral sphingomyelinase activity. We therefore investigated
whether the protein we purified as SM synthase could actually be the
previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze
phosphatidylcholine and SM in P. aeruginosa. First, the purified SM
synthase preparation contained a 78-kDa protein that reacted with monoclonal
antibodies raised against purified PlcH. Second, purified PlcH showed SM
synthase activity. Third, using different knockout mutant strains for the
PlcH operon, PlcH was found to be necessary for SM synthase
activity in P. aeruginosa. Interestingly, SM synthase activity was
specific to the Pseudomonas PlcH as other bacterial phospholipases
did not display SM synthase activity. Biochemical studies on the
Pseudomonas SM synthase confirmed that it is a transferase, similar
to the mammalian enzyme, that specifically recognizes the choline head-group
and the primary hydroxyl on ceramide. This SM synthase did not have reverse
transferase activity. In conclusion, the Pseudomonas PlcH also exerts
SM synthase activity; therefore, for the first time, we have identified a
structural gene for a SM synthase. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Important biological roles have been clearly established for ceramide and
DAG (substrate and product of SM synthase) in the regulation of fundamental
cellular functions such as proliferation and apoptosis (for reviews, see Refs.
3–8).
Therefore, it has been hypothesized that the cellular role of SM synthase goes
beyond the production of SM. In fact, SM synthase could represent a key
mechanism in the control of the cellular levels of ceramide and DAG and
therefore would influence functions mediated by these bioactive lipids.
Indeed, a number of studies have documented regulation of SM synthase
activity. It has been shown, for instance, that activity of SM synthase is
enhanced in conditions of increased proliferation such as regenerating rat
liver (9), SV-40 transformation
of human fibroblasts (10),
highly malignant hepatoma (10,
11), and treatment of
astrocytes with bFGF (12).
Additionally, it has been shown that SM synthase activity is inhibited by
tumor necrosis factor 
in Kym-1 rhabdomyosarcoma cells before the onset
of tumor necrosis factor-induced apoptosis and that this inhibition is a
caspase-dependent event (13).
Finally, DAG formed through activation of SM synthase has been associated with
activation of NF-
B in SV-40-transformed human fibroblasts
(14). Despite the biological
importance of SM synthase, understanding of the molecular mechanisms of its
regulation is limited by the fact that no successful purification of the
protein has been achieved and the gene(s) encoding for this activity remain
elusive.
Although sphingolipids are primarily present in eukaryotes, some prokaryotic cells express and secrete enzymes with sphingolipid-metabolizing capability. For example, the bacteria Bacillus cereus and Staphylococcus aureus produce and secrete a phospholipase C that recognizes SM as substrate at neutral pH, and this enzyme is responsible for their hemolytic activity (15, 16). Importantly, analysis of the nucleotide sequence of the neutral sphingomyelinase (N-SMase) from B. cereus has led to the identification of at least three homologous genes: two in mammalian cells (nSMase1/lysoPAF-PLC and nSMase2) (17, 18) and one in yeast (ISC1) (19). The products of these genes have all proven to hydrolyze SM in vitro at neutral pH and therefore to have neutral SMase activity.
Other bacteria known to express and secrete sphingolipid-metabolizing enzymes are Pseudomonas species. SMases have been isolated from Pseudomonas aeruginosa (20), Pseudomonas fluorescens (21), and Pseudomonas cepacia (22), although sequence and substrate specificity might be different. The SMase from P. aeruginosa (PlcH) is a phospholipase C that recognizes both SM and PC as substrates, whereas the SMase from P. fluorescens only recognizes SM. In addition, an alkaline ceramidase (CDase) has been purified, characterized and identified from the culture media of P. aeruginosa AN17 (23). Importantly this bacterial CDase shares significant homology to the mammalian mitochondrial CDase (24, 25).
In this study we report the existence of SM synthase activity in the culture medium of P. aeruginosa. We have purified and characterized this bacterial SM synthase activity and have found it to correspond to the previously identified PlcH.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Cultures of Pseudomonas for Purification—A starting culture of P. aeruginosa cells, PAK strain (kind gift from Dr. Joan Olson, Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC), from a frozen stock was incubated with vigorous shaking at 30 °C in LB for 14 h. A 1:500 dilution of the starting culture was incubated at 30 °C overnight in 6 liter of synthetic medium containing 8.2 mM NH4Cl, 2.5 mM K2HPO4, 74.9 mM NaCl, 21.8 mM glucose, 2.5 g/liter LB, pH 7.2. The culture was centrifuged at 6500 x g for 25 min at 4 °C. The supernatant was then precipitated with 60% ammonium sulfate for 3–4 h at 4 °C with gentle swirling, and subsequently centrifuged at 2500 x g for 30 min at 4 °C. The pellet was resuspended in buffer A (20 mM MES, pH 6.5, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.005% Triton X-100). The suspension was clarified through a 0.45-µm syringe filter.
Butyl-Sepharose—The filtered solution was adjusted to 1 M ammonium sulfate and applied to a 20-ml HiTrap butyl-Sepharose column (1.2 mg of proteins/ml of column) previously equilibrated with buffer B (buffer A + 1 M ammonium sulfate) at a flow rate of 0.5 ml/min. The bound proteins were washed with 5 column volumes of buffer B at 1 ml/min flow rate. Proteins were eluted by decreasing the ammonium sulfate concentration from 1 M to zero applying a 6-column volume linear gradient of buffer A. Fractions of 3 ml were collected, and SM synthase activity (50 µl/fraction) and protein assay were performed. The butyl-Sepharose column was then washed with 5 column volumes of double distilled water (ddH2O) at a rate of 2 ml/min, and fractions of 5 ml were collected. Water wash fractions were also tested for SM synthase activity and protein concentration, and fractions with peak activity were combined with fractions with peak activity from the elution step of the butyl-Sepharose.
Octyl-Sepharose—Pooled fractions with peak activity from the butyl-Sepharose column were adjusted to 1 M ammonium sulfate and loaded on a 10-ml octyl-Sepharose column (0.7 mg of proteins/ml of column) previously equilibrated with buffer B. The loosely associated proteins were washed at 0.5 ml/min for 3 column volumes of buffer B, followed by a 2.5-column volume step elution at 55% buffer B. Proteins were further eluted at a flow rate of 1 ml/min with a 1.5-column volume linear gradient of buffer B (from 55 to 0%), and the column was further washed with 2.4 column volumes. Fractions of 1 ml were collected, and SM synthase activity (50 µl) and protein concentration were determined.
Blue Sepharose—Fractions with peak activity from the octyl-Sepharose column were pooled, adjusted to pH 6.0, and loaded (0.3 mg of proteins/ml of column) at 0.5 ml/min on a 2-ml Blue Sepharose high performance column equilibrated with buffer C (20 mM MES, pH 6.0, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.005% Triton X-100). A step elution with 7.5 column volumes of 12% buffer D (buffer C + 1.5 M NaCl) was followed by an increasing linear gradient of NaCl (from 12 to 75% buffer D) and a column wash with 75% buffer D of 11 column volumes each. One-ml fractions were collected, and the SM synthase activity (50 µl) and protein concentration were determined. The fractions with peak activity were then pooled, desalted, and buffer-exchanged with buffer E (20 mM Tris, pH 7.4, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.005% Triton X-100) on PD-10 desalting columns.
Mono P—The desalted, buffer-exchanged fractions were applied to a 1-ml Mono P column equilibrated with Buffer E. The column was washed at 0.5 ml/min with 7 column volumes of Buffer E, and then eluted by decreasing the pH with Buffer F (1:8 dilution of Polybuffer 74, pH 4.0). Fractions of 0.5 ml were collected and neutralized with concentrated Tris buffer, pH 7.4–9.0. SM synthase activity (50 µl) was determined.
Mono S—The fractions with peak activity from the Mono P column were combined and adjusted to 50 mM sodium acetate, pH 4.0, then loaded onto a 1 ml Mono S column previously equilibrated with Buffer G (50 mM sodium acetate, pH 4.0, 0.2 mM phenylmethylsulfonyl fluoride, 0.005% Triton X-100). A column wash at 0.5 ml/min with 7 column volumes of Buffer G preceded the elution step with Buffer H (Buffer G + 1.5 M NaCl) consisting of a linear gradient (0–0.75 M NaCl) of 10 column volumes and 20 column volumes of 1.5 M NaCl. One-ml fractions were collected in tubes containing 0.1 ml of 1.5 M Tris, pH 9.0. SM synthase assay was performed on 100 µl/fraction, and active fractions were concentrated on YM-10 Microcon centrifugal filters, separated on an 8% Tris-glycine gel, and visualized by silver staining.
SM Synthase Assay—The SM synthase assay was performed using aliquots (50–100 µl depending on the column) from the fractions of interest. The substrate was prepared as a 2x mixture of 40 µM NBD-C6-ceramide and 240 µM PC resuspended in 100 mM Tris, pH 7.4, 50 mM KCl, 1 mM EDTA by sonication and vortexing until clear. The substrate was diluted 1:1 with the protein, and the incubation was carried out for 1 h in the dark at 37 °C. The reaction was stopped on ice by addition of a volume of chloroform:methanol (1:1, v/v) 3 times the assay volume. After vortexing, the phases were clarified by centrifugation at 2500 x g for 5 min. The lower phase was transferred to new tubes, dried down, and lipids were resuspended with 30 µl of chloroform:methanol (2:1, v/v). NBD-C6-sphingomyelin was separated from the substrate by thin layer chromatography in chloroform, methanol, and 15 mM CaCl2 (60: 35:8). Fluorescence was measured by using a Storm 860 Imaging Analysis System from Amersham Biosciences. Data were analyzed using ImageQuant software from Amersham Biosciences.
Characterization Experiments—PlcHR2 was purified
as previously described (26)
and diluted to 0.1 µg/ml in 50 mM Tris, pH 7.4 for the SM
synthase assay. The reaction mixture contained 16.7 pg/µl of purified
protein, 75 mM Tris, pH 7.4, 100 µM
NBD-C6-ceramide, and 120 µM PC in a total volume of
100 µl and was carried out for 15 and 30 min at 30 °C. Formation of
NBD-C6-SM was determined as previously indicated. In some
experiments, radioactive PC was used instead of fluorescent
C6-ceramide as the labeled substrate. Briefly, a 2x substrate
mixture containing 200 µM C6-ceramide and 240
µM [14C-choline]PC (specific activity of
45 nCi/nmol) was resuspended in 0.1 M Tris, pH 7.4. The
substrate was diluted 1:1 with the protein (200 µl final reaction volume)
and incubated for 30 min at 30 °C in the dark. The reaction was stopped
with 1.5 ml of chloroform:methanol (2:1, v/v) and 0.2 ml of ddH2O.
After vortexing, the samples were centrifuged at 2500 x g for 5
min to separate the phases. The lower phase was collected and dried down, then
resuspended with 40 µl of chloroform:methanol (2:1, v/v), and separated by
thin layer chromatography in chloroform, methanol, and 15 mM
CaCl2 (60:35:8). The plate was analyzed by autoradiography, the
[14C-choline]C6-SM band was identified by
comparison with an authentic standard, and the radioactivity associated with
the band of interest was quantified by scintillation counting.
Neutral SMase Activity—Neutral SMase activity was determined using radiolabeled substrate delivered in a mixed micelle system. Briefly, the reaction mixture contained 0.1 M Tris, pH 7.4, 2.5 mM MgCl2, 0.1% Triton X-100, 10 nmol of sphingomyelin, 100,000 cpm of 14C-labeled sphingomyelin and an aliquot from the fractions to be tested (10–50 µl) in a final volume of 200 µl. The incubation was carried out at 37 °C for 30 min in a water bath. The reaction was stopped by addition of 1.5 ml of chloroform:methanol (2:1, v/v) and 0.2 ml of water. After vortexing and centrifugation at 1500 x g for 5 min, 400 µl from the upper phase were counted in 4 ml of scintillation fluid.
PC-PLC Activity—Purified PlcHR2 (40–140 pg/µl) was incubated with 20 mM p-nitrophenylphosphorylcholine (p-NPPC) in 100 mM Tris, pH 7.4, and 25% glycerol, in a total volume of 200 µl. Incubation was carried out at 37 °C for 1 h in a 96-well plate, and production of the chromogenic product p-nitrophenyl was measured at 405 nm (26).
BCA Protein Determination—Protein concentration was determined according to the instructions from the manufacturer (Pierce).
Western Blotting—The Western blot analysis for PlcHR2 was performed as previously described (26). Proteins were separated on a 7% PAGE-SDS gel and transferred onto nitrocellulose at 80 V for 1 h. The membranes were blocked with 5% milk in phosphate-buffered saline containing 0.05% Tween, and blotted with monoclonal antibodies (H952, 1:250 dilution) in the blocking solution. After extensive washing, the membranes were blotted with secondary anti-mouse horseradish peroxidase-conjugated antibodies (1:4000 dilution) in blocking medium for 1 h. The signal was visualized by enhanced chemiluminescence (Amersham Biosciences).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
SM synthase present in the medium of an overnight culture was concentrated
by incubation with 60% ammonium sulfate, and then the precipitated proteins
were resuspended in 20 mM MES, pH 6.5. The suspension was filtered,
adjusted to a final concentration of 1 M ammonium sulfate, and
subjected to hydrophobic interaction chromatography using a butyl-Sepharose
column previously equilibrated with the same buffer containing 1 M
ammonium sulfate (buffer B) (Fig.
1A). In these conditions, most of the activity was found
to bind to the matrix whereas the bulk of the proteins (84% of the total
proteins), represented mainly by small size proteins and/or peptides, were
found in the flow-through. Elution of the activity was observed in two
distinct peaks upon decrease of the ammonium sulfate concentration, a first
peak at 0.43 M ammonium sulfate and a second peak at 100
mM ammonium sulfate. Moreover, when the column was regenerated with
a wash of double distilled water, 30% of the initial SM synthase activity
and 10% of proteins were eluted from the column. The fractions containing the
two peaks of activity were combined with the activity recovered in the water
wash, and the buffer and salt concentration were adjusted to 20 mM
MES, pH 6.5, and 1 M ammonium sulfate. The total volume was applied
to an octyl-Sepharose column (Fig.
1B) previously equilibrated with the same buffer. The SM
synthase activity was found to bind to the column, as did most of the other
proteins. Elution of SM synthase was then achieved by decreasing the ammonium
sulfate concentration similar to what was observed with the butyl-Sepharose
column. The bulk of the proteins eluted at 50 mM ammonium
sulfate (although this value has been at times lower), whereas detachment of
SM synthase from the column was mostly obtained at zero salt. This column
therefore almost completely desalted the preparation obtained from the
previous column and allowed an additional step in the purification process
(2–3-fold). Also in this case, 20% of the loaded activity and
proteins were still bound to the matrix after the elution step. The
water-eluted enzyme was not purified further, as it behaved differently in the
next column from the one eluted with 20 mM MES, pH 6.5, and no
salt, underscoring the possibility of the existence of SM synthase in
different isoforms or at different stages of denaturation and/or
aggregation.
|
Fractions with peak activity were pooled (typically fractions
228–238; Fig.
1B), the pH was adjusted to 6.0, and the total volume was
applied to a Blue Sepharose affinity column previously equilibrated with 20
mM MES, pH 6.0 (Fig.
1C). The bulk of the proteins did not bind to the column,
whereas SM synthase did, and it was eluted at 0.4 M NaCl as a
single peak by application of an increasing linear gradient of salt (from 0.18
M to 1.13 M NaCl). To elute the SM synthase activity in
the middle of the gradient as a defined peak without too much tailing, it was
important that the sample that was loaded had a conductivity between 0.6 and 3
millisiemens. In this step of purification, SM synthase was enriched
20–25-fold.
Next, fractions containing peak activity were pooled, desalted, and
buffer-exchanged with 20 mM Tris, pH 7.5, using PD-10 columns. The
enzyme was further purified by application to a Mono P chromatofocusing column
(Fig. 1D) previously
equilibrated with 20 mM Tris, pH 7.5. Proteins were eluted by
decreasing the pH with an increasing linear gradient of Poly-buffer 74
adjusted to pH 4.0. The isoelectric point of the protein was determined to be
4.7–4.9. Because the low amount of protein applied to this and to
the next column (Mono S) did not allow an accurate protein determination in
the fractions, the efficacy of the columns was determined by silver
staining.
Fractions at pH 4.7–4.9 were pooled together (typically a total of 2
ml), adjusted to pH 4.0 with sodium acetate (at a final concentration of 20
mM), and applied to a cation exchange Mono S column
(Fig. 1E). In these
conditions, SM synthase bound to the column, and it was eluted with 0.34
M NaCl, applied as an increasing linear gradient. By analysis with
silver staining of the proteins separated on a SDS gel, fractions with peak SM
synthase activity contained only two bands, one at 75 kDa and one at
30 kDa, and both correlated with the activity (data not shown).
Purified SM Synthase Also Shows N-SMase Activity— P.
aeruginosa PA01 is known to express and secrete a N-SMase activity
(encoded by the plcH gene) for which the theoretical isoelectric
point and molecular mass are 5.0 and 78 kDa, respectively
(20), similar to what was
observed with the purified SM synthase. Because SM synthase activity was
observed in both PAK and PA01 strains, and the gene encoding for N-SMase
activity in PAK is identical to the plcH gene in
PA01,2 we wondered
whether the purified preparation of SM synthase from PAK also contained
N-SMase activity. To this end, N-SMase activity was measured in the fractions
collected from the Mono P chromatofocusing column
(Fig. 2A). As shown in
the figure, not only was N-SMase activity detected in the fractions but this
activity mimicked the profile of the SM synthase activity
(Fig. 1D). In
contrast, no significant acid SMase activity was detected (data not shown).
N-SMase activity was then measured in fractions collected from the last column
of the purification protocol, the cation exchange chromatography
(Fig. 2B). Also in
this case, N-SMase activity paralleled exactly SM synthase activity.
|
To confirm the existence of a N-SMase in the highly purified SM synthase preparation, a Western blot analysis was performed on the fractions with peak activity for SM synthase from a Blue Sepharose column using monoclonal antibodies raised against purified PlcH from P. aeruginosa, strain PA01 (26) (Fig. 3). As shown in the figure, fractions from the elution step with peak SM synthase activity (fractions 42–46, Fig. 3A) tested positive for the presence of a 78-kDa protein, which reacted with the specific antibodies against PlcH and that run similarly to the purified PlcH from PA01 (Fig. 3B). These results show that the preparation of SM synthase purified from PAK indeed contained also a protein homologous to the PlcH isolated and purified from strain PA01. Therefore, these results raised the question of whether the SM synthase from PAK is identical to this N-SMase.
|
PlcHR2 Is Sufficient and Necessary for SM Synthase Activity—Given the high homology between N-SMase from PAK and PlcH from PA01, we utilized purified PlcH from PA01 to determine whether this enzyme also had SM synthase activity in vitro (Fig. 4). The protein responsible for the N-SMase activity in PA01 (PlcH) is secreted as a complex with two accessory proteins (PlcR1 and PlcR2), believed to regulate PlcH secretion (26, 27). In the purification process, however, only PlcR2 maintains its association with PlcH, forming the PlcHR2 complex. Therefore, purified PlcHR2 was incubated with vesicles containing phosphatidylcholine and NBD-C6-ceramide, and the formation of NBD-C6-sphingomyelin was monitored as described under "Experimental Procedures." As shown in Fig. 4, purified PlcHR2 was able to synthesize SM in a protein-dependent (Fig. 4A) and time-dependent (Fig. 4B) manner. Therefore, these results clearly show that PlcHR2 is sufficient for SM synthase activity.
|
Next, we asked whether PlcH was necessary for SM synthase activity in
P. aeruginosa, PA01. To this end, SM synthase activity was measured
in different cultures of mutant strains lacking the genes responsible for the
synthesis and secretion of PlcH. Because the plcR1,2 genes
are located 3' to plcH and are expressed from in-phase
overlapping genes, the single deletion of plcH causes a block of
transcription of the plcR1,2, thus delivering in fact a
plcH/plcR double knock-out. In the following experiments,
strains were used that were lacking in either
plcR1,2(
R) or plcH and
plcR1,2(HR). As a control we employed a strain that
was deficient in the neutral/alkaline ceramidase (CDase) gene, which is
adjacent to the PlcHR operon (Fig.
5). As expected, the R strain secreted significantly less
N-SMase activity in the media compared with the parental strain (PA01)
(left top panel) retaining the activity in the cells (left bottom
panel). The lack of the PlcHR operon (HR) caused an
almost total disappearance of N-SMase activity in both medium and cells. On
the other hand, absence of the CDase gene did not affect
significantly N-SMase activity in either medium or cells, demonstrating that
the observed effects on N-SMase activity are specific to the
PlcH/R1,2 genes. When SM synthase activity was
measured in these same cultures, an almost identical profile was observed; the
R strain retained the activity in the cells, and lack of the
PlcHR operon caused an almost total disappearance of SM synthase in
both cells and medium. Moreover, no significant difference between the
parental strain and the CDase in both cells and medium was observed
when measuring the SM synthase activity, thus confirming also in this case the
specificity of the results (Fig.
5). These results therefore support the necessity of the
expression of the PlcH for SM synthase activity in P.
aeruginosa, PA01.
|
Free PlcR2 Enhances SM Synthase Activity of Free PlcH—As mentioned, PlcR1,2 chaperones PlcH out of the cells where their interaction is maintained. More recently it has also been shown that the interaction of free PlcR2 with free PlcH increased the phospholipase activity of PlcH (26). Therefore we investigated the effect of free PlcR2 on SM synthase activity of free PlcH (Fig. 6). As shown in the figure, addition of increasing amount of free PlcR2 to a constant amount of free PlcH significantly augmented PlcH ability to synthesize NBD-C6-sphingomyelin. No significant SM synthase activity was detected from free PlcR2 alone (data not shown). These results suggest that in the case of SM synthase, as for PC-PLC, PlcH may represent the catalytic subunit of the PlcHR2 complex whereas PlcR2 could represent a possible regulatory subunit.
|
Characterization of pH Dependence and Effects of Cations on SM Synthase Activity of PlcHR2—To characterize and further optimize the conditions for the SM synthase activity of PlcHR2, the pH optimum of the reaction was determined using purified PlcHR2 (Fig. 7A). As shown in the figure, purified PlcHR2 showed significant activity over a wide range of pH (from 7 to 9.5) with peak activity observed between 7.5 and 8.5. At pH lower than 6.0 and higher than 9.5, activity was almost undetectable.
|
Next, the effects of cations were studied. It has been described that some divalent cations significantly affect PC-PLC activity of PlcHR2 (26); therefore, the effects of these cations on SM synthase activity were investigated (Fig. 7B). As shown in the figure, SM synthase activity was effectively inhibited by zinc and to a lesser extent by nickel, thus demonstrating effects similar to those observed on PC-PLC activity. Manganese significantly inhibited SM synthase activity but at higher concentrations than zinc and nickel, also reproducing the effects observed with PC-PLC activity. Calcium, magnesium, and low concentrations of manganese slightly but reproducibly activated SM synthase, whereas they did not significantly affect PC-PLC, suggesting a possible difference in the regulation of these activities.
SM Synthase from P. aeruginosa Is a Transferase—PlcH has been previously described as a phospholipase C that hydrolyzes both PC and SM, releasing phosphorylcholine (20). In mammalian cells, SM synthesis occurs upon transfer of a phosphorylcholine (choline-P) moiety onto the primary hydroxyl of ceramide through a transferase-like reaction (2). Therefore, it became important to determine the biochemical nature of the bacterial SM synthase activity. A screen with different donors of choline-P was set up, and the possibility of each compound to be used as substrate for the synthesis of SM was tested (Fig. 8A). As shown in the figure, free phosphorylcholine (Ch-P) was not a substrate for SM synthesis (as compared with PC). Moreover, neither CDP-choline (CDP-Ch) nor p-NPPC (often used as substrate for the phospholipase C activity in vitro) was used as substrate for SM synthesis. On the other hand, PC, platelet activating factor, and their respective lyso-forms were efficiently recognized as substrates. SM itself and its lyso-counterpart were also used as donors of the phosphorylcholine group but with significantly less efficiency. These results suggest that the bacterial SM synthase is a transferase that requires at least a fatty acyl chain on the choline-P donor molecule to be recognized as a substrate. Next, the possibility that the bacterial SM synthase would use phospholipids other than PC as substrates was investigated. As shown in Fig. 8B, none of the phospholipids tested (phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylglycerol) other than PC induced formation of SM. These results suggest that SM synthase specifically recognizes the choline head group on its substrate.
|
On the other hand, we then determined the specificity of the
phosphorylcholine-acceptor molecule (i.e. ceramide). First, the
stereospecificity of the transferase reaction when ceramide was used as
substrate was evaluated by directly measuring the conversion of
14C-fatty acid-labeled C6-ceramide stereoisomers to
their respective short chain sphingomyelins. As shown in
Fig. 9A, the enzyme
best operated when the erythro-C6-ceramide isomers were
used as substrates, whereas the threo isomers showed lower activity.
Importantly, these results suggest that the transferase reaction is
stereoselective. Then the ability of C6-dihydroceramide to be
recognized as substrate was evaluated as compared with the ceramide isomers
(Fig. 9A), and it was
found to be significantly lower than the erythro isomers and similar
to the threo isomers. This observation might implicate the relevance
of the presence of the 4–5 double bond on the ceramide molecule to be
efficiently recognized as a substrate. Next, the specificity of the reaction
was further tested using short chain diacylglycerol, a molecule biochemically
similar to short chain ceramide, and in particular having an accessible
hydroxy-group in the sn-3 position. As shown in
Fig. 9A, it is
apparent that the bacterial enzyme does not recognize diacylglycerol as
acceptor molecule for the transferase reaction. This result would suggest that
the transferase reaction discriminates among acceptor hydroxyls. To further
verify this hypothesis, other alcohols were tested as substrates for the
bacterial enzyme (Fig.
9B) in competition with NBD-C6-ceramide (30
µM; Km = 20 µM). In this
experiment, L-erythro- and
L-threo-C6-ceramides were used as controls. As
expected from the results in Fig.
9A, L-erythro-C6-ceramide,
being a good substrate for the transferase activity, efficiently competed with
the NDB-C6-ceramide for the formation of NBD-C6-SM
(80 and 90% inhibition at 60 and 90 µM, respectively)
whereas L-threo-C6-ceramide was not as
effective (20 and 50% inhibition at 60 and 90 µM,
respectively). Importantly, none of the alcohols used in the assay, namely
ethyl, lauryl, or stearyl alcohol, were able to compete with the
NBD-C6-ceramide. Thus, they were neither inhibitors nor substrates
because, according to this approach, a substrate would act as a substrate
competitor of NBD-C6-ceramide. These results therefore strengthen
the hypothesis that the transferase reaction discriminates among acceptor
hydroxyl groups.
|
Altogether, these results show that the transferase reaction is selective in the choice of the phosphorylcholine acceptor molecule at different levels; the reaction is stereoselective, it favors the presence of the 4–5 double bond on the ceramide, and it exchanges only with the ceramide primary hydroxyl and not other hydrophobic alcohols.
SM Synthase from P. aeruginosa Has No Reverse Activity—It has been suggested that the enzyme responsible for SM synthesis in Madin-Darby canine kidney cells also has reverse activity (28). Therefore, we investigated whether this was also the case for the bacterial SM synthase. By using radiolabeled sphingomyelin in the choline moiety and diacylglycerol as substrates, formation of radioactive phosphatidylcholine was monitored. To determine the production of phosphatidylcholine, the lipids were extracted with 1.5 ml of chloroform:methanol (2:1, v/v) and 200 µl of ddH2O, and then processed with two alternative protocols. First, the lower phase was base-hydrolyzed (causing liberation of radioactive choline-P from the newly synthesized PC), and, after a second lipid extraction, aliquots of the aqueous phase were counted (Table I). Alternatively, the whole lower phase from the first extraction was dried down and lipids separated by thin layer chromatography (TLC) (Fig. 10A). As shown in the table and in the figure, no formation of radioactive PC and therefore transferase activity was detected by either protocol, even though hydrolysis of SM occurred (Table I).
|
|
The optimum pH for SM synthesis by PlcHR2 is between 7.5 and 8.5 (Fig. 7A); however, the reverse reaction may demonstrate a different pH optimum (a condition seen with neutral ceramidase; Ref. 29). Therefore, PC synthesis was examined at different pH by TLC analysis (Fig. 10B). Also in this case, no formation of PC from SM could be observed. Altogether, these results suggest that PlcHR2 performs only the synthesis of SM and not the reverse transferase reaction.
SM Synthase Activity Is Specific to the Phospholipase C of P. aeruginosa—Other bacterial phospholipases C have been identified, such as sphingomyelinase and PC-specific phospholipase C from B. cereus (15, 30) or S. aureus (16). These enzymes show similar substrate preferences (PC or SM) to the P. aeruginosa PlcH, and, therefore, they were evaluated for SM synthase activity. Purified sphingomyelinase (bSMase) or PC-PLC (bPC-PLC) from B. cereus, which gave comparable N-SMase (cpm) or PC-PLC activity to the PlcHR2 from Pseudomonas were tested for SM synthase activity. As shown in Table II, neither bSMase nor bPC-PLC showed detectable SM synthase activity. Additionally, several other dilutions of the two enzymes were tested for SM synthase and found to have no activity under any condition (data not shown). It has been shown that magnesium activates bSMase and upon magnesium activation bSMase would also act as a PC-PLC. Therefore, the effects of magnesium on possible SM synthase activity of bSMase were examined (Table II). Whereas PC-PLC activity could be clearly measured under these conditions (Table II), there was no detectable SM synthase activity. These results clearly show that the ability to synthesize SM is specific to the Pseudomonas PlcH.
|
SM Synthase Activity of P. aeruginosa Is Inhibited by D609— Mammalian SM synthase has been previously shown to be inhibited by an inhibitor of the bacterial PC-PLC, D609 (10, 12). Therefore, the effect of D609 on the Pseudomonas SM synthase was also tested. As shown in Fig. 11, preincubation of the enzyme with different concentrations of D609 significantly inhibited the reaction in a dose dependent manner. The extent of inhibition is comparable with that observed with mammalian SM synthase and PC-PLC.3 These results provide an additional level of similarity between mammalian and bacterial SM synthase.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
On the other hand, the bacterial SM synthase is a soluble protein, whereas the mammalian enzyme has been described as a tightly membrane-bound enzyme mainly residing in the Golgi apparatus (32–37), but also present in the plasma membrane (28, 31, 32, 38–41) and in the nucleus (42, 43). Interestingly, however, during the purification protocol, the bacterial SM synthase was found to bind strongly to butyl- and octyl-Sepharose matrices, highlighting an important hydrophobic component of this soluble protein. This aspect is not surprising given the lipid-metabolizing activity of the enzyme, and thus the necessity for a lipid/protein interaction exemplified by the requirement of at least one fatty acyl chain in the donor molecule for SM synthesis.
The difference in the solubility between sphingolipid-metabolizing enzymes from Pseudomonas and the mammalian counterparts is not unique to the SM synthase protein. In fact, a similar situation is observed in the case of both neutral/alkaline ceramidase and neutral SMase. Both are secreted proteins from P. aeruginosa (23) and P. fluorescens (21), respectively, whereas their mammalian counterparts, the mitochondrial/neutral ceramidase and the nSMase2, are associated with membranes. Despite this difference, a significant homology between the amino acidic sequences can be observed. For instance, the Pseudomonas ceramidase and the mammalian mitochondrial ceramidase share 35% identity and 53% similarity. Importantly, a similar correspondence could support the possibility of finding a mammalian homologue to the bacterial SM synthase.
The bacterial SM synthase was found to be the previously isolated PlcH, a phospholipase C known to hydrolyze phosphatidylcholine and sphingomyelin, and responsible for the hemolytic activity of P. aeruginosa. The results from this work, by using a combination of biochemical and genetic approaches, demonstrate that PlcH is sufficient and necessary for SM synthase activity. First, the purified protein from PAK strain showed neutral SM hydrolytic activity and similar molecular weight and isoelectric point to the PlcH from PA01 strain. Second, the purified SM synthase from PAK specifically reacted with monoclonal antibodies raised against purified PlcH from PA01. Third, purified PlcH from PA01 was able to synthesize SM. Fourth, SM synthase activity is abolished when the PlcH gene in P. aeruginosa PA01 is deleted.
Interestingly, SM synthase activity is associated only with PlcH and not PlcN, a second phospholipase C from P. aeruginosa. Deletion mutants for PlcN did not display any significant defect in SM synthesis (data not shown). Although PlcH and PlcN share 40% identity in their amino acidic sequences, they show different substrate preference. PlcH recognizes PC and SM whereas PlcN hydrolyzes PC and phosphatidylserine (PS) (20). This observation brings up the possibility that the ability of PlcH to recognize SM might be a requirement for SM synthase activity.
The ability of PlcH to hydrolyze PC (PC-PLC) and at the same time to synthesize SM (SM synthase) raises the interesting issue of the potential identity of the mammalian PC-PLC/SM synthase. Based on the biochemical similarities between the two reactions (hydrolysis of PC and formation of DAG, and inhibition by D609) and possible biological phenotypes (increased activity in transformation), we previously proposed that the PC-PLC and SM synthase reactions could be regulated by the same enzyme (10). The results presented in this study importantly confirm the validity of this hypothesis, at least for the Pseudomonas enzyme.
The possibility of PlcH to catalyze different reactions (PC-PLC, SMase, and SM synthase) raises the interesting question on how these reactions might be regulated. Interestingly, several ceramidases (yeast, mammalian, and bacterial) have been reported to have dual activity, hydrolytic (ceramidase) and synthetic (ceramide synthase) (23, 29, 44, 45). Biochemical characterization has shown that regulation of these reactions might involve different mechanisms such as different pH optima, product inhibition (sphingosine on ceramidase), and differential effects of lipid cofactors (cardiolipin and phosphatidylglycerol promote ceramidase activity and inhibit the ceramide synthase). In the case of PlcH, hydrolytic and synthetic reactions occur at similar pH optima excluding a differential regulation at different pH. Interestingly, it has been previously reported that PlcH and its chaperone PlcR2 bind to calcium and that the latter predicted to contain four EF-hands (26). Therefore, one possible regulatory mechanism might occur through the interaction of specific cations, in particular calcium. Although no effect of calcium has been reported on PC-PLC activity and a slight but reproducible activation has been now shown with SM synthase, we found that the neutral SMase activity of PlcH was significantly inhibited by calcium in the absence of detergent.3 This observation might suggest that PlcH hydrolyzes PC and SM through a different catalytic mechanism/site. On the other hand, it has not yet been determined whether PC-PLC and SM synthase reactions share a common hydrolytic site for PC.
Importantly, bacterial SM synthase activity is specific to P. aeruginosa as it has been found neither in other bacteria such as E. coli nor in other purified phospholipases C with similar substrate preference to PlcHR2. This observation is not surprising, given the lack of sequence homology between, for instance, PC-PLC and N-SMase from B. cereus and S. aureus and PlcH, which appears to belong to a distinct family of enzymes. Also this observation would lead to the prediction that the mammalian phospholipases homologue to these bacterial enzymes (such as nSMase1) should not exhibit SM synthase activity.
In conclusion, this study identifies the first structural gene for a SM synthase, providing the first molecular tool for the study of this class of enzymes. Importantly, it may potentially lead to the identification of the elusive mammalian SM synthase.
|
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grants
HL43707 (to Y. A. H.) and HL62608 (to M. L. V.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
|| Present address: Diabetic Microangiopathy Research Unit,
Merck-Santé/INSERM U352, INSA-Lyon, 69621 Villeurbanne, France.
To whom correspondence should be addressed: Dept. of Biochemistry and
Molecular Biology, Medical University of South Carolina, STB Rm. 652,
Charleston, SC 29425. Tel.: 843-876-5214; Fax: 843-876-5172; E-mail:
luberto{at}musc.edu.
1 The abbreviations used are: SM, sphingomyelin; PlcH, hemolytic
phospholipase C; N-SMase, neutral sphingomyelinase; SMase, sphingomyelinase;
PC-PLC, phosphatidylcholine-specific phospholipase C; p-NPPC,
p-nitrophenylphosphocholine; DAG, diacylglycerol; ddH2O,
double distilled H2O; MES, 4-morpholineethanesulfonic acid; CDase,
ceramidase; NBD, (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)).
2 M. L. Vasil, unpublished observations.
3 C. Luberto, E. Collins, and Y. A. Hannun, unpublished observations.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
