(Received for publication, April 27, 1995; and in revised form, June 29, 1995)
From the
After uptake by various cells (human skin fibroblasts, rat
neuroblastoma B 104, human neuroblastoma SHSY5Y, murine cerebellar
cells), a radioactive and a fluorescent analog of a nondegradable
glucosylceramide with sulfur in the glycosidic link were glycosylated
to a cell-specific pattern of glycolipid analogs. These results, for
the first time, show that a glucosylceramide analog can be conveyed
from the plasma membrane of cultured cells to those Golgi compartments
that function in the early glycosylation steps of glycolipids. This
observation is further confirmed by the fact that the cationic
ionophore monensin, known to impede membrane flow from proximal to
distal Golgi cisternae, inhibited the formation of complex ganglioside
analogs but not those of lactosylceramide, sialyl lactosylceramide
(G), and disialyl lactosylceramide (G
).
On the cell surface of vertebrate cells, glycosphingolipids
(GSL) ()form cell-specific patterns which change
specifically with cell differentiation, morphogenesis, and oncogenic
transformation (for review see (1) and (2) ). Although
being surmised to be the place for the functional role of GSL, the
plasma membrane is not the site of their metabolism. Rather, GSL are
synthesized in the Golgi complex by sequential addition of
monosaccharide units to ceramide and are degraded in lysosomes by the
sequential removal of glycosyl residues starting from the nonreducing
end. Thus, the maintenance of a balanced glycolipid profile requires a
stringent control of metabolism and transport of GSL (for review see (3) and (4) ).
We have observed labeled and cell-specific glycosylation products when a radioactive or fluorescent analog of glucosylceramide had been administered to cultured cells. These products (analogs of globosides and gangliosides) could have been formed in the Golgi complex by direct glycosylation of the labeled glucosylceramide analogs or from the labeled ceramide analogs; the latter resulting from deglucosylation, most likely in lysosomes, of the former. Therefore, we have studied the metabolism of glucosylceramide analogs that contain sulfur in the glycosidic bond (Fig. 1) and are thus resistant to enzymatic deglucosylation. We will present data showing unambiguously that a direct glycosylation of these analogs of glucosylceramide takes place in various cultured cells leading to a cell-specific pattern of labeled glycolipid analogs.
Figure 1: Structures of labeled glucosylthioceramides. * denotes position of the radiocarbon.
Figure 2:
Postendocytotic glycosylation of
[C]C
-Glc-S-Cer in various
cell types. Human skin fibroblasts, rat neuroblastoma B 104, human
neuroblastoma SHSY5Y, and murine cerebellar cells were incubated with
[
C]C
-Glc-S-Cer as described
under ``Experimental Procedures.'' In cases where monensin
was used, this drug was present in a 1 µM concentration at
all times of incubation. The incubation media were saved, and the cells
were harvested with a rubber policeman. The lipids of cells and media
were desalted and combined prior to separation by TLC using
chloroform/methanol/15 mM calcium chloride (60:35:8, by
volume) as developing system. The radioactive lipids were visualized by
exposure to x-ray-sensitive film. In the right-hand margin,
the mobilities of
[
C]C
-Glc-S-Cer and its
glycosylation products, if produced in the respective cell type, is
denoted by the standard abbreviation for GSL as outlined in Footnote 1. x
, x
, x
,
and x
denote the sulfoxides of
[
C]C
-Glc-S-Cer and the
putative sulfoxides of the corresponding thioglycosides of LacCer,
GbOse
Cer, and G
, respectively. Lane
1, reference lipids from top to bottom:
[
C]C
-Glc-S-Cer,
-Lac-S-Cer, -GbOse
Cer,
[
C]C
-G
,
-G
, -G
, -G
; lane 2,
lipid extract of fibroblasts; lane 3, lipid extract of
fibroblasts that were incubated in the presence of monensin; lane
4, lipid extract of B 104 cells; lane 5, lipid extract of
B 104 cells that were incubated in the presence of monensin; lane
6, lipid extracts of SHSY5Y cells; lane 7, lipid extracts
of SHSY5Y cells that were incubated in the presence of monensin; lane 8, lipid extract of murine cerebellar cells; lane
9, lipid extract of murine cerebellar cells that were incubated in
the presence of monensin.
Figure 4:
Enzymatic degradation of cell lipid
extracts with glycohydrolases. Lipid extracts obtained as for Fig. 2were treated with the enzymes as described under
``Experimental Procedures.'' For a second degradation step,
one-half of the assay mixture was treated with a second enzyme for an
additional 10 h. Thereafter, the degradation products were separated by
TLC and visualized as for Fig. 2. In the right-hand
margin, the mobilities of
[C]C
-Glc-S-Cer and of its
glycosylation products are denoted by the standard abbreviation for GSL
as outlined in Footnote 1. x
, x
, x
, and x
denote the sulfoxides of
[
C]C
-Glc-S-Cer and the
putative sulfoxides of the corresponding thioglycosides of LacCer,
GbOse
Cer, and G
, respectively. The character y denotes a putative sulfone of
[
C]C
-Glc-S-Cer. Lane
1, reference lipids from top to bottom:
[
C]C
-Glc-S-Cer,
-Lac-S-Cer, -GbOse
Cer,
[
C]C
-G
,
-G
, -G
, -G
; lane 2,
lipid extract of fibroblasts after treatment with
-hexosamidases; lane 3, lipid extract of fibroblasts after treatment with
-hexosamidases and subsequent treatment with
-galactosidase; lane 4, lipid extract of B 104 cells after treatment with
sialidase; lane 5, lipid extract of SHSY5Y cells after
treatment with
-hexosamidases; lane 6, lipid extract of
SHSY5Y cells after treatment with
-hexosamidases and further
treatment with sialidase; lane 7, lipid extract of murine
cerebellar cells after treatment with sialidase; lane 8, lipid
extract of murine cerebellar cells after treatment with sialidase and
additional treatment with
G
-
-galactosidase.
Figure 6:
Oxidation of
[C]C
-Glc-S-Cer to its
sulfoxide and its reduction back to
[
C]C
-Glc-S-Cer. Lane
1, [
C]C
-Glc-S-Cer; lane 2, isolated sulfoxide (x
) of
[
C]C
-Glc-S-Cer; lane
3, the sulfoxide (x
) of
[
C]C
-Glc-S-Cer treated with
trimethylsilyl iodide; lane 4, sulfoxide (x
) of
[
C]C
-Glc-S-Cer obtained by
oxidation of [
C]C
-Glc-S-Cer
with hydrogen peroxide; lane 5, sulfone (y) of
[
C]C
-Glc-S-Cer obtained by
oxidation of [
C]C
-Glc-S-Cer
with hydrogen peroxide.
Figure 3:
Glycosylation products of
[C]C
-Glc-S-Cer in
fibroblast pellets and incubation media. Human skin fibroblasts were
incubated with
[
C]C
-Glc-S-Cer as for Fig. 2. The lipids of cells and media were desalted and
separated by TLC as for Fig. 2. To test for any
glycosyltransferase activity in the incubation media, these media were
incubated in the presence of
[
C]C
-Glc-S-Cer and
nucleotide sugars as described under ``Experimental
Procedures.'' The radioactive lipids were visualized by exposure
to x-ray-sensitive film. In the right-hand margin, the
mobilities of
[
C]C
-Glc-S-Cer and of its
glycosylation products is denoted by the standard abbreviation for GSL
as outlined in Footnote 1. x
denotes the sulfoxide
of [
C]C
-Glc-S-Cer. Lane
1, [
C]C
-Glc-S-Cer as
used for the incubation studies; lane 2, lipid extract of
incubation medium that was tested for glycosyltransferase activity; lane 3, lipid extract of medium that was used in a fibroblast
experiment; lane 4, lipid extract of a fibroblast
pellet.
Figure 5:
Hydrolysis of radioactive
globotriaosylceramide analog with -galactosidase. The radioactive
GbOse
Cer analog was isolated from TLC plates, treated with
-galactosidase as described under ``Experimental
Procedures,'' and analyzed by TLC in chloroform/methanol/water
(65:25:4, by volume). Lane 1, radioactive GbOse
Cer
analog after treatment with
-galactosidase; lane 2,
radioactive GbOse
Cer analog after treatment with
heat-inactivated
-galactosidase. In the right-hand margin are indicated the mobilities of the GbOse
Cer analog
and its degradation product as well as its putative sulfoxides x
and x
,
respectively.
The
thick band just below glucosylthioceramide in all lipid extracts
(denoted by x in Fig. 2, 3, and 4) is
formed predominantly in the process of cell incubation. After isolation
from the TLC plate, its structure was shown by FAB-MS to be the
oxidation product, i.e. the sulfoxide of
[
C]C
-Glc-S-Cer. When
treated with trimethylsilyl iodide according to (17) , the
sulfoxide was reduced to
[
C]C
-Glc-S-Cer (Fig. 6, lane 3). This sulfoxide could also be obtained
by treating glucosylthioceramide with methanolic hydrogen peroxide.
Under this condition, the corresponding sulfone is also produced. Both
products could be separated by TLC (Fig. 6, lane 4 and 5), and their structure proven by FAB-MS. The faint bands seen
between [
C]C
-Glc-S-Cer and
its sulfoxide in Fig. 4(denoted by y in lanes 6 and 7) may well represent this sulfone. The sulfoxide of
[
C]C
-Glc-S-Cer also seems
to be prone to glycosylation as most clearly demonstrated by the
prominent band just below the G
analog for the B 104 cell
extract (denoted by x
in Fig. 2, lane
4).
Figure 7:
Postendocytotic glycosylation of
NBD-C-Glc-S-Cer in various cell types. Human skin
fibroblasts, rat neuroblastoma B 104, human neuroblastoma SHSY5Y, and
murine cerebellar cells were incubated with
NBD-C
-Glc-S-Cer as described for Fig. 2.
The fluorescent lipids were photographed on Polaroid film under UV
light. In the slot, the mobilities of
NBD-C
-Glc-S-Cer and of its glycosylation products,
if produced in the respective cell type, is denoted by the standard
abbreviation for GSL as outlined in Footnote 1. x
, x
, and x
denotes the
sulfoxides of NBD-C
-Glc-S-Cer and the putative
sulfoxides of the corresponding thioglycosides of LacCer and
GbOse
Cer, respectively. Lane 1, reference lipids
from top to bottom:
NBD-C
-Glc-S-Cer, -Lac-S-Cer,
-GbOse
Cer, NBD-C
-G
,
-G
, -G
, -G
; lane 2,
lipid extract of fibroblasts; lane 3, lipid extract of
fibroblasts that were incubated in the presence of monensin; lane
4, lipid extract of B 104 cells; lane 5, lipid extract of
B 104 cells that were incubated in the presence of monensin; lane
6, lipid extracts of SHSY5Y cells; lane 7, lipid extracts
of SHSY5Y cells that were incubated in the presence of monensin; lane 8, lipid extract of murine cerebellar cells; lane
9, lipid extract of murine cerebellar cells that were incubated in
the presence of monensin.
The band closest to the
fluorescent glucosylthioceramide (x in Fig. 7) has been identified by FAB-MS and chemical reduction as
the corresponding sulfoxide.
In this paper we present, for the first time, an unambiguous
proof for direct glycosylation of glucosylceramide analogs in various
cultured cell types. In human fibroblasts, rat neuroblastoma B 104
cells, and murine cerebellar cells, the glycosylation pattern obtained
was identical with the pattern that is obtained by metabolic labeling
of these cells(14) . Human neuroblastoma SHSY5Y cells also
yielded a glycosylation pattern which completely agreed with the
endogenous glycolipid pattern(12) . The amount of the
fluorescent glycosylation products was, however, much less than that of
the radioactive anabolites (Table 2) indicating that the
fluorescent tag somehow interferes with glycosylation and/or transport.
This is most obvious for fibroblasts when comparing the patterns of
labeled analogs showing that more fluorescent G than
fluorescent globotriaosylceramide was formed (compare Fig. 7, lane 2, to Fig. 2, lane 2). In contrast, more
of the radioactive analog of globotriaosylceramide than that of
G
was synthesized, thus exactly mimicking the endogenous
glycolipid pattern. The NBD group may render the fluorescent analogs
more polar. This may explain the high degree of fluorescent lipids
extracted into the incubation medium and may also explain why less
fluorescent than radioactive glycosylation products have been produced.
Owing to the hydrophilic glucosyl head group, no spontaneous
diffusion across the plasma membrane can occur, and the internalization
of the labeled analogs at 37 °C very likely takes place by
vesicular membrane flow. Hence we have to assume that vesicles carrying
these glucosylceramide analogs eventually fuse with Golgi cisternae (or
other compartments active in glycosylation) that contain the enzymes
for lactosylceramide, G, and G
synthesis. We
do not yet know from where these vesicles are derived and if endosomes
and/or lysosomes are intermediate stations in this transport. It has
been shown clearly, however, that these glucosylceramide derivatives
participate in the biosynthetic pathway of the respective cell-specific
glycosphingolipids.
For glycosphingolipids, the Golgi apparatus is
the well accepted site for their biosynthesis. It is believed that
lactosylceramide and gangliosides G and G
are
synthesized presumably in proximal Golgi cisternae, and that the more
complex glycosphingolipids are synthesized in distal Golgi
membranes(23) . The membrane flow from proximal to distal Golgi
membranes is inhibited by monensin (for review, see (24) ).
Thus, from our experiments with monensin, we infer that the
glucosylthioceramides were conveyed to Golgi cisternae that function in
the early glycosylation steps in glycolipid biosynthesis, i.e. the formation of lactosylceramide and gangliosides G
and G
. The formation of analogs of lactosylceramide
and G
was not impaired by monensin whereas the production
of the complex glycosylation products was severely decreased.
Our
findings lend support to observations made by others who found
indication for a glycosylation of glucosylceramide in cultured
fibroblasts derived from patients with Gaucher disease(25) . It
has to be kept in mind, however, that these fibroblasts may contain
enough residual glucocerebrosidase activity (26) to degrade an
appreciable amount of the exogenously supplied glucosylceramide and
that its labeled degradation product may have been used for de novo synthesis of glucosylceramide and more complex glycolipids. Also,
the mode of uptake of the exogenously supplied glucosylceramide via
liposomes may have directed this lipid to cellular compartments active
in another nonlysosomal glucocerebrosidase that is not deficient in
Gaucher disease (27) or active in unspecific -glucosidases
that fall within the normal range of activity in patients with all
forms of Gaucher disease(28) .
An important question is whether native glucosylceramide and perhaps other glycosphingolipid molecules with long acyl chains would participate in the transport process we have observed for the labeled glucosylthioceramides. If so, would this have implications in terms of regulation of glycolipid biosynthesis? To address this question, we intend to perform studies employing undegradable analogs of glucosylceramide and other glycosphingolipids carrying long acyl chains in their ceramide portion. Even if the short chain glucosylceramide analogs should not mimic the endocytotic pathway of the endogenous glycolipids, they are valuable tools for revealing membrane transport that otherwise may be overlooked and that may be important for cellular events. Further experiments are needed to clarify this point.