(Received for publication, November 13, 1995; and in revised form, January 22, 1996)
From the
This paper describes the homeostasis of glycosphingolipid (GSL)
on the cell surface as revealed for the first time by an application of
endoglycoceramidase (EGCase) capable of hydrolyzing the linkage between
the oligosaccharide and the ceramide of various GSLs. When cell-surface
GSLs of B16 melanoma cells were hydrolyzed by the action of EGCase, the
synthesis of GSLs was found to increase transiently, possibly due to
the activation of UDP-glucose:ceramide glucosyltransferase. As a
result, the cell-surface GSL content was restored quickly to exactly
the same level found without the EGCase treatment, if EGCase was
removed from the cell culture. Treatment of erythrocytes with EGCase
was found to increase the ceramide content of the plasma membrane.
Surprisingly, however, in B16 cells the increase of membrane ceramide
by EGCase caused the suppression of de novo ceramide
production, resulting in maintenance of the ceramide content of B16
cells at the same level even after EGCase treatment. The signal for
homeostatic regulation could be the ceramide released by the action of
EGCase, since C-ceramide was found to mimic in part the
action of EGCase; it suppressed de novo production of ceramide
and was directly converted to GSL, NeuAc
2,3Gal
1,4Glc
1,1 N-acetylsphingosine (C
-ceramide GM
).
Our finding demonstrates a novel form of homeostatic regulation coupled
to the GSL-synthesizing system in mammalian cells for maintaining the
contents of both cell-surface GSLs and free ceramide. Since many
opportunistic pathogens were found to produce EGCase extracellularly,
this restoration mechanism could also be present as a defense mechanism
against microbial EGCase.
Glycosphingolipids (GSLs) ()are characteristic
constituents of plasma membranes of mammalian cells and may modulate
cell proliferation, differentiation, and cell-cell
interaction(1) . Although both the GSL content and the
composition were found to change drastically during cellular
differentiation and oncogenic transformation(2) , under static
conditions they could be kept constant in individual organelles, cell
types, and organs, in spite of a continuous lipid flow between plasma
membranes and intracellular organelles(3, 4) .
However, the molecular mechanism for maintaining the content and
composition of cell-surface GSLs is presently not well understood.
Endoglycoceramidase (EGCase) is an enzyme that specifically
hydrolyzes the linkage between oligosaccharide and ceramide of various
GSLs(5, 6) . By using EGCase with the assistance of
its protein activator(7, 8) , cell-surface GSLs of
erythrocytes were found to be hydrolyzed specifically without damaging
other cell membrane components(9, 10) . We observed
that the decrease of the GM(NeuAc) content of B16 melanoma
cells during EGCase treatment was much slower than
GM
(NeuGc) of equine erythrocytes, although the initial
velocity of EGCase toward GM
(NeuAc) is exactly the same as
that toward GM
(NeuGc)(10) . This observation
motivated us to undertake this study. We report here a novel form of
homeostatic regulation of cell-surface GSLs coupled to a
GSL-synthesizing system that may include the activation of
UDP-glucose:ceramide glucosyltransferase.
Treatment of erythrocytes with EGCase was found to increase the ceramide content of the plasma membrane(9) . Ceramide (11, 12, 13, 14) and its metabolites, ceramide 1-phosphate(15) , sphingosine(16) , sphingosine 1-phosphate (17) and N,N-dimethylsphingosine(18) , were found to evoke various physiological effects on various types of cells. Surprisingly, however, we also observed that in B16 cells the increase of membrane ceramide by EGCase causes the suppression of de novo ceramide production, with the result that the ceramide content in B16 cells is maintained at the same level even after EGCase treatment. This study clearly demonstrates the presence of a novel form of homeostasis at the cellular level, which maintains the content of both cell-surface GSLs and ceramide in mammalian cells.
Figure 1:
Hydrolysis of cell-surface GM by EGCase and its restoration after removal of the enzyme. A, hydrolysis of cell-surface GM
by EGCase. B16
cells (1
10
) or equine erythrocytes (1
10
) were incubated at 37 °C for the time indicated in
400 µl of 20 mM PBS, pH 7.2, with 40 milliunits of EGCase
in the presence of 20 nmol of activator. The hydrolysis of cell-surface
GM
was determined by the method described under
``Experimental Procedures''; B, B16 cells (1
10
) were incubated at 37 °C for 6 h in 400 µl of
MEM containing 5% FCS with 40 milliunits of EGCase in the presence of
20 nmol of activator. At time 0, the medium was replaced with fresh
medium without the enzyme and then incubated at 37 °C in a CO
incubator for the time indicated. Values are the mean for
duplicate determinations. The range of deviation of all measured
results was within 5%.
Figure 2:
Restoration of cell-surface GM
of B16 cells revealed by cytofluorometric analysis. B16 cells (5
10
) were incubated with 20 milliunits of EGCase in
the presence of 10 nmol of activator in 200 µl of MEM containing 5%
FCS in a CO
incubator at 37 °C for 16 h. Cells with or
without EGCase treatment were incubated with M2590 monoclonal antibody
followed by a second incubation with FITC-conjugated goat anti-mouse
IgM and analyzed by flow cytometry as described under
``Experimental Procedures.'' A, without EGCase
treatment (positive control); B, without first antibody
(negative control); C, EGCase treatment in the presence of 40
µMD-threo-PDMP; D, EGCase
treatment; E, after EGCase treatment, washing and re-culturing
in fresh medium for 3 h; F, the same as E, but
re-cultured for 6 h.
Figure 3:
De novo synthesis of GSLs of B16
cells after EGCase treatment. After treatment of EGCase under the same
conditions as indicated in the legend of Fig. 2, B16 cells were
washed with fresh medium and metabolically labeled with 1 µCi of
[C]Gal for 3 h in fresh medium without EGCase.
GSLs were extracted and separated with TLC. The determination was
performed with an imaging analyzer as described under
``Experimental Procedures.'' Values are the mean ±
S.D. for triplicate determinations. CMH, ceramide
monohexoside. Photostimulated luminescence.
Figure 4:
Transient increase of de novo synthesis of GM and GlcTase activity after EGCase
treatment. A, GlcTase activity; B, de novo synthesis of GM
. C, control experiment
without EGCase treatment; E, EGCase treatment under the same
conditions as indicated in the legend of Fig. 2; R,
after EGCase treatment, B16 cells were washed with fresh medium and
incubated for an additional 6 h in the fresh medium without EGCase.
Cell-surface GM
was restored as indicated in Fig. 2F. Details are described under
``Experimental Procedures.'' Values are the mean ±
S.D. for triplicate determinations. Photostimulated
luminescence.
This restoration mechanism could be present universally in mammalian cells, since an increase of GSL synthesis after EGCase treatment was observed not only in B16 cells but also in HL60 myelocytic leukemia cells and Swiss 3T3 fibroblasts.
Figure 5:
Release of ceramide from B16 cells and
suppression of de novo production of ceramide by EGCase
treatment. A, release of ceramide from B16 cells by EGCase.
B16 cells were incubated with 1 µCi of
[C]Ser for 2 days, washed with 10 µM cold Ser, suspended in fresh medium, and treated with EGCase under
the same conditions as indicated in the legend of Fig. 2. B, the experiment was conducted in the same manner as A, except that EGCase treatment was carried out in the
presence of 1 µCi of [
C]Ser. C, de novo synthesis of ceramide with or without EGCase
treatment. B16 cells were treated with EGCase under the same conditions
as indicated in the legend of Fig. 2. After EGCase treatment,
the cells were washed with fresh medium and then 1 µCi of
[
C]Ser was added. De novo production of
ceramide was determined after 3 h. C, control without EGCase; E, EGCase treatment; Cer, ceramide; DM,
N,N-dimethylsphingosine; Sph,
sphingosine.
Figure 6:
Effects of C-ceramide on de novo ceramide production and GSL synthesis. A, de novo synthesis of ceramide with or without
C
-ceramide treatment. B16 cells (1
10
)
were incubated with (5 µM) or without
C
-ceramide at 37 °C in a CO
incubator for 3
h in 200 µl of MEM containing 2 µCi of
[
C]Ser and 5% FCS.
C-Labeled
ceramide was extracted from cells and determined by an imaging
analyzer. Upper and lower represent the ceramide of
upper and lower bands on TLC, respectively. Values are the mean
± S.D. for triplicate determinations and are expressed as the
percentage of the upper band in the control experiment. B,
fluorography showing de novo synthesis of
C
-ceramide GM
after addition of 5 µM C
-ceramide. The labeling experiment was conducted by
the same method as described in A. In this experiment,
[
C]Gal was used for the precursor instead of
[
C]Ser. Lane 1, control; lane
2, 5 µM C
-ceramide. C,
fluorography showing the release of
[
C]sialyllactose from both
C-labeled normal GM
(lane 2) and
C
-ceramide GM
(lane 4) by EGCase. Lanes 1 and 3 represent GM
and
C
-ceramide GM
, respectively. Details are
described under ``Experimental
Procedures.''
These results strongly suggest that the ceramide released from GSLs by EGCase might be a signal for the homeostatic regulation of ceramide and cell-surface GSLs.
The reason why mammalian cells possess a restoration system
of cell-surface GSLs as described in this study remains to be
clarified. EGCase has been isolated not only from
microorganisms(5, 24) , but also from
leeches(25) , earthworms(26) , and clams(27) .
The functional significance of this system, however, may only be
clarified after the presence of EGCase in mammals is clearly
demonstrated, although the presence of the enzyme in rabbit mammary
glands has been suggested(28, 29) . We can indicate
another possibility, given the fact that several strains of
opportunistic pathogens were recently found to secrete EGCase into
culture media. ()The microorganism isolated as an EGCase
producer from land soil (5) was identified as Rhodococcus
equi according to physiological, biochemical, and chemotaxonomic
studies(30) . Recently it was found that an authentic strain of R. equi ATCC 6939 retained the ability to produce EGCase. R. equi is a Gram-positive actinomycete originally associated
with a severe, often fatal pneumonia in foals. More recently, it was
identified as an opportunistic pathogen in humans infected with the
AIDS virus(31) . Thus we examined the possibility of whether
other opportunistic microorganisms can produce EGCase extracellularly.
Surprisingly, many bacteria and actinomycetes, including opportunistic
pathogens such as Rhodococcus erythropolis, Rhodococcus
rhodochrous, Corynebacterium hoagii, Corynebacterium
mediolanum, Arthrobacter aurescens, Brevibacterium
sterolicum, and Nocardia globerula produce EGCase
extracellularly. If such microbes succeeded in entering mammalian
tissue, cell-surface GSLs might be exposed to the action of EGCase.
Cells therefore might possess the homeostatic regulation system for
maintaining GSL content that was demonstrated in this study as a
defense mechanism against microbial EGCase. Further investigations
should reveal the relationship between pathogenicity and EGCase.
EGCase was found to efficiently hydrolyze the cell-surface GSLs of intact erythrocytes without damaging other cell membrane components (9, 10) and has recently been used for the analysis of functions of endogenous GSLs of A431 cells (21) and cultured cortical neurons(32) . However, for cultured cells, EGCase appeared to hydrolyze cell-surface GSLs very slowly in comparison with erythrocytes (10) or the plasma membrane fraction of cultured cells(21) . This paper may clarify the reason for this, i.e. the hydrolysis of cell-surface GSLs by EGCase evoked an increase of de novo GSL synthesis, possibly due to the activation of GlcTase, and thus cell-surface GSL supply could be reinforced markedly during EGCase treatment, preventing the loss of cell-surface GSLs.
Since ceramide is involved in cell
regulation(12) , its intracellular levels must be carefully
regulated. We found in this study that the intracellular level of
ceramide was maintained at the same level even after EGCase treatment
due to the suppression of de novo synthesis of ceramide. The
signal might be the ceramide released by the action of EGCase, since a
cell-permeable analog of ceramide, C-ceramide, also
suppressed the de novo synthesis of ceramide. Furthermore,
C
-ceramide was found to be converted to
C
-ceramide GM
. This result strongly suggests
that at least a part of the ceramide released from GSLs by EGCase might
be transported directly to the Golgi apparatus where it could be
converted to GM
and thus could be finally recycled to
plasma membrane. This hypothesis is consistent with the fact that the
intracellular ceramide released by EGCase could not be converted to
sphingosine or N,N-dimethylsphingosine (Fig. 5). Slife et al. (33) have also reported that sphingosine was
generated from sphingomyelin by sphingomyelinase treatment of rat liver
plasma membranes, but not from GSLs by EGCase, although ceramide was
produced from both enzyme treatments. Whether the intracellular
metabolism of ceramide released from GSLs by EGCase in intact cells is
different from that of sphingomyelin by sphingomyelinase should be
carefully clarified, and this study is currently in progress in our
laboratory.
Cell-permeable, synthetic short-chain ceramide was found
to exert various physiological effects on different cell types; it
induced the differentiation of HL-60 cells into monocyte-like
cells(11) , the cell-cycle arrest of Molt-4 cells(34) ,
the programmed cell death of U937 cells(35) , and it inhibited
the endocytosis of Chinese hamster ovary cells(14) . However,
little is actually known of the intracellular metabolism of the
short-chain ceramide. Interestingly, it was revealed in this study that
C-ceramide could be converted to C
-ceramide
ganglioside GM
in B16 cells. We have also confirmed that
C
-ceramide could be converted to C
-ceramide
GM
in B16 cells. The synthesis of these GSLs with
short-chain ceramide and the kinetics for their intracellular formation
have been reported(36) . These results lead us to the
hypothesis that the physiological effects of short-chain ceramide
reported so far may be attributed in part to the intracellular
formation of short-chain ceramide gangliosides.