Glycobiology vol. 10 no. 10 pp. 1025–1032, 2000
Evidence supporting a late Golgi location for lactosylceramide to ganglioside GM3
conversion
Maria Laura Allende1,2, Jianghong Li2, Douglas S.Darling,
Christopher A.Worth4 and William W.Young Jr.3
Departments of Molecular, Cellular, and Craniofacial Biology and
Biochemistry, Schools of Dentistry and Medicine and 4James G. Brown
Cancer Center, University of Louisville, Louisville, KY 40292, USA
Received on March 3, 2000; revised on April 21, 2000; accepted on April 25,
2000
Ganglioside GM2 synthase and other enzymes required for
complex ganglioside synthesis were localized recently to the
trans Golgi network (TGN). However, there are conflicting
reports as to the location of GM3 synthase; originally this
enzyme was detected in the early Golgi of rat liver but a
recent report localized it to the late Golgi. We have used
chimeric forms of ganglioside GM2 synthase to determine
if the location of lactosylceramide (LacCer) to GM3
conversion in Chinese hamster ovary (CHO) cells was the
early or late Golgi. Our approach tested whether GM3
could be utilized as a substrate by GM2 synthase chimeras
which were targeted to compartments earlier than the trans
Golgi, i.e., GM3 produced in the cis Golgi should be utilized
by GM2 synthase located anywhere in the Golgi whereas
GM3 produced in the trans Golgi should only be used by
GM2 synthase located in the trans Golgi or TGN. Comparison of cell lines stably expressing these chimeras revealed
that the in vivo functional activity of GM2 synthase
decreased progressively as the enzyme was targeted to
earlier compartments; specifically, the percentage of GM3
converted to GM2 was 83–86% for wild type enzyme, 70%
for the medial Golgi targeted enzyme, 13% for the ER and
cis Golgi targeted enzyme, and only 1.7% for the ER
targeted enzyme. Thus, these data are consistent with a late
Golgi location for LacCer to GM3 conversion in these cells.
Key words: ganglioside/glycosyltransferase/GM2 synthase/
Golgi
Introduction
Gangliosides are of importance as targets for cancer immunotherapy (Livingston, 1998) and for treatment of disorders of
the nervous system including spinal cord injury (Geisler, 1998)
1Present
address: NIH, Building 10 Room 9-D16, 9000 Rockville Pike,
Bethesda, MD 20892
2These authors contributed equally to this work
3To whom correspondence should be addressed at: Dental School,
University of Louisville, 501 S. Preston St., Louisville, KY 40292
© 2000 Oxford University Press
and Parkinson’s disease (Schneider et al., 1998). Recent
reports (Lannert et al., 1998; Giraudo et al., 1999) have
clarified several aspects of ganglioside biosynthesis which for
the “a” series of gangliosides follows the pathway of: Cer →
GlcCer → LacCer → GM3 → GM2 → GM1 → GD1a. The
lipid moiety of all sphingolipids, ceramide, and GlcCer are
produced in the cytoplasmic leaflets of the ER (Mandon et al.,
1992; Rother et al., 1992) and Golgi (Coste et al., 1986;
Futerman and Pagano, 1991; Jeckel et al., 1992; Schweizer et
al., 1994), respectively. Stepwise conversion of GlcCer first to
LacCer and then to ganglioside GM3 and higher gangliosides
occurs on the lumenal leaflet of Golgi membranes (Lannert et al.,
1998). Although initial reports had described GM3 synthase as
an early Golgi resident (Trinchera and Ghidoni, 1989;
Trinchera et al., 1990; Iber et al., 1992), recently both LacCer
synthase and GM3 synthase were localized to the late Golgi of
rat liver (Lannert et al., 1998). Furthermore, GM2 synthase
was localized to the trans Golgi network (TGN) (Lannert et al.,
1998; Giraudo et al., 1999). Thus, according to the current
model (Lannert et al., 1998), all steps of de novo ganglioside
biosynthesis beyond GlcCer production occur in the distal
Golgi, in contrast to the steps of synthesis of the N-linked
chains of glycoproteins which are spread throughout the Golgi
cisternae (Kornfeld and Kornfeld, 1985). In the CHO cells we
used in the present study, GM3 is the only ganglioside endogenously produced (Yogeeswaren et al., 1974; Briles et al.,
1977), but following transfection with GM2 synthase, GM2,
GM1, and GD1a are produced (Rosales Fritz et al., 1997).
In the present study we tested if the location for LacCer to
GM3 conversion is the early or late Golgi in CHO cells. Our
experimental approach was to target GM2 synthase to the
medial Golgi, the cis Golgi, and the ER and then to determine
the relative ability of the enzyme in each of these locations to
convert GM3 to GM2. If LacCer to GM3 conversion occurred
in the cis Golgi, then GM3 produced in that early compartment
could be utilized as a substrate to produce GM2 if GM2
synthase were targeted to any Golgi cisternae. In contrast if
LacCer to GM3 conversion occurred in the trans Golgi and if
we assume minimal retrograde trafficking of gangliosides to
the early Golgi (see Discussion), then GM2 could be produced
efficiently by GM2 synthase targeted to the trans Golgi or
TGN, but GM2 synthesis would be progressively less efficient
as GM2 synthase was targeted to the medial Golgi, cis Golgi,
and ER, respectively. Previously, we reported that conversion
of GM3 to GM2 occurred when GM2 synthase was targeted to
the medial Golgi but not when it was targeted to the ER
(Jaskiewicz et al., 1996b). However, our earlier analysis of
ganglioside synthesis was performed on lipids metabolically
labeled with [3H] palmitate for 4 h, and it did not reveal that the
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M.L.Allende et al.
Table I. Polypeptide domains used for the construction of chimeric GM2 synthase variants
Amino acid range
Targeting sequence donor
Target
Donor
Acceptor (GM2 synthase/myc)
Clone expressing chimera
GnTI
medial Golgi
1–41
28–544
B5
ppGaNTase
ER/cis Golgi
1–97
27–544
E5
Iip33
ER
1–47
5–544
E6
product GM2 could be further converted to GM1 and GD1a by
endogenous enzymes as was subsequently shown by metabolic
labeling with [3H] galactose (Rosales Fritz et al., 1997). Therefore, in the present report we examined the chemical quantities
of gangliosides produced in cells expressing these GM2
synthase chimeras in order to analyze the entire ganglioside
profile. In addition we tested a new construct which targeted
GM2 synthase to the ER and cis Golgi. This targeting approach
was based on the localization of endogenously expressed
UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase
I (ppGaNTase) (Homa et al., 1993) to the cis Golgi (Roth et al.,
1994). The results of this study support a late Golgi location for
LacCer to GM3 conversion in CHO cells.
Results
Cis Golgi-targeted chimera characteristics
We targeted GM2 synthase to the cis Golgi by constructing a
ppGaNTase/GM2 synthase/myc chimera (Table I). Immunofluorescence analysis with anti-myc of clones stably
expressing this chimera revealed strong Golgi staining plus
additional weak staining of the ER (data not shown). To identify
the region of the Golgi occupied by this chimera, we conducted
digestions with endoglycosidase H. Cell extracts of clone E5
expressing ppGaNTase/GM2 synthase/myc produced a single
anti-myc reactive Western blot band having an apparent
molecular weight of ∼76,000 (Figure 1). This value is
consistent with the predicted molecular weight of a chimera
consisting of the lumenal domain of GM2 synthase which
includes three N-linked carbohydrate chains (Haraguchi et al.,
1995), the myc epitope, and the ppGaNTase transmembrane
domain and flanking regions which include one N-linked chain
(Homa et al., 1993). In contrast wild type GM2 synthase/myc
from clone C5 cells produced an anti-myc reactive doublet
which we have shown previously to be due to an endo H
resistant upper band present in the Golgi and an endo H sensitive
lower band present in the ER and Golgi (Figure 1 and refs.
(Jaskiewicz et al., 1996a; Zhu et al., 1997). Endo H digestion
of the clone E5 cell extract produced a single band ∼8 kDa
smaller than the band prior to digestion (Figure 1). This endo H
sensitivity indicated that this protein had not moved to the point
where N-linked chains become endo H resistant, generally
thought to be the medial Golgi. These results, when combined
with additional evidence for Golgi targeting described below,
indicated that ppGaNTase/GM2 synthase/myc was targeted to
the ER and cis Golgi.
We previously described the release of a soluble form of
wild type GM2 synthase/myc as a result of proteolytic
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Fig. 1. Cellular ppGaNTase/GM2 synthase/myc is endo H-sensitive. Clone C5
(GM2 synthase/myc) and clone E5 (ppGaNTase/GM2 synthase/myc) cell
extracts (C) and conditioned media (M) were treated with (+) or without (-)
endo H and Western blotted with anti-myc under reducing conditions.
cleavage near the border between the transmembrane and
lumenal domains (Jaskiewicz et al., 1996a). The ppGaNTase/
GM2 synthase/myc chimera produced by clone E5 cells was
also released into the culture medium (Figure 1). Previously
we found that when GnTI/GM2 synthase/myc was targeted to
the medial Golgi (clone B5, Table I), it was subsequently
cleaved and released whereas the Iip33/GM2 synthase/myc
chimera targeted to the ER (clone E6, Table I) was not released
(Jaskiewicz et al., 1996a). Therefore, release of a soluble form
of GM2 synthase from clone E5 cells (Figure 1) provides
supporting evidence that this ppGaNTase/GM2 synthase
chimeric enzyme was in fact targeted to the Golgi.
To further establish that ppGaNTase/GM2 synthase/myc
was targeted to the Golgi, we performed subcellular fractionation
on sucrose gradients (Figure 2). Western blotting with antimyc indicated that chimeric GM2 synthase was present in both
Golgi and ER fractions (peak fractions 3 and 9, respectively,
Figure 2A); quantification of the GM2 synthase monomer
Western bands indicated that the Golgi fractions contained
6.6% of the total in all fractions. In contrast, the Golgi fractions
contained 30.4% of the total cellular GM2 synthase activity
(Figure 2B); the fact that the anti-myc staining of the Golgi
fractions was a much lower percentage of the staining of the
ER fractions (Figure 2A) indicates the presence of less active
enzyme in the ER. Furthermore, the ER fractions also
contained significant staining of lower molecular weight bands
resulting from degradation of chimeric GM2 synthase. In
Late Golgi location for LacCer to GM3 conversion
contrast the Golgi fractions contained primarily a single band
which comigrated with full length chimeric GM2 synthase
present in the cell extract, thus indicating that membrane
bound enzyme occupied the Golgi. The absence of the breakdown bands in the Golgi fraction also indicated that the presence
of full length chimeric GM2 synthase in the Golgi fractions
was not simply due to contamination of the Golgi fractions
with ER membranes. In summary these results indicate that
active, membrane bound, full length chimeric GM2 synthase
occupied the Golgi in clone E5 cells.
In vivo GM2 synthesis by chimeric enzymes
CHO cell clone C5 which stably expresses a high level
(Table II) of wild type GM2 synthase/myc converted 86% of
its ganglioside GM3 to GM2 and more complex gangliosides
(Figure 3, lane 5; Table II). Previously we showed by immunoelectronmicroscopy that GM2 synthase was present in all Golgi
cisternae and the TGN of clone C5 cells (Jaskiewicz et al.,
1996b). Giraudo et al. (1999) recently reported that GM2
synthase is a TGN-located enzyme when correctly targeted
whereas overexpression, such as occurs in clone C5, causes
saturation of the targeting mechanism and mislocalization to
the cis, medial, and trans Golgi cisternae. In contrast to the
overexpression in clone C5, clone R12 expresses wild type
GM2 synthase at a level (Table II) which is similar to the level
of endogenous GM2 synthase expressed by two murine
lymphoma cell lines (Lutz et al., 1994) and tenfold less than
the level expressed in clone C5. Despite their difference in
activity levels, clone R12 was similar to clone C5 in that a high
percentage of GM3 (83.7%) was converted to GM2 and higher
gangliosides in R12 cells (Table II and Figure 3, lane 4).
Therefore, the expression level did not have a major effect on
the extent of conversion of GM3 to GM2.
To begin to test the effect of GM2 synthase location on GM2
production, we analyzed clone B5 cells. These cells express
the chimeric GnTI/GM2 synthase/myc enzyme (Table I) which
we showed previously by immunoelectronmicroscopy was
restricted to the medial Golgi (Jaskiewicz et al., 1996b). In
clone B5 GM2 was the major ganglioside and in addition there
was considerable conversion of GM2 to GM1 and GD1a
(Figure 3, lane 3; Table II). Clone B5 cells were less efficient
than clone R12 cells at converting GM3 to GM2 in that only
70.3% of GM3 was converted to GM2 and more complex
gangliosides in clone B5 (Table II). This reduction in ability to
convert GM3 to GM2 cannot be explained simply by there
being insufficient enzyme activity in clone B5 cells because
the specific activity in these cells was more than 5-fold greater
than that of wild type clone R12 (Table II). Thus, these results
indicated that GM2 synthase located in the medial Golgi could
produce considerable GM2 but at a reduced extent as
compared to wild type enzyme. If the targeting of Golgi
enzymes were absolutely restricted to individual cisternae,
then these results might support an early Golgi location for
GM3 synthase. However, according to the cisternal maturation
model (Glick and Malhotra, 1998), the distributions of Golgi
resident enzymes take the form of concentration gradients
which are centered over given regions of the Golgi. Therefore,
if GM3 synthase is centered over the trans Golgi, then some
amount of this enzyme must be expected in the medial Golgi,
and this overlap could account for the GM2 produced in clone
B5 cells. In fact considerable GM3 synthase activity was
detected in fractions of rat liver corresponding to medial Golgi
(Lannert et al., 1998). Similarly, although we detected GnTI/
GM2 synthase by immunoelectronmicroscopy in clone B5
cells only in the medial Golgi (Jaskiewicz et al., 1996b), it is
likely that some portion of this chimeric enzyme would be
present in the trans Golgi at least transiently as has shown to be
the case for GnTI in HeLa cells (Nilsson et al., 1993; Rabouille
et al., 1995). Therefore, it is likely that GM3 produced by GM3
synthase centered over the trans Golgi would be capable of
acting as substrate for GM2 production by a GM2 synthase
chimera centered over the medial Golgi and furthermore that
GM2 production in this case would be less efficient than that
produced by wild type enzyme; in fact that is what our data
suggest.
We next tested clone E5 cells in which GM2 synthase was
targeted to the ER and cis Golgi by the ppGaNTase/GM2
synthase/myc construct. If GM3 synthase were located in the
Table II. GM2 synthase activities and ganglioside profiles of CHO cells expressing chimeric GM2 synthase
Specific activityb
% of total gangliosidesc
Cell clone
Targeting sequence
Target
(nmol/mg/h)
GM3
GM2
GM1
E6
Iip33
ER
3.08 ± 0.26
98.3
NDd
ND
GD1a
E5
ppGaNTase
ER/cis Golgi
3.08 ± 0.18
87.3 ± 4.3
2.6
2.1
B5
GnTI
medial Golgi
3.81 ± 0.28
29.7 ± 2.7
38.2 ± 2.7
9.8 ± 0.6
22.3 ± 0.9
R12
Wild type
TGNa
0.7 ± 0.02
16.3 ± 2.8
45.1 ± 1.8
8.0 ± 1.9
30.6 ± 1.1
C5
Wild type
Golgia
7.3 ± 0.25
13.9 ± 2.0
63.7 ± 4.0
4.7 ± 1.0
17.7 ± 5.2
1.7
8.1 ± 1.8
aGM2
synthase is a TGN-located enzyme (Giraudo et al., 1999) that when overexpressed as in clone C5 is mislocalized to the cis, medial, and trans Golgi
cisternae (Jaskiewicz et al., 1996b).
bSpecific activity data are presented as mean ± SEM for two separate experiments; the number of assay points was 7 for clones R12, C5, and E5 and 4 for clones
B5 and E6.
cThe gangliosides shown in Figure 3 were quantitated by the comparative dilution method (Siddiqui and Hakomori, 1970). Data are presented as mean ± SEM for
three separate determinations; where no SE is indicated, the values are the average of duplicate determinations.
dND, not detectable.
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M.L.Allende et al.
Fig. 3. Thin layer chromatography pattern of the gangliosides from CHO cells
expressing GM2 synthase chimeras. Gangliosides were visualized by
resorcinol staining. Aliquots of cell extracts applied to the plate were
equivalent to 0.05 ml of packed cells. Lanes: 1, clone E6 (Iip33/GM2
synthase/myc); 2, clone E5 (ppGaNTase/GM2 synthase/myc); 3, clone B5
(GnTI/GM2 synthase/myc); 4, clone R12 (GM2 synthase/myc); and 5,
clone C5 (GM2 synthase/myc).
Fig. 2. Cellular ppGaNTase/GM2 synthase/myc copurifies with both Golgi and
ER membranes. Clone E5 cells were homogenized and centrifuged on a
discontinuous sucrose gradient. Fractions were analyzed by Western blotting
with anti-myc (A), for GM2 synthase activity (B), and (C) for α-glucosidase II
(ER marker enzyme; open circles) and galactosyl transferase (Golgi marker
enzyme; solid circles).
cis Golgi, its distribution should maximally overlap with
ppGaNTase/GM2 synthase/myc whereas if GM3 synthase
were centered over the trans Golgi then the chance of overlap
with ppGaNTase/GM2 synthase/myc would be less than for
the medial Golgi-targeted chimera. Clone E5 expressing the
ER and cis Golgi targeted ppGaNTase/GM2 synthase/myc
chimera converted 12.7% of GM3 to GM2 and higher gangliosides (Figure 3, lane 2; Table II). Thus, the extent of conversion
was markedly reduced as compared to clones C5 and R12
expressing wild type enzyme and clone B5 expressing the
GnTI/GM2 synthase chimera. A trivial explanation for the
reduced in vivo functional activity of the ppGaNTase/GM2
synthase/myc chimera in clone E5 cells might be that the
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amount of enzyme in the Golgi was too low for efficient GM2
production, i.e., even if there were compartmental overlap of
GM3 and GM2 synthase, the GM2 synthase activity in the
Golgi might be below the minimum necessary to produce
greater GM2 synthesis in vivo. In fact, however, the specific
activity of clone E5 was 4.4-fold greater than that of wild type
clone R12 (Table II). Subcellular fractionation (Figure 2) indicated that 30.4% of GM2 synthase activity was present in the
Golgi of clone E5. Therefore, the amount of active enzyme in
the Golgi in clone E5 cells was at least 1.3-fold greater than in
clone R12, even when we make the conservative assumption
that all GM2 synthase is located in the Golgi in R12 cells.
Therefore, we can rule out the possibility that the amount of
chimeric enzyme expressed in the Golgi of clone E5 cells was
too small for efficient conversion. Instead, these data support
the alternate interpretation that compartmental separation
prevented GM3 produced in the late Golgi from being available to act as a substrate for GM2 synthase targeted to the ER
and cis Golgi. These results provide strong evidence that GM3
synthase is centered over the late Golgi and not the cis Golgi in
these cells.
Finally, clone E6 stably expresses Iip33/GM2 synthase/myc
(Table I) which we showed previously was restricted to the ER
(Jaskiewicz et al., 1996b). In clone E6 only 1.6% of GM3 was
converted to higher gangliosides (Table II; Figure 3, lane 1).
This minimal capability to produce GM2 cannot be explained
simply by there being insufficient enzyme in clone E6 cells
because the specific activity in these cells was more than 4-fold
greater than that of wild type clone R12 (Table II). The Iip33
motif has been shown to be a signal for retrieval of proteins
bearing the motif from the early Golgi to the ER (Teasdale and
Jackson, 1996). If the distribution of GM3 synthase were
centered over the cis Golgi, then Iip33/GM2 synthase/myc and
GM3 synthase should coexist in the same compartments at
least transiently and produce GM2. The low amount of GM2
produced in clone E6 cells thus provides additional evidence
Late Golgi location for LacCer to GM3 conversion
against a cis Golgi location for GM3 synthase and, therefore,
supports a late Golgi location for GM3 synthase in CHO cells.
Ganglioside patterns produced by chimeric enzymes
In clones C5 and R12 which expressed high and low levels of
wild type enzyme, respectively, and in clone B5 expressing
GnTI/GM2 synthase, GM2 was the major ganglioside (Figure 3,
Table II). In contrast, we found previously that GM2 was not
detectable on the surface of clone E6 cells expressing Iip33/
GM2 synthase (Jaskiewicz et al., 1996b), and the same was
true for clone E5 expressing ppGaNTase/GM2 synthase
(Figure 4A). However, flow cytometry using fluoresceinated
cholera toxin (Figure 4B) revealed the presence of ganglioside
GM1 on the surface not only of clone E5 cells expressing ER
and cis Golgi targeted ppGaNTase/GM2 synthase but also on
clone E6 cells expressing Iip33/GM2 synthase. Thus, for clone
E5 expressing the ER and cis Golgi targeted ppGaNTase/GM2
synthase and for clone E6 expressing the ER targeted Iip33/
GM2 synthase, the small amount of GM2 that was produced
was utilized to a large extent to produce GM1 and GD1a as
judged by both chemical analysis (Table II) and flow cytometry
Fig. 4. CHO cells expressing cis-Golgi targeted GM2 synthase and ER
targeted GM2 synthase display GM1 but not GM2 on the cell surface.
(A) Cells were incubated with anti-GM2 antibody 10–11 followed by FITCconjugated goat anti-mouse Ig. Background staining in the first decade was
obtained for primary antibody control staining of cells expressing GM2
synthase chimeras (data not shown). (B) Cells were stained with FITC-cholera
toxin. Surface staining was analyzed by flow cytometry; relative fluorescence
is in arbitrary logarithmic units.
(Figure 4); i.e., in these cells GM2 functioned primarily as a
metabolic intermediate and did not accumulate significantly
(see Discussion).
Discussion
GM3 synthase has been cloned (Ishii et al., 1998; Kono et al.,
1998; Fukumoto et al., 1999; Kapitonov et al., 1999), but its
precise distribution within the Golgi is controversial. Subcellular
fractionation of rat liver localized this enzyme to the cis Golgi
in early reports (Trinchera and Ghidoni, 1989; Trinchera et al.,
1990) but to the trans Golgi more recently (Lannert et al.,
1998). The latter authors proposed that the concentration of
detergent used for sialyltransferase assays in the earlier studies
masked the true GM3 synthase activity profile. In addition
GM3 synthase was localized to the early Golgi in primary
cultured cerebellar neurons of 6-day-old mice (Iber et al.,
1992). However, this difference in distribution from that
reported recently for rat liver has been attributed to alterations
in the organization of the secretory pathway in the developing
brain (Lannert et al., 1998). In the present study we adopted a
chimeric enzyme targeting approach, the results of which
provide evidence supporting the conclusion of Lannert et al.
(1998) that GM3 synthase is located in the late Golgi and not
the cis Golgi as originally proposed (Trinchera and Ghidoni,
1989; Trinchera et al., 1990). In addition our results extend the
findings of Lannert et al., 1998, because subcellular fractionation
only indicated where GM3 synthase activity was detectable by
in vitro assay and could not indicate the site(s) where GM3 is
produced in the living cell. In contrast our approach indicates
where GM3 becomes available to act as a substrate for GM2
synthase in whole cells. In the cisternal maturation model for
Golgi structure, which has recently received new support
(Glick and Malhotra, 1998), new Golgi cisternae appear by the
coalescence of ER-derived membranes, cisternae move
progressively across the stack in the cis to trans direction with
all Golgi resident enzymes recycling by retrograde vesicles to
earlier compartments, and the TGN finally fragments into
secretory vesicles. In this model the distributions of Golgi
resident enzymes take the form of concentration gradients
rather than being the result of strict and absolute targeting to
individual cisternae. Thus, our finding of progressively less
GM2 being produced as GM2 synthase was targeted to earlier
compartments is consistent with significant overlap of GM3
synthase activity centered over the trans Golgi with GnTI/
GM2 synthase/myc centered over the medial Golgi in clone
B5, less overlap with ppGaNTase/GM2 synthase/myc centered
over the ER and cis Golgi in clone E5, and essentially no
overlap with Iip33/GM2 synthase/myc targeted to the ER in
clone E6.
An alternative explanation for the limited GM2 production
by our chimeric enzymes would be that GM2 synthesis occurs
following retrograde traffic of GM3. The subcellular fractionation performed by Lannert et al. (1998) provided elegant data
about glycosyltransferase distribution but by its very nature
could not be informative about the dynamics of ganglioside
synthesis which includes not only de novo biosynthesis but also
ganglioside synthesis via recycling pathways (Schwarzmann and
Sandhoff, 1990; Riboni et al., 1997; Gillard et al., 1998). In
contrast, here we analyzed the chemical patterns of ganglioside
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M.L.Allende et al.
profiles which are the net result of all synthetic pathways. In
many cell lines recycling pathways produce the majority of
gangliosides, particularly the more complex structures (Gillard
et al., 1998). The recycling pathways which have been
analyzed consist of: (1) recycling of gangliosides from the
plasma membrane via endosomes to the Golgi where they are
subsequently utilized for the synthesis of more complex
gangliosides; and (2) transfer of gangliosides from the plasma
membrane to the lysosomes where the gangliosides are
degraded to their sphingosine or sphinganine moiety which is
then reutilized in the ER for new sphingolipid synthesis. In
addition the Golgi cisternal maturation model offers the
untested possibility of retrograde trafficking of gangliosides
within the Golgi. At present it is not known if these retrograde
vesicles are targeted only to the adjacent “younger” cisterna or
whether they may reach all earlier cisternae. If GM3 produced
in the trans Golgi were carried by these retrograde vesicles to
all earlier cisternae, then the outcome of our present analysis
would have been the production of high levels of GM2, regardless
of the location of GM2 synthase. Similarly, the endocytotic
recycling pathway potentially could transport GM3 from the
cell surface to the early Golgi or even to the ER because it is
clear from studies of cholera toxin, shiga toxin, and ricin that
endocytosis can deliver material to the ER (Sandvig and Van
Deurs, 1999). If high amounts of cell surface to ER transport of
GM3 had occurred in our cells, we would have detected high
levels of GM2 regardless of the location of GM2 synthase.
Clearly our results do not support either of these extreme cases
because we found a progressive decrease in GM2 production
as GM2 synthase was targeted to earlier compartments.
However, the limited amount of GM2 produced by our
chimeras could have been the result of either intra-Golgi
retrograde trafficking of GM3 only to the adjacent cisterna or
endocytotic recycling of GM3 from the cell surface which was
most efficient for delivery to the late Golgi and progressively
less efficient to earlier compartments.
Recently Grabenhorst and Conradt constructed chimeras of
α1,3-fucosyltransferase by replacing its transmembrane and
flanking regions with those of late and early acting Golgi glycosyltransferases and determined the functional sublocalization
of these chimeras by assessing the structures of the N-linked
chains that were produced in transfected cells (Grabenhorst
and Conradt, 1999). Here we have adopted a similar approach
with GM2 synthase. The experimental design we employed
included cis Golgi targeting of GM2 synthase via the transmembrane domain and flanking regions of ppGaNTase I.
Previously endogenous ppGaNTase I was localized to the cis
Golgi (Roth et al., 1994). More recently, however, epitope
tagged ppGaNTase I in transfected HeLa cells was found to be
distributed throughout the Golgi stack rather than being only in
the cis Golgi (Rottger et al., 1998). The apparent discrepancy
between these results may be due to cell type differences,
differences due to overexpression of the transfected enzyme
and/or epitope tagging, or the possibility that the antiserum
used to detect endogenous ppGaNTase I may have recognized
other members of the ppGaNTase family. Regardless of the
explanation for this point of controversy, our data indicate that
ppGaNTase/GM2 synthase hybrid molecules were targeted to
the ER and cis Golgi as revealed by subcellular fractionation
(Figure 2), which showed the presence of membrane-bound
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enzyme in the ER and Golgi, combined with endo H sensitivity
(Figure 1).
An assumption in our experimental design is that the
compartments to which our chimeras were targeted would
allow GM2 synthesis if substrate GM3 were present. Clearly,
the cis Golgi can provide the UDP-GalNAc required for GM2
synthesis because it has been identified as the site for GalNAc
addition that initiates protein O-glycosylation (Roth, 1984;
Piller et al., 1990). A precedent for another GalNAc transferase being able to function in the ER was provided by
Rottger et al., 1998, who reported that O-linked specific
GalNAc transferase 1 and 2 were capable of initiating protein
O-glycosylation when they were relocated to the ER (Rottger
et al., 1998).
In the present study GM2 accumulated in cells in which
GM2 synthase was targeted to the medial and late Golgi
(clones B5, C5, and R12). However, in cells expressing GM2
synthase targeted to the ER and cis Golgi or to the ER alone
(clones E5 and E6, respectively; Figures 3 and 4; Table II)
GM2 did not accumulate but instead functioned primarily as a
biosynthetic intermediate. Ruan et al. (1999) found that
neuroblastoma cell lines which expressed moderate to high
levels of GM2 synthase activity (which also catalyzes the
conversion of GD3 to GD2 (Pohlentz et al., 1988) and low
levels of GD3 synthase activity contained high levels of GD2
but surprisingly low levels of GD3, i.e., a very high percentage
of the intermediate GD3 was converted to GD2. The situation
with our cis Golgi- and ER-targeted clones is similar in that the
intermediate GM2 does not accumulate; however, in our case
there is no correlation between the level of GM2 synthase activity
and the accumulation of GM2. The lack of accumulation of GM2
in cells expressing GM2 synthase targeted to the early Golgi
may simply be due to the amount of GM2 being below the
level at which GM1 synthase is saturated. In the case of wild
type clone R12 which expresses an even lower level of wild
type enzyme (Table II) but accumulates GM2, one explanation
for GM2 accumulation could be saturation of GM1 synthase.
Alternatively, a portion of the GM2 produced in the TGN by
wild type enzyme might proceed to the cell surface without
being acted upon by GM1 synthase and remain there without
significant endocytosis and utilization for GM1 production. In
the case of clone B5 expressing medial Golgi-targeted GnTI/
GM2 synthase/myc, saturation of GM1 synthase seems the
likely explanation for GM2 accumulation because all GM2
produced in the medial Golgi should encounter GM1 synthase
in the TGN on its way to the cell surface.
Material and methods
Chimeric constructs
CHO cell clone C5 expressing GM2 synthase/myc, clone
R12 expressing GM2 synthase/myc/6His, clone B5 expressing
N-acetylglucosaminyltransferase I (GnTI)/GM2 synthase/myc,
and clone E6 expressing Iip33/GM2 synthase/myc were
described previously (Jaskiewicz et al., 1996a,b). ppGaNTase/
GM2 synthase/myc was constructed by fusing the sequence for
the cytoplasmic and transmembrane domain plus the first 69
amino acids of the lumenal domain of bovine UDP-GalNAc:polypeptide GalNAc transferase I (ppGaNTase) to the lumenal
domain of GM2 synthase/myc. The sequence of ppGaNTase, a
Late Golgi location for LacCer to GM3 conversion
gift of Dr. A. Elhammer, The Upjohn Co., was amplified by
PCR using the following primers: sense: 5′-GCTAAAGCTTGCCAGGATGAGAAAATTTGCATACTGC and antisense:
5′-CTAGCCCCGGGTAGATCTGTTGAGTGCAAT. The PCR
product was digested with HindIII and XmaI and cloned into
the pCDM8 plasmid containing the GM2 synthase/myc
construct using the same enzymes. Wild type CHO cells were
transfected, and clone E5 stably expressing ppGaNTase/GM2
synthase/myc was selected by limiting dilution and anti-myc
immunofluorescence screening as previously described
(Jaskiewicz et al., 1996a,b).
Flow cytometry
Staining of transfected CHO cells with monoclonal IgM antiGM2 10–11 (Zhu et al., 1998). Briefly, cells were removed
from monolayer culture with trypsin-EDTA and incubated for
30 min at 4°C with the primary antibody in PBS-BSA. The
cells were then washed in cold PBS-BSA and incubated for
30 min at 4°C in PBS-BSA containing fluorescein-conjugated
goat anti-mouse Ig. For staining with FITC-cholera toxin,
transfected CHO cells were trypsinized and incubated for
30 min on ice with 10 µg/ml FITC-cholera toxin diluted in
PBS-BSA. After washing, the cells were resuspended in PBSBSA, and analyzed on an Epics Elite flow cytometer (Coulter,
Hialeah, FL).
Endo-glycosidase treatment, subcellular fractionation, and
glycolipid analysis
Digestion of cell extracts with endo-β-N-acetylglucosaminidase H (endo H) and Western blotting with anti-myc
were described previously (Jaskiewicz et al., 1996a,b). For
subcellular fractionation cells from 15 150 cm2 flasks were
homogenized, and Golgi and ER fractions were separated on
sucrose gradients as described previously (Jaskiewicz et al.,
1996a). Fractions (1.3 ml per fraction) were collected from the
sucrose gradients and the following aliquots taken for each
assay: 0.008 ml for anti-myc Western blotting, 0.02 ml for
GM2 synthase activity, 0.1 ml for α-glucosidase II, and 0.055 ml
for GalT. The in vitro assay for GM2 synthase activity was
performed as described elsewhere (Jaskiewicz et al., 1996b)
except that the assay mixture contained 0.1 mM UDP-[3H]
GalNAc and 10 mM CDP-choline and the product GM2 was
isolated by butanol-water partitioning. Details of glycolipid
analysis were described previously (Nichols et al., 1986) with
the modification that large scale cell culture was achieved in
roller bottles using the serum-free medium CHO-S-SFM II
(Life Technologies) according to Kolhekar et al. (1997).
Lipids were extracted, Folch partitioned, and upper phase
lipids desalted on Bond Elut C18 columns (Analytichem,
Harbor City, CA; Schnaar, 1994). Samples were separated on
HPTLC plates in CHCl3:CH3OH:0.25% KCl in H2O, 50:40:10,
v/v, and ganglioside bands were visualized by resorcinol.
Acknowledgments
We thank Drs. K.Furukawa and A.Elhammer for plasmids,
K.O.Lloyd for anti-GM2 10–11, and S.-U.Gorr for reading the
manuscript. This work was supported by RO1 GM42698 from
the NIGMS and NSF Grant EPS-9874764.
Abbreviations
Cer, ceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; CHO, Chinese hamster ovary; PBS-BSA, phosphate
buffered saline pH 7.4 containing 1% bovine serum albumin.
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