In vertebrates, glycosphingolipids (GSLs) (
)are
located on the outer leaflet of the plasma membranes and may function
as mediators of cell-cell interaction, attachment, proliferation, and
differentiation(1) . Lyso-GSLs, which are GSLs N-deacylated in the ceramide moiety, have been detected in
normal tissues at very low levels but are accumulated in inherited
sphingolipid storage diseases(2) . Recently, several lines of
evidence have suggested the biological significance of lyso-GSLs in
cell activities. Tyrosine-specific autophosphorylation of the epidermal
growth factor receptor of A431 cells is inhibited by lyso-GM3 as well
as by GM3, and both of these are detected in the cells (3) .
Lyso-GSLs inhibit protein kinase C, and this may be responsible for the
pathogenesis of sphingolipidoses(4) . Furthermore, sphingosine,
a deglycosylated form of lyso-GSLs, has been found to modulate protein
kinase C-dependent cell functions (5) as well as a number of
other systems(6) .
Although one possible mechanism by which
intracellular lyso-GSLs may be removed by direct N-acylation
has been proposed(7) , the molecular mechanism of lyso-GSL
generation in situ remains unclear. Recently, Hirabayashi et al. reported the presence of lyso-GSL-generating hydrolase
activity in actinomycetes(8) . The enzyme was, however,
difficult to solubilize from the cells, and thus the enzyme protein has
not yet been characterized. In this paper we report that the novel
enzyme, purified as an apparently homogeneous protein from a newly
isolated bacterium, cleaves the N-acyl linkage of ceramides in
various GSLs as well as sphingomyelin to produce their lyso forms. This
is the first report describing the generation of lysosphingomyelin from
sphingomyelin by a specific hydrolase. Lysosphingomyelin has been shown
to exert potent mitogenic activity (9) and to modulate
cytosolic protein phosphorylation(10) . The enzyme, tentatively
designated sphingolipid ceramide N-deacylase, should
facilitate the further study of sphingolipid as well as
lysosphingolipid functions.
EXPERIMENTAL PROCEDURES
Materials
A mixture of gangliosides was prepared
from bovine brain using a method described previously(11) . GM1
and asialo GM1 were prepared from a mixture of gangliosides by
digestion with neuraminidases isolated from Clostridium perfringens (Sigma) and Arthrobacter ureafaciens (Nakarai Chemical
Co., Japan), respectively, followed by purification with DEAE-Sepharose
and Iatrobead column chromatography. Other GSLs were purchased from
Iatron Laboratories, Inc. (Japan). Sphingomyelin and ceramide from
bovine brain were purchased from Matreya, and Triton X-100 was from
Sigma. Precoated Silica Gel 60 TLC plates were obtained from Merck
(Germany). Endoglycoceramidase was prepared as described previously (12, 13) or purchased from Takara Shuzo Co. (Japan).
Enzyme Assay
The activity of sphingolipid ceramide N-deacylase was measured using asialo GM1 as the substrate as
described below. The reaction mixture contained 10 nmol of asialo GM1
and an appropriate amount of the enzyme in 20 µl of 20 mM sodium acetate buffer, pH 5.0, containing 0.8% Triton X-100.
Following incubation at 37 °C for 30 min, the reaction was stopped
by heating in a boiling water bath for 3 min. The reaction products
were freeze-dried by a Speed Vac concentrator (Savant Instruments,
Inc.), redissolved in 5 µl of chloroform/methanol (1:2, v/v), and
analyzed by TLC using chloroform, methanol, 10% acetic acid (5:4:1,
v/v/v) as the developing solvent. GSLs and lyso-GSLs were visualized by
spraying the TLC plates with orcinol-H
SO
reagent and scanning them with a Shimadzu CS-9300 chromatoscanner
with the reflectance mode set at 540 nm. The extent of hydrolysis was
calculated as follows: hydrolysis (%) = (peak area for
lysoasialo GM1 generated)
100/(peak area for remaining asialo
GM1 + peak area for lysoasialo GM1 generated). One enzyme unit was
defined as the amount capable of catalyzing the release of 1 µmol
of lysoasialo GM1/min from the asialo GM1 under the conditions
indicated above. Exoglycosidases and proteases were assayed using p-nitrophenylglycosides (14) and azocoll(15) ,
respectively, as substrates.
TLC Analysis
Lysosphingolipids and sphingolipids
were analyzed by TLC using chloroform, methanol, 10% acetic acid
(5/4/1, v/v/v) as the developing solvent. Lyso-GSLs were visualized
with either orcinol-H
SO
reagent or ninhydrin.
Lysosphingomyelin was visualized with either Coomassie Brilliant Blue (16) or ninhydrin. Oligosaccharides released from the product
by endoglycoceramidase were analyzed by TLC using n-butyl
alcohol/acetic acid/H
O (2/1/1, v/v/v) (17) and
visualized with orcinol-H
SO
reagent.
FAB-MS Analysis
Lyso-GSLs were analyzed by
negative FAB-MS using a JEOL JMS HX-100 mass spectrometer (JEOL Ltd.,
Japan) with triethanolamine as the matrix. For lysosphingomyelin,
analysis was conducted in the positive mode using diethanolamine as the
matrix(18) .
SDS-Polyacrylamide Slab Gel Electrophoresis and Protein
Assay
SDS-Polyacrylamide gel electrophoresis was conducted on a
slab gel with 10% acrylamide according to Laemmli(19) . The
sample was heated at 100 °C for 3 min before electrophoresis except
for detection of the activity. For this purpose, the sample was left at
room temperature for 10 min. The duplicate gel was cut into 4-mm slices
without staining. Each gel slice was crushed with a glass bar in an
Eppendorf tube containing 1 ml of 20 mM sodium acetate buffer,
pH 5.0, containing 0.3% Triton X-100 and shaken at 4 °C for 2 h.
After centrifugation at 10,000 rpm for 10 min, the supernatant was
dialyzed against 2 mM sodium acetate buffer, pH 6.0, in order
to remove SDS, and this was found to be effective for restoration of
enzyme activity. The enzyme activity was determined by the method
described under ``Experimental Procedures'' using asialo GM1
as the substrate. The incubation time for this experiment was 16 h. The
protein was stained with Coomassie Brilliant Blue, and the protein
content at each step of purification was determined by the
bicinchoninic acid protein assay (Pierce) with bovine serum albumin as
the standard.
Sugar Composition Analysis
Lysoasialo GM1 (100
nmol) was hydrolyzed with 2.5 N trifluoroacetic acid at 100
°C for 6 h and analyzed using a Dionex HPLC system with a Carbo Pac
PA column (Dionex) and pulsed amperometric detection(20) .
Isolation and Cultivation of Pseudomonas sp.
TK-4
A strain (TK-4) capable of producing sphingolipid ceramide N-deacylase was isolated from pond water using a synthetic
medium containing gangliosides as the sole source of carbon. The
bacterium was assigned to the genus Pseudomonas on the basis
of morphological and biochemical characteristics, which will be
reported in detail elsewhere. In order for this strain to retain its
ability to produce the enzyme, it must be maintained in a medium
containing gangliosides (0.5% polypeptone, 0.1% yeast extract, 0.2%
NaCl, 0.1% bovine brain gangliosides, and 1.6% agar, pH 7.0), as is
presently being done at our laboratory. For preparation of sphingolipid
ceramide N-deacylase, inocula from an agar slant of the strain
TK-4 were introduced into a cotton-plugged 50-ml flask containing 20 ml
of sterilized liquid medium (0.5% polypeptone, 0.1% yeast extract, 0.2%
NaCl, and 0.1% bovine brain gangliosides) and incubated at 25 °C
for 1 day with vigorous shaking. The culture was then transferred to a
cotton-plugged 5000-ml flask containing 1000 ml of the same medium and
incubated at 25 °C for 3 days with vigorous shaking.
Purification of Sphingolipid Ceramide N-Deacylase from
the Culture Supernatant
The supernatant obtained (1,800 ml) was
adjusted to 75% saturation with solid ammonium sulfate and allowed to
stand overnight. The precipitate was collected by centrifugation and
dissolved in 72 ml of 20 mM sodium acetate buffer, pH 6.0,
containing 0.1% (w/v) Lubrol PX (buffer A). A 15-ml aliquot of the
enzyme solution from the ammonium sulfate precipitation step was
applied to a DEAM column (2.2
15 cm; Yamazen Co., Japan),
previously equilibrated with buffer A, using a BPLC-600FC HPLC system
(Yamazen Co., Japan). The column was washed with 5 bed volumes of
buffer A, and sphingolipid ceramide N-deacylase was then
eluted from the column with a linear salt gradient generated by buffer
A and 1 M NaCl in the same buffer. The active fractions were
pooled, concentrated by an Amicon concentrator using a YM10 membrane,
and dialyzed against buffer A. The enzyme solution was applied to a gel
filtration column of HW-55F (4.4
30 cm; Yamazen Co., Japan)
using a BPLC-600FC HPLC system. The column was equilibrated and eluted
with buffer A containing 0.2 M NaCl. The flow rate was 5
ml/min, and fractions of 5 ml were collected. The active fractions were
pooled, concentrated, and applied to a Phenyl-5PW column (8
75
mm; Tosoh, Japan) using a GT1 gradient HPLC system (Pharmacia Biotech
Inc.). The column was equilibrated with buffer A, and the enzyme was
eluted from the column at a flow rate of 0.5 ml/min with a linear
gradient generated using 20 mM sodium acetate buffer, pH 6.0,
and the same buffer containing 2% Lubrol PX. The enzyme was finally
purified by a DEAE-5PW column (8
75 mm; Tosoh, Japan) using a
GT1 gradient HPLC system (Pharmacia). The column was equilibrated with
buffer A, and the enzyme was eluted from the column at a flow rate of
0.5 ml/min with buffer A. Contaminating proteins were adsorbed on a
column and eluted with buffer A containing 1 M NaCl.
Purification of Lysosphingolipids
Products from
asialo GM1 after sphingolipid ceramide N-deacylase treatment
were purified by reverse phase HPLC using an ODS column (2
300
mm; Tosoh, Japan) as described in (21) . Monitoring of
lyso-GSLs was conducted by TLC as described above. For products
originating from GalCer and sphingomyelin, a silica gel 60 column was
used instead of an ODS column. Lysosphingolipids were eluted from the
column using a solvent system composed of chloroform/methanol/water
(5:4:1, v/v/v).
RESULTS
Purification of Sphingolipid Ceramide
N-Deacylase
In a typical experiment, the enzyme was purified
about 300-fold from a culture filtrate of the newly isolated Pseudomonas sp. TK4 strain with 5% recovery. The purified
enzyme preparation was completely free from the following enzyme
activities:
- and
-galactosidases,
-N-acetylhexosaminidase,
-N-acetylgalactosaminidase,
-N-acetylglucosaminidase,
-L-fucosidase,
- and
-mannosidases,
- and
-glucosidases,
sialidase, endoglycoceramidase, sphingomyelinase, and proteases. The
enzyme preparation showed a single protein band corresponding to a
molecular mass of 52 kDa on SDS-polyacrylamide slab gel electrophoresis
after staining with Coomassie Brilliant Blue (Fig. 1). The
duplicate gel was cut into 4-mm slices, the protein was eluted and
dialyzed, and the enzyme activity was measured as described under
``Experimental Procedures.'' The activity was detected only
at the position corresponding to the 52-kDa band.
Figure 1:
Analysis of the purity of sphingolipid
ceramide N-deacylase by SDS-polyacrylamide gel
electrophoresis. The standard proteins (molecular masses in
parentheses) are as follows: phosphorylase b (94 kDa), bovine serum
albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and
soybean trypsin inhibitor (20.1 kDa). The enzyme activity was detected
at a position corresponding to a 52-kDa band. Details are given under
``Experimental Procedures.''
General Properties
The general properties of the
enzyme are as follows: optimal activity at pH 5.0-6.0 and stable
between pH 4.0 and 9.0; potently inhibited by Hg
,
Cu
, and Zn
(2 mM) but not
by Ca
, Mn
, Mg
,
and EDTA, all at the same concentration. The enzyme retained 80% of its
activity when kept at 60 °C for 30 min and can be kept at -85
°C for 2 months without any loss of activity. Addition of Triton
X-100 at a concentration of 0.4-0.8% (w/v) increased the enzyme
activity about 10-fold in comparison with that in the absence of the
detergent.
Characterization of Enzymatic Digestion
Products
To elucidate the action mode of the enzyme, asialo GM1,
GalCer, and sphingomyelin were digested with the enzyme, and the
digestion products were separately purified by HPLC followed by TLC
analysis. The digestion products migrated on the TLC plate more slowly
than native sphingolipids and were stained with either
orcinol-H
SO
(those from asialo GM1 and GalCer; Fig. 2A, lanes2 and 4) or
Coomassie Brilliant Blue (that from sphingomyelin; Fig. 2B, lane2). The product from
GalCer released by the enzyme was identical to the
galactosylsphingosine (psychosine) standard on TLC (Fig. 2A, lanes4 and 5).
All sphingolipids tested were changed to ninhydrin-positive substances
after enzyme treatment (Fig. 2A, lanes7 and 9, and Fig. 2B, lane4), whereas the parental sphingolipids were not stained
with ninhydrin (Fig. 2A, lanes6 and 8, and Fig. 2B, lane3).
This demonstrated the generation of free amino groups in sphingolipids
by the enzyme treatment. Sugar composition analysis of the product from
asialo GM1 revealed that the sugar chain was intact even after enzyme
treatment (GalN:Gal:Glc = 1.01:2.0:0.87). This result was
confirmed by the fact that the sugar chain released from the product by
endoglycoceramidase, which releases sugar chains from both GSLs and
lyso-GSLs(12) , had the same mobility on TLC as that from the
parental asialo GM1 released by endoglycoceramidase (Fig. 2C, lanes2 and 4).
The product from asialo GM1 released by the enzyme was stained with
ninhydrin (Fig. 2C, lane5), but the
oligosaccharide released from the product by endoglycoceramidase was
not (Fig. 2C, lane6), suggesting
that the acetyl group at the C-2 position in GalNAc could not be
removed by the enzyme, i.e. the enzyme is specific to the N-amide linkage in ceramide but not to that in the
carbohydrate moiety. Sphingosine, which was stained by ninhydrin, was
generated from the product by endoglycoceramidase treatment (Fig. 2C, lane6). Finally, the
products released from asialo GM1, GalCer, and sphingomyelin were
identified using a FAB-MS. As shown in Fig. 3, A and B, the characteristic pseudomolecular ions
(M-H)
were found at m/z 989
for the product released from asialo GM1 (M
1256;
C18:0, d 18: 1), and m/z 461 for the product released
from GalCer (M
728; C18:0, d 18:1) using the
negative ion mode. On the spectra of the product released from asialo
GM1, fragment ions m/z 827 (corresponding to
lysoasialo GM2) and m/z 624 (corresponding to
lysoasialo GM3) were also observed (Fig. 3A). For the
product released from sphingomyelin (M
732;
C:18:0, d 18:1), (M + H)
was found at m/z 467 using the positive ion mode (Fig. 3C). Using the negative ion mode, however, the
(M-H)
ion was not detectable, while the
characteristic ion triplet for choline-containing lipids was observed
at m/z 451, 406, and 380 (18) (data not
shown). These triplet ions were characterized as
(M-CH
)
,
(M-HN(CH
)
)
, and
(M-CH
CHN(CH
)
)
,
respectively. FAB-MS analysis indicated that these products were lyso
forms of the respective parental sphingolipids having a d
sphingosine base as the major molecular species. In summary, it
was concluded that the enzyme cleaves the N-acyl linkage
between the fatty acid and sphingosine base in ceramides of
sphingolipids. We tentatively designate this enzyme sphingolipid
ceramide N-deacylase, whose systematic name should be
sphingolipid N-acylsphingosine amidohydrolase, based on its
unique specificity. It should be emphasized that the enzyme cleaves the N-acyl linkage of ceramides in sphingomyelin to produce
lysosphingomyelin, since the sphingomyelinase (EC 3.1.4.12) reported so
far cleaves the linkage between ceramide and phosphorylcholine but
never that between sphingosine and fatty acid in ceramide of
sphingomyelin(22) . Therefore, this paper is the first to
report a lysosphingomyelin-generating hydrolase.
Figure 2:
TLC analysis of products released from
asialo GM1, GalCer, and sphingomyelin by sphingolipid ceramide N-deacylase. A, products released from asialo GM1 and
GalCer by the enzyme. Lanes1 and 6, asialo
GM1; lanes2 and 7, product from asialo GM1; lanes3 and 8, GalCer; lanes4 and 9, product from GalCer; lanes5 and 10, psychosine standard. Lanes1-5,
orcinol-H
SO
staining; lanes6-10, ninhydrin staining. Gal and Lac, galactose and lactose, respectively. B, product
released from sphingomyelin by the enzyme. Lanes1 and 3, sphingomyelin; lanes2 and 4, product from sphingomyelin. Lanes1 and 2, Coomassie Brilliant Blue staining; lanes3 and 4, ninhydrin staining. C, oligosaccharide
released from the product by endoglycoceramidase; lanes1 and 5, product from asialo GM1; lanes2 and 6, oligosaccharide released from the product by
endoglycoceramidase; lanes3 and 7, asialo
GM1; lanes4 and 8, oligosaccharide released
from asialo GM1 by endoglycoceramidase; lane9,
sphingosine standard. Lanes1-4,
orcinol-H
SO
staining; lanes5-9, ninhydrin staining. Chloroform, methanol, 10%
acetic acid (5:4:1, v/v/v) was used as the developing solvent for TLC
of A and B, and n-butyl alcohol/acetic
acid/H
O (2:1:1, v/v/v) was used for TLC of C.
Figure 3:
FAB-MS analysis of the products released
from sphingolipids by sphingolipid ceramide N-deacylase. A, product released from asialo GM1 by the enzyme; B,
product released from GalCer by the enzyme; C, product
released from sphingomyelin by the enzyme. Analysis was conducted using
a negative mode for A and B and a positive mode for C. Details are given under ``Experimental
Procedures.''
Specificity of Enzyme
Fig. 4shows the time
course for the degradation by this enzyme of asialo GM1, GM1,
globotetraosylceramide, GalCer, sphingomyelin, and ceramide. The time
course of the degradation rates of asialo GM1 by the enzyme was similar
to that of GM1, suggesting that this enzyme acts on both neutral and
acidic GSLs at the almost same reaction velocity. In contrast to the
enzyme from actinomycetes(8) , this enzyme can hydrolyze GalCer
as well as glucosylceramide (Fig. 4, Table 1). However,
the enzyme shows hardly any activity on ceramides, indicating that it
is completely different from ceramidase (EC 3.5.1.23)(23) . The
extent of hydrolysis of various sphingolipids after exhaustive
digestion with the enzyme is summarized in Table 1. This enzyme
shows quite wide specificity, i.e. it hydrolyzes both neutral
and acidic GSLs, including sulfatide, and also a range from simple GSLs
(cerebrosides) to complex polysialogangliosides (GQ1b). Furthermore,
the enzyme hydrolyzes not only GSLs but also sphingomyelin. It was
notable, however, that the enzyme did not hydrolyze completely all
sphingolipid substrates tested even after prolonged incubation. The
reason for this is unknown at present but may be due to feedback
inhibition of the enzyme by the fatty acids generated, since addition
of stearic acid to the reaction mixture inhibited the enzyme activity
(data not shown).
Figure 4:
Time course of degradation of various
sphingolipids by sphingolipid ceramide N-deacylase.
--
, asialo GM1;
--
, GM1;
--
, globotetraosylceramide;
--
, GalCer;
--
,
sphingomyelin;
--
, ceramide. Sphingolipids (200
nmol each) were incubated separately at 37 °C with 40 milliunits of
the enzyme in 200 µl of 20 mM sodium acetate buffer, pH
5.0, containing 0.8% Triton X-100. At the times indicated, 10 µl of
the reaction mixture was withdrawn for measurement of hydrolysis of
samples by TLC as described under ``Experimental
Procedures.'' Each value is the mean from three independent
experiments. The range of deviation of all the measured results was
within 5%.
DISCUSSION
Lysosphingolipids are present at low levels in normal tissues
but are abnormally accumulated in cells in various lysosomal storage
diseases(2) . For example, in Gaucher's disease, which is
caused by a deficiency of glucosylceramidase, abnormal accumulation of
glucosylceramide as well as its lyso form, glucosylsphingosine, is
observed(24) . Intracellular generation of psychosine is seen
in cases of Krabbe's disease(25) , which is a progressive
and fatal neurogenic disorder. Both psychosine and glucosylsphingosine
are considered to be synthesized by the glycosylation of sphingosines in situ, although Yamaguchi et al.(26) reported very recently that glucosylsphingosine is
formed not only through the glycosylation of sphingosine but also
through the deacylation of glucosylceramide in cultured fibroblasts.
They suggested the participation of acidic ceramidase in
glucosylsphingosine formation. The enzyme presented here, however,
seems to be completely different from the ceramidase reported so far,
since the enzyme hydrolyzes various sphingolipids efficiently but
hardly acts on ceramide (Fig. 4), which is the most favored
substrate for ceramidase(23) . Therefore, we tentatively
designate the novel enzyme sphingolipid ceramide N-deacylase
or sphingolipid N-acylsphingosine amidohydrolase to
distinguish it from the known ceramidase. The mode of action of
sphingolipid ceramide N-deacylase on asialo GM1 along with
that of endoglycoceramidase is presented in Fig. 5. It should be
noted that sphingolipid ceramide N-deacylase hydrolyzes
various sphingolipids including cerebrosides and sphingomyelin, both of
which are completely resistant to hydrolysis by endoglycoceramidase (12) .
Figure 5:
Mode of action of sphingolipid ceramide N-deacylase on asialo GM1. Sphingolipid ceramide N-deacylase hydrolyzes the N-acyl linkage between
fatty acids and sphingosine bases in ceramides of sphingolipids, while
endoglycoceramidase hydrolyzes the glycosidic linkage between ceramides
and oligosaccharides in GSLs(12) .
In addition to monohexosylsphingosines,
lysogangliosides (sialic acid-containing lyso-GSLs) have been found in
mammalian cells. Lyso-GM2 has been detected in the brain of
Tay-Sach's patients, but it has not been detected in the normal
brain(27) . Hannun and Bell have reported that not only
sphingosine but also lyso-GSLs including lysogangliosides potently
inhibit protein kinase C and have suggested that the accumulation of
lyso-GSLs would eventually lead to cell death due to dysfunction of the
signal transduction system(4) . Besides inherited lysosomal
disease, lyso-GM3 has also been found in a human epidermoid carcinoma
cell line, A431, and was shown to inhibit EGF-dependent EGF receptor
phosphorylation(3) . However, the mechanism of lysoganglioside
formation in situ has not yet been elucidated. Whether
sphingolipid ceramide N-deacylase (or a similar enzyme)
responsible for lysoganglioside generation is present in mammalian
tissues should be clarified carefully.
The discovery of sphingolipid
ceramide N-deacylase should provide advantages for the study
of sphingolipids as well as lysosphingolipids. The preparation of
lysosphingolipids will become much easier by using this enzyme. To
date, the preparation of lyso GSLs has been done using purely chemical
procedures(28) , which are somewhat troublesome,
time-consuming, and give a low yield, especially in the case of
polysialogangliosides. Using the sphingolipid ceramide N-deacylase we were able to obtain easily the lyso forms of
all species of GSLs tested without any alternation of their
carbohydrate and sphingoid moieties, allowing preparation of new GSL
derivatives containing appropriately labeled fatty acids. Furthermore,
utilizing the amino groups newly generated in lyso-GSLs, they can be
coupled with either appropriate proteins or gel matrix for the affinity
column. In addition to the GSLs, the fact that the enzyme can be
applied to sphingomyelin should be noted.