(Received for publication, July 10, 1995; and in revised form, October 31, 1995)
From the
We present a method for solubilizing and purifying
UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide
synthase (GCS)) from rat liver and present data on its substrate
specificity. A Golgi membrane fraction was isolated, washed with N-lauroylsarcosine, and subsequently treated with
3[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate
to solubilize the enzyme. GCS activity was monitored throughout
purification using UDP-Glc and a fluorescent ceramide analog as
substrates. Purification of GCS was achieved via a two-step dye-agarose
chromatography procedure using UDP-Glc to elute the enzyme. This
resulted in an enrichment >10,000-fold relative to the starting
homogenate. The enzyme was further characterized by sedimentation on a
glycerol gradient, I labeling, and SDS-polyacrylamide gel
electrophoresis, which demonstrated that two polypeptides (60-70
kDa) corresponded closely with GCS activity. Purified GCS was found to
require exogenous phospholipids for activity, and optimal results were
obtained using dioleoyl phosphatidylcholine. Studies of the substrate
specificity of the purified enzyme demonstrated that it was
stereospecific and dependent on the nature and chain length of the N-acyl-sphingosine or -sphinganine substrate. UDP-Glc was the
preferred hexose donor, but TDP-glucose and CDP-glucose were also
efficiently used. This study provides a basis for molecular
characterization of this key enzyme in glycosphingolipid biosynthesis.
Glycosphingolipids (GSLs) ()are amphipathic molecules
that contain the hydrophobic moiety, ceramide, and a hydrophilic
oligosaccharide residue. They are found in the plasma membranes of all
eukaryotic cells and play important roles in cell recognition, cell
proliferation and differentiation, immune recognition, and signal
transduction (for reviews, see (1, 2, 3, 4, 5) ).
The biochemical pathways for GSL synthesis are well established (reviewed in (6, 7, 8, 9) ), but not all of the enzymes involved in GSL synthesis have been purified and/or cloned (for review, see (10) ). One such enzyme is UDP-Glc:ceramide glucosyltransferase (EC 2.4.1.80; glucosylceramide synthase (GCS)), which catalyzes the formation of glucosylceramide (GlcCer) from ceramide and UDP-Glc(11) . This enzyme is of particular interest for a number of reasons. First, it has been shown that there is a correlation between tumor progression and cell surface GSLs(1, 12) . Since many complex acidic and neutral GSLs are derived from GlcCer, regulation of GCS activity could have a profound effect on cell growth activity. Indeed, Radin and colleagues (13) have developed a GCS inhibitor (1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)) and demonstrated some remarkable effects of this compound both in vitro and in vivo. Recently, a defect in GCS activity was also characterized in a mutant melanoma cell line and associated with altered growth properties(14) . Second, GCS activity is concentrated at the Golgi complex(15, 16) , but its precise distribution is not known. Unlike sphingomyelin synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis is more widely distributed within the Golgi. Thus, it is of interest to learn what parts of the GCS molecule are responsible for its unique intracellular distribution. Finally, it should be noted that GlcCer synthesis occurs at the cytosolic surface of intracellular membranes(15, 16, 17) . However, formation of complex GSLs from GlcCer is believed to occur by glycosylation reactions taking place on the lumenal surface of the Golgi apparatus. Thus, GlcCer synthesis must be accompanied by transbilayer movement or ``flip-flop'' of the lipid during or after its synthesis.
To further study some of these problems, it will be necessary to purify, characterize, and eventually clone and sequence GCS. In the present paper, we describe methods for the solubilization and purification of GCS from rat liver Golgi membranes as a step toward this goal. We also provide novel information about the enzymatic properties of GCS.
Figure 5:
Effect of D-threo-PDMP
on GCS activity. Dye-agarose-purified GCS was preincubated with 2.5
µmol of UDP-Glc, 0.05% CHAPSO, and 0.1 mM DOPC for 5 min
at 37 °C in the presence of D-threo-PDMP. The
reaction was initiated by addition of 5 nmol of C-NBD-Cer,
and the results were quantitated as described in Table 2and
under ``Experimental Procedures.'' The data are means of
triplicate determinations and are expressed as a percent of control
values.
We used rat liver Golgi
membranes as a starting material for the purification of GCS because
our previous studies showed that these membranes were highly enriched
in GCS activity(16) . The Golgi fractions prepared in the
present work were enriched 60-80-fold in galactosyltransferase, a trans-Golgi marker protein, with 35% recovery of this
enzyme. In addition, electron microscopy of the Golgi samples revealed
numerous intact stacks comprised of 4 and 5 cisternae (data not shown).
Thus, these Golgi preparations were similar in enrichment and
morphology to highly purified Golgi fractions prepared by established
methods (for review, see (37) ).
Golgi membrane fractions
were enriched >60-fold in GCS activity compared to crude rat liver
homogenate with a yield of >50% (Table 1). Since our enzyme
assay used a fluorescent ceramide analog, which is also a substrate for
sphingomyelin synthase(18, 38, 39) , we were
also able to determine that the Golgi membranes were enriched
35-40-fold in sphingomyelin synthase activity with a recovery of
40% (data not shown). This observation is in agreement with the
differential location of GCS and sphingomyelin synthase in the Golgi
apparatus(16, 17, 21, 40) .
Figure 1: Chromatography of rat liver Golgi GCS on dye-agarose. The detergent-solubilized enzyme was loaded onto a dye-agarose column (A) in the presence of 1 mM UDP-Glc as described under ``Experimental Procedures.'' After washing with 50 ml of buffer B and 50 ml of 0.15 M KCl in buffer B without UDP-Glc, the column was eluted with 1 M KCl in the same buffer as indicated by the arrow. The unbound fractions, indicated by the horizontal bar, were pooled, subjected to gel filtration, and loaded onto a second dye-agarose column equilibrated in buffer B without UDP-Glc (B). The column was then washed in the same buffer and sequentially eluted (at arrows) with 0.15 M KCl (1), 20 mM UDP-Glc plus 20 mM NADH (2), and 1 M KCl (3) in buffer C.
Figure 2:
SDS-PAGE
analysis of GCS at various steps of purification. Protein samples from
different steps in the purification of GCS from rat liver Golgi
membranes were subjected to SDS-PAGE under reducing conditions using a
10% gel. Following electrophoresis, the gel was either silver stained (lanes 1-4) or I-labeled proteins were
visualized by autoradiography (lanes 5 and 6). Lane 1, CHAPSO extract; lane 2, unbound fraction from
dye-agarose (column I); lane 3, unbound fraction from
dye-agarose (column II); lanes 4 and 5, 0.15 M KCl elution from dye-agarose (column II); lane 6,
UDP-Glc/NADH elution from dye-agarose (column II). Approximately 5
µg of protein was loaded in each of lanes 1-3 and
1.5 µg in lane 4. Arrows indicate the
positions of molecular weight standards.
In an attempt to further purify GCS following
dye-agarose chromatography, UDP-Glc-eluted GCS, including a
radiolabeled aliquot, was centrifuged through a glycerol gradient.
Preliminary experiments showed that only 50% of the starting GCS
activity was preserved after a 12-h ultracentrifugation in a glycerol
gradient with 0.5 mM UDP-Glc present, preventing a
quantitative evaluation of GCS enrichment. Nevertheless, the discrete
peak of GCS activity in the glycerol gradient allowed us to assess the
apparent relative size of the enzyme under nondenaturing conditions.
Based on a standard curve of marker enzymes sedimented in the same
tube, we estimated the sedimentation coefficient of GCS as
4.2 s (Fig. 3A). The peak of GCS activity migrated to
position immediately preceding malate dehydrogenase, which has a
molecular mass of 70 kDa. Fig. 3B shows the glycerol
gradient distribution of the radiolabeled 45-, 60-, and 66-kDa
polypeptides present in dye-agarose purified GCS. The intensity of the
45-kDa polypeptide peaked earlier in the glycerol gradient than the
peak of GCS activity (Fig. 3, B and C). Most
of the radioactivity associated with the 66-kDa band was found to be
rat albumin by immunoprecipitation; however, this technique did not
completely remove the radioactivity in this region of the gel (data not
shown). Furthermore, both the 60- and the 66-kDa bands peaked in
intensity at fraction 11, which coincided with the peak of GCS activity (Fig. 3, B and C). Thus, although we cannot
definitively rule out the possibility that another polypeptide is
responsible for GCS activity, our data suggest that the
60- and/or
66-kDa polypeptides are the GCS protein.
Figure 3:
Glycerol gradient sedimentation of
purified GCS. A concentrated sample of dye-agarose-purified GCS (250
µl) including an aliquot (4
10
cpm), which
had been radiolabeled with
I, was layered onto the top of
a 6-25% glycerol density gradient (5 ml) and centrifuged at
250,000
g for 12 h as described under
``Experimental Procedures.'' The gradient was then
fractionated into 30 samples, and the fractions were assessed for
polypeptide composition, GCS activity, and the positions of marker
proteins (cytochrome C, 14 kDa; malate dehydrogenase (MDH), 70
kDa; lactate dehydrogenase (LDH), 140 kDa; and catalase, 250
kDa) as described under ``Experimental Procedures.'' A, standard curve showing the migration of marker proteins and
GCS activity. B, 10% SDS-PAGE of glycerol gradient fractions
in which
I-labeled proteins were visualized by a
phosphorimager. AS, applied sample. Numbers beneath
the gel correspond to glycerol gradient fractions. Position of
molecular mass markers are shown at the right. Arrow at left marks position of the
60-kDa polypeptide. C, relative GCS activity plotted versus glycerol
gradient fraction number. Also shown plotted versus fraction
number are the relative intensities of the 45-,
60-, and 66-kDa
polypeptides determined by phosphorimager
analysis.
Figure 4:
Effect of phospholipid concentration on
GCS activity. Dye-agarose-purified GCS, eluted in the absence of
exogenous phospholipids, was incubated in the presence of various
concentrations of synthetic (di-C, di-C
and C
, C
) phosphatidylcholines as
described in Table 2.
Next we examined the specificity of GCS
for various acceptor substrates. Among the ceramide analogs tested, the D-erythro- and L-erythro- isomers
of C-NBD-Cer were the best substrates (Table 3).
Glucosylation of C
-NBD-Cer from a commercial source, which
contains a mixture of both isomers, gave similar results to the pure
stereoisomers (Table 3). Neither D- nor L-threo-NBD-Cer were used as substrates by the
enzyme, showing that the enzyme is stereospecific. D-erythro-C
-NBD-dihydroceramide was
glucosylated to about 25% of control values obtained with
C
-NBD-Cer (Table 3). Among the different
C
-NBD-Cer tested, the hexanoyl- and octanoyl- analogs were
the best GCS substrates, while little or no measurable activity was
found toward shorter (propanoyl- and pentanoyl-) and longer
(tetradecanoyl-) ceramide analogs (Table 3). Similarly, GCS was
4.5 times more active toward short chain
[
C]C
-Cer than toward a longer chain
[
C]C
-Cer (data not shown). (
)However, the glucosylation yield of the radioactive
C
-Cer was only
25% of that of C
-NBD-Cer
(data not shown). By contrast, C
-DMB-Cer containing a
different fluorophore was a poor substrate with 21% glucosylation
compared to C
-NBD-Cer (Table 3). We also tested the
effects of D-threo-PDMP, a known specific inhibitor
of GCS (43) . PDMP inhibited the activity of purified GCS
45% at 1 µM and 85% at 10 µM (Fig. 5).
Finally, we evaluated the ability of GCS to
use various donor substrates to glycosylate C-NBD-Cer. Of
the UDP-hexoses, GCS was able to utilize UDP-Glc efficiently but had
little or no activity using UDP-glucuronic acid, UDP-galactose,
UDP-N-acetylglucosamine, UDP-mannose, or UDP-xylose as hexose
donors (Table 4). UDP-Glc was the best glucose donor among
diphosphoglucose nucleotides, but TDP-glucose and CDP-glucose also were
efficient glucose donors (Table 4). Surprisingly, ADP-glucose was
also used as a substrate by GCS, leading to about 6% glucosylation of
C
-NBD-Cer compared to UDP-Glc.
We report here for the first time a method for the
purification of GCS. Several features of the method were critical for
the successful purification of this enzyme. First, the modifications
that we introduced in the homogenization and Golgi fractionation
procedure (see ``Experimental Procedures'') significantly
improved the enrichment and recovery of GCS activity relative to our
previous work(16) . Second, we found that inclusion of UDP-Glc
and DOPC as protective agents improved the stability of GCS. Similarly,
UDP and phospholipids were reported to stabilize
UDP-Glc:dolichyl-phosphate glucosyltransferase(44) ; however,
UDP had no protective effect on GCS activity (data not shown). Finally,
we found that green dye-agarose could be used in a two-step procedure
to purify GCS based on the selective binding of GCS to the dye-agarose
in the absence, but not in the presence, of UDP-Glc. This procedure led
to a 20-fold enrichment of GCS in the UDP-Glc-eluted fractions and
was the only chromatographic procedure that we tried that produced any
enrichment in the enzyme. Dye-agaroses have been used previously in the
purification of numerous proteins such as dehydrogenases, kinases, and
serum proteins (for review, see (45) ) as well as
UDP-Glc:dolichyl-phosphate glucosyltransferase(46) . The
observation that the binding of GCS to dye-agarose is inhibited by
UDP-Glc suggests a competition between UDP-Glc and dye molecules for
the active site of the enzyme. Similar results were described for
another glycosyltransferase purified on dye-agarose(47) .
Using the solubilized and purified GCS, we also obtained new
information about the enzymatic characteristics of this protein. First,
we found that purified GCS had almost no activity in the absence of
exogenous phospholipid. Activity was restored by the addition of
phospholipids, with the highest enhancement of activity observed with
low concentrations (0.1 mM) of unsaturated, long chain
(C or C
) phosphatidylcholine (Table 2, Fig. 4). In contrast, phospholipids had little
effect on GCS activity in the Golgi or CHAPSO-solubilized Golgi
membranes. Presumably, endogenous phospholipids present in Golgi
membranes were sufficient to stimulate maximal activity in the Golgi
fractions. These endogenous phospholipids may have been depleted during
purification of the enzyme by dye-agarose chromatography, causing an
almost complete loss of GCS activity, which could be restored by
exogenous phospholipids. A stimulating effect of exogenous
phospholipids on glycosyltransferase activities was documented
previously(42, 48, 49, 50) . More
recently, stimulation of solubilized GCS activity by
phosphatidylcholine was reported(51) ; however, the
concentrations reported for optimal activity were
100-fold higher
(8-10 mM) than the value that we report here (0.1
mM).
Our results on the specificity of purified GCS toward
ceramide analogs are in good agreement with previously published
studies concerned with GCS activity in cultured cells or membrane
preparations. First, we found that GCS is stereospecific, utilizing erythro- but not threo-C-NBD-Cer as a
substrate, similar to results reported using cultured
fibroblasts(18) . We also found that D-erythro-C
-NBD-dihydroceramide was
glucosylated to about 25% of control values obtained with
C
-NBD-Cer (Table 3). This result supports recent
observations on the metabolism of C
-Cer analogs in Chinese
hamster ovary cells(52) . Second, we found that C
-
and C
-ceramides were better substrates than ceramides with
longer or shorter N-acyl chains, confirming observations
reported for GCS activity in microsomal preparations(41) .
Finally, we found that C
-NBD-Cer is a better substrate for
GCS than is [
C]C
-Cer. By contrast,
C
-DMB-Cer, containing a different fluorophore, was poorly
glucosylated relative to C
-NBD-Cer (Table 3),
consistent with previous findings in cultured cells(53) . We
also examined the nucleotide specificity of GCS and found that,
surprisingly, GCS is able to efficiently utilize CDP-Glc and TDP-Glc as
glucose donors (see Table 4). While CDP-Glc and TDP-Glc are not
naturally occurring glucose donors, earlier studies have also described
UDP-Glc:glucosyltransferases that are able to utilize CDP-Glc and
TDP-Glc(54, 55) .
In summary, we have presented for
the first time a method for the purification of GCS, applied this
method to isolate a 10,000-fold enriched GCS fraction from rat
liver, identified two polypeptides (60-70 kDa) as likely
candidates for the GCS protein, and further characterized the purified
enzyme. This information provides a basis for future molecular studies
of this key enzyme in GSL biosynthesis.
Note Added in Proof-Recently Ichikawa et al. (34) have isolated a cDNA encoding human GCS by expression cloning using GM-95 cells lacking the enzyme as a recipient. The open reading frame encodes a protein containing 394 amino acids (predicted molecular mass of 44.9 kDa).