Sphingolipid metabolism Mefr
Lev,
biosynthesis in Bacteroides
known. Sphingolipids are characteristic compoofthe membranes ofall eukaryotic cells where they exist as two classes, the carbohydrate-containing sphingoipids, such as gangliosides which function as receptors for certain hormones and a number of toxins (7), and the phosphosphingolipids, such as sphinnents
gomyelin. The medical importance of the study these lipids lies in the cerebral lipidosesgenetic diseases that are usually fatal and caused by deficiencies in degradative zymes of the gangliosides (7). Effect of vitamin metabolism
K depletion
of are en-
on cell
The approach used to the study of vitamin K mode of action was to starve the cells of this microorganism for the vitamin, deterAmerican
K
Ph.D.
At this time the vitamin K requirement of certain strains of Bacteroides melaninogenicus is well known. Studies of this requirement stem from our observations of cross-feeding between a rumen isolate of B. me/aninogenicus and a strain of Proteus (1). Since then studies by MacDonald et al. (2) showed vitamin K dependence in oral B. me/aninogenicus strains and vitamin K is now routinely added to media for isolation of anaerobic bacteria (3). The characterization of a new class of lipids-the sphingolipids from B. melaninogenicus by White and colleagues-added an important tool for the study of this class of compounds (4-6). Although sphingoipid biosynthesis has interested neurobiobogists for more than a century, a number of important questions remains unanswered, most notably related to the biosynthesis of sphingomyein-the principal phospholipid in brain. Moreover the function of these phosphosphingolipids in the membranes of cells is not
The
and vitamin
Journal
ofClinical
Nutrition
32: JANUARY
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mine the biochemical lesion and then to correct the defect specifically by vitamin K addition. On vitamin K depletion, cells of B. melaninogenicus elongate from 0.4 to 0.6 tm up to 14 jzm in length. This inability to divide suggested a membrane defect (8). We had found that B. melaninogenicus could be grown in the absence of vitamin K in the presence of 2 x l0_2 M sodium succinate. Under these conditions the microorganism grows slowly and the growth rate of the culture is increased after the addition of vitamin K (9). Succinate grown cells are useful in providing vitamin K-depleted bacteria since their growth is more predictable than that obtained simply by the incubation of cells in a vitamin K-free medium. In all experiments on metabolism, the modified anaerobicjar (10) was used that permits removal and additions to cultures with minimal disturbance of the Eh. The effect of vitamin K depletion on macromobecular synthesis was then studied and it was shown that vitamin K depletion had no effect on the incorporation of a number of amino acids into protein and further that vitamin K depletion had no primary effect on DNA and RNA synthesis. The first insight into the mode of action of vitamin K came from studies on the uptake of labelled succinate by vitamin K-fed and vitamin K-depleted cells. The vitamin K depleted culture incorporated succinate at 6 x the rate of the vitamin K-fed cells (Fig. 1) and analysis ofthe distribution oflabeb in cell fractions showed a significantly higher proportion in the lipids of the vitamin K-dcpleted culture than in the lipids of a vitamin K-supplemented culture (9). The lipids of B. melaninogenicus have been
1 From the nology, Albert York 10461.
1979,
pp.
179-186.
Department of Microbiology Einstein College ofMedicine,
Printed
in U.S.A.
and ImmuBronx, New
179
LEV
180 CPM 0
2000
1 800
-
1600
-
400
-
1 200
-
0
which
-
600
-
0
Pathways 100
40C-
OD FIG. with
(0)
1. Uptake and
without
occurred
40 min
after
the addition
of
the vitamin to the culture (Fig. 2). In contrast to the effect on the sphingolipids, vitamin K had no effect on incorporation into PE and incorporation into PS was increased only after 90 mu of vitamin K addition at which time the culture was growing at an enhanced rate characteristic of the vitamin K supplemented culture. However, the stimulation of sphingolipid synthesis occurred before the rapid growth-phase, that is, before there was an increase in general cell metabolism (1 1).
1000-
800
taken at certain time intervals and incorporation into the lipids determined after extraction and chromatography. After the addition of vitamin K there was a linear increase in incorporation of 32P into CPE and CPG
660
(#{149}) vitamin
m
by cells
synthesis
The first step in the sphingolipid thesis pathway is the condensation coenzyme A (CoA) with serine
200
of [2,3-’4C]succinate
for phc
biosynof an acyl to form
grown
K (1 1).
characterized by White and coworkers (4-6) who demonstrated that in addition to phosphatidyb serine (PS) and phosphatidyl ethanolamine (PE), these bacteria contained phosphosphingolipids. These lipids are cxtremely rare in bacteria and to date they have been described only in certain species of Bacteroides although their presence in other bacteria will no doubt be demonstrated. The two major phosphosphingolipids were ceramide phosphorybethanolamine (CPE), an analogue of sphingomyebin and ceramide phosphorylglycerol (CPG). These sphingolipids comprise more than 50% of the phosphoipid content of the cells. Because of the enhanced incorporation of labelled succinate into the lipid fraction of vitamin K-depleted cells, the lipid metabolism of this microorganism was studied. It was found that incorporation of succinate or 32p into the sphingolipids was significantly reduced in vitamin K-depleted cultures whereas incorporation into PE or PS was not significantly affected. The effect of vitamin K on sphingoipid synthesis was shown directly by the addition of vitamin K to a vitamin K-depleted culture to which 32P had been added. Samples were
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0 0
0 I 0
0 I
0.
aU
OF PULSE
DURATION
FIG.
(Minutes)
2. Stimulation of synthesis of ceramide phosphorylethanolamine and ceramide phosphorylglycerol after addition of vitamin K at time 0 to a K (-) + succinate culture. The values are total cpm for each phospholipid spot from the thin-layer plate. Vitamin Ksupplemented culture: 0 ceramide phosphorylethanolamine; L ceramide phosphorylglycerol. Vitamin K-dcficient control culture: #{149} , ceramide phosphorylethanolamine; A ceramide phosphorylglycerol (I 1).
,
SPHINGOLIPID
an
The
cepted
sequence
3KDS
is reduced
AND
3-ketodihydrosphingosine
intermediate,
(3KDS).
BIOSYNTHESIS
following
is a generally
in sphingomyelin
by NADPH
ac-
synthesis.
to form
dihy-
drosphingosine and a double bond is inserted by an unknown mechanism to form sphingosine which is then acylated to ceramide. The choline moiety is then added as shown below. NADPH
3KDS
VITAMIN
K
cation little or no activity the cell envelopes. Supernatants prepared cells
grown
under
181
was
associated
by
differing
with
sonication cultural
of condi-
tions were examined from synthetase activity. These included cells grown with vitamin K; cells depleted of vitamin K by inoculation into a vitamin K-unsupplemented medium and vitamin K-depleted cells prepared by the acylCoA
.
>
METABOLISM
dihydrosphingosine
)
sphingosme
{
ceramide cytidine
di-
phosphate
choline? sphingomyein Although 3KDS is the recognized first step there is some doubt concerning the biosynthetic sequence of the subsequent steps and since CPE is a close analogue of sphingomyein, differing in that CPE possesses an ethanolamine residue in place ofcholine in sphingomyelin, it follows that study of CPE synthesis can serve as a useful model for spin-
gomyelin
biosynthesis.
Activity supplemented
of 3KDS Synthetase and -depleted
in vitamin cells.
K-
The experiments quoted above had shown that spingolipid synthesis was markedly reduced in vitamin K depleted cells and that following addition of vitamin K a specific enhancement of sphingoipid synthesis occurred (1 1). It was interest, therefore, to determine which biosynthetic step was influenced by vitamin K. We examined the activity of the enzyme involved in the first step in sphingolipid biosynthesis 3KDS synthetase which catalyses:
of the bacteria in the abK but in the presence of 2 102 (9). In contrast to the good activity found in extracts of B. melaninogenicus that had been grown in a vitamin K-supplemented medium, only low synthetase activity was found in extracts of the two vitamin K-depleted cubtures (Fig. 3). Vitamin K added in vitro to extracts from the vitamin-depleted cultures did not enhance enzyme activity. This result suggests that vitamin K is not a coenzyme for the synthetase. continuous sence of
x
After tamin
II serine
relatively
easily
and
following
addition
of vitamin
K to a vi-
NH2OH
I
I
R-C-C--
CH2
+
CO2
3KDS
The activity ofthe synthetase was determined by usingonebabeledsubstrate and determining the incorporation into the product following extraction, thin-layer chromatography, and radioautography. The enzyme preparation used was a 100,000 x g supernatant of sonicated cells. The bacterial system differs from those of eukaryotic cells in that the bacterial synthetase can be dissociated from the membranes
the
K-depleted
0 +
culture vitamin M succinate
culture, enhanced enzyme activity was found in extracts of the cells. Experiments were then performed to determine whether inhibitors of protein and RNA synthesis could effect the vitamin K-mediated induction of the synthetase. When a low level of vitamin K, (0. 1 tg/ml fmal concentration) was added to a vitamin K-depleted culture, a linear increase in enzyme activity was found in extracts of cells 15 to 40 mm after addition
0 R-C-SCoA acyl CoA
lecithin?
soni-
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4). In the increase in enzyme activity leveled off after 30 mm suggesting that protein synthesis was a requirement for vitamin K induction of enzyme activity. A similar result was obtained with an inhibitor of RNA synthesis, rifampicin. These experiments with inhibitors indicate vitamin K induced de novo synthesis of the enzyme (12). of
vitamin
presence
K to the of 50 tg/ml
culture (Fig. puromycin,
182
LEV
--
I
FIG. palmitoyl
2
3. Radioautogram
of enzymically
4
3
synthesized
3-[’4C]
5
ketodihydrosphingosine
from L-Senfle and [14C] mixtures (37C, 30 mm) extract (0.5 ml). Products K-depleted and succinate (KD) (12).
CoA. Thin-layer chromatogram of lipid products recovered from reaction containing L-serine (3 moles), (‘4C] pabmitoyl CoA. (0.2 .tmole, 0.5 tCi), and bacterial from mixtures I, 3, and 5 made with extracts from cells grown with vitamin K, vitamin grown-vitamin K-depleted cells, respectively; 2 and 4, authentic 3-ketodihydrosphingosine
Purification synthetase
and
properties
of 3KDS
tions). mine action
This mainly thetase
enzyme
has
not
because
attempts
to date to
been
solubilize
purified
the syn-
the membranes of eukaryotic cells results in loss of activity although in a preliminary report a 10 to 15x purification from rat brain microsomes was obtained (13). The bacterial enzyme possesses a unique advantage for purification studies since it is easily solubilized by sonication of the cells. Brady et al. (14) and Braun and Snell (15, 16) have shown that this enzyme is pyridoxab phosphate dependent and we have found that preincubation of a 20,000 x g supernatant of sonicated cells from a mid log-phase culture results in an approximately 100% increase in the activity of the enzyme. Using this preincubated extract we have purified this synthetase >40x and the enzyme elutes as a symetrical
with and
from
peak
from
an approximate A. F. Milford,
a
gel-filtration
MW of 48,000 unpublished
column
(M. Lev observa-
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Experiments kinetic data of the
are in progress and to study the
enzyme
with
the
to determode of purified mate-
rial. In
the
scheme the reduced
quoted
above,
3KDS
is
Dihydrosphingosine
for
sphingolipid synthesis product of the enzyme, to dihydrosphingosine.
is stable
compared
to
3KDS and, therefore, would be useful, for example, in the selection of mutants defective in the synthetase. However, this compound was found to be toxic and at a low (4 ELM) level, dihydrosphingosine completely inhibited the growth of B. melaninogenicus. This growth inhibition could be the result of inhibition of 3KDS synthetase and the addition ofdihydrosphingosine to a mid-log phase culture resulted in a drop in synthetase activity to minimal levels (Fig. 5). In contrast to these results, dihydrosphingosine added at 5x the level used in the above experiments (20 .LM) had no effect on the activity of 3KDS synthetase which had been previously solu-
bilized
by sonication.
To explain
these
results
SPHINGOLIPID
BIOSYNTHESIS
AND
we should consider that 3KDS synthetase is a membrane associated enzyme and the dihydrosphingosine taken up by the cells exerts its effect following permeation of the membrane. With a solubilized enzyme preparation, the concentration of dihydrosphingosine (added as an emulsion) in the water phase is probably too low to affect activity (17). The product of the synthetase reaction, 3KDS is not toxic for B. me/aninogenicus. The toxicity of dihydrosphingosine in contrast to 3KDS may indicate that the former may not be an intermediate as previously supposed and that reduction of the sphingo-
lipid step
base
may
occur
at a later
biosynthetic
(18).
VITAMIN
K METABOLISM
requiring
Energy
step
183
in sphingolipid
biosynthesis In studying the sphingolipids mine the steps
the
biosynthetic pathways for it is of importance to deterafter 3KDS formation. In early experiments considerable difficulty was encountered when attempts were made to utilize this compound for synthesis of the complete sphingolipids or the formation of other intermediates using washed cells or cell-free cxtracts. This inability of washed cells to utilize 3KDS was probably due to the lack of a suitable energy source since growing cultures do convert 3KDS into CPE and CPG. Since the basal medium used for growth of B. mel-
0
0
x
_-4
-I
-.
45
TIME FIG.
4. Effect
of puromycin
on the
induction
of 3KDS
75
60
90
105
MIN. synthetase.
Vitamin
K1 (0.1 g/ml
final
concn)
and
puromycin (50 &g/mb final ooncn) were added at time zero to culture of vitamin K-depleted cells successively subcultured with lO2 M sodium succinate, which had been grown to a turbidity of ca. 150 Klett units. A control culture received vitamin K only. At the times indicated, 20 ml of culture was withdrawn, the cells washed and sonicated. Enzyme determinations were made as described in the text. In the same experiment, 40 Ci [‘4C1 leucine was added to each culture at the same time as the antibiotic and the vitamin K. In the culture containing puromycin, little incorporation of leucine into trichboroacetic acid-precipitable material occurred during the experimental period (12).
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LEV
184
of
B.
me/aninogenicus
are
permeable
to
NADH. To elucidate
source,
m;
that
I
g
(Hr)
aninogenicus consists of amino acids and peptides supplied in the Trypticase component, we examined a number of amino acids for their growth stimulating capability when added at the 0.5% level to a nonsupportive medium. Glutamine, asparagine and serine stimulated growth under these conditions.
and
serine
was inactive.
asparagine
when
the glutamine
3KDS parent
no conversion
was
similar
strain
occurred.
cells grown vitamin conversion of 3KDS
to
whereas
On the
K-depleted when either
in vitamin cells and strain S has effective
in
Use of penicillin biosynthesis The inability melaninogenicus
complete inability nously, which
driving the conversion of K-depleted and -supplealso the cytochrome-free a requirement for vitamin
spheroplasts
in sphingolipid
of intact, washed cells of B. to convert 3KDS into the
sphingolipids could be due to the of this precursor, supplied exogeto reach the is the site of
Penicillin
cytoplasmic phospholipid
spheropbasts,
membrane synthesis.
in contrast
to intact
cells, do convert 3KDS into CPE in the absence of an energy source and A. F. Milford, unpublished
and CPG (M. Lev observations). However, after incubation, this capacity is lost and can be restored by glutamine, asparagine, and NADH. In addition, ATP
included
plus 3KDS stimulated insubstrate into CPE + CPG;
That
hand, good
3KDS mented mutant K.
Dihydrosphingosine inhibition of 3KDS activity in an actively growing culture. Dihydrosphingosine (4 LM final concentration) was added to a flask containing a mid log phase culture of B. melaninogenicus. Samples (20 ml) were removed at intervals from this and a control culture for enzyme determinations (18). 0 , control culture; L, plus dihydrosphingosine.
cells ofthe
NADH
other show
the
equally
5.
Glutamine
with
of
with
1
0
with washed corporation
conversion found
glutamine or NADH were used (Table 2). It would appear, therefore, that vitamin K does not participate in the election transport chain of B. melaninogenicus since NADH is
I.-
FIG. synthetase
glutamine
2 :D
TIME
K in the
.
I 4
3
of vitamin
Lu
I
z
action
stimulated incorporation of 3KDS, experiments were performed with strain “5”, a mutant that grows independently of added heme, does not make the b type cytochrome found in the parent strain but requires vitamin K (20). With glutamine as the energy
I
5
in I-
the
stim-
ubated incorporation mediated reaction to dinitrophenol, carbodiimide and
of 3KDS was an energy was shown by its sensitivity azide, N,N -dicycbohexylcarbonyl cyanide m-chborophenythydrazone (Table 1). Aerobic incubation of the reaction mixture completely abolished the glutamine driven 3KDS conversion (19). Beside glutamine and asparagine, NADH was active in stimulating conversion of 3KDS to CPE plus CPG; apparently the membranes
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TABLE
1
Effect of inhibitors of 3KDS into CPE
on glutamine-driven (20)
Inhibitor
incorporation
Concentration
Inhibition %
DNP
mM
Azide
50 nmi
DCCDb
20zM
64 68 100
DCCD
l0M
82
CCCPC Aerobic conditions Activity is expressed
20tM
56 100 of inhibition
a
glutamine control(2.98 dicycbohexylcarbodiimide.
chlorophenylhydrazone.
as a percentage nmol
ofCPE formed). Carbonyl cyanide
b
of
N,N’m-
SPHINGOLIPID
TABLE
BIOSYNTHESIS
AND
2
Incorporating activities of washed cells of B. melaninogenicus strain I, strain S (cytochrome-free), and SB (vitamin K depleted) with glutamine or NADH 3KDS
converted
Strain CPG
CPE nmole
2.98 1.5
1.21 0.21
2.74