(Received for publication, June 2, 1995; and in revised form, June 19, 1995)
From the
The synthesis of the oligosaccharide of gangliosides is carried
out in the Golgi complex by successive sugar transfers to proper
glycolipid acceptors. To examine how the product of one glycosylation
step couples with the next transfer step, the endogenous gangliosides
of Golgi membranes from 14-day-old chick embryo retina were labeled
from CMP-[H]NeuAc or
UDP-[
H]GalNAc or UDP-[
H]Gal
in conditions which do not allow vesicular intercompartmental
transport. After saturation of the endogenous acceptor capacity,
labeling was mostly in the immediate acceptors of the corresponding
labeled sugars. However, some labeled intermediates progressed to more
glycosylated gangliosides if the membranes were incubated in a second
step in the presence of the necessary unlabeled sugar nucleotides. This
was particularly evident in the case of membranes incubated with
UDP-[
H]Gal, in which most of the
[
H]Gal-labeled lactosylceramide synthesized in
the first step was converted to GM3 and GD3, or to GM2 or to GD1a in a
second incubation step in the presence of unlabeled CMP-NeuAc alone, or
together with UDP-GalNAc, or together with UDP-Gal plus UDP-GalNAc,
respectively. Conversion was time dependent and dilution-independent.
Since prior reports using brefeldin A indicate that transfer steps
catalyzed by GalNAc-T, Gal-T2, and Sial-T4 localize in the trans-Golgi network (TGN), our results lead to the following
major conclusions: (a) transfer steps catalyzed by GalNAc-T,
Gal-T2, and Sial-T4 colocalize and are functionally coupled in the TGN; (b) proximal Golgi Gal-T1, Sial-T1, and Sial-T2, and their
corresponding glycolipid acceptors, extend their presence to the TGN,
and (c), GalNAc-T and Sial-T2 compete for a common pool of
acceptor GM3 in the synthesis of GM2 and GD3.
The synthesis of the oligosaccharide of gangliosides occurs by successive transfers of sugar moieties from the corresponding sugar nucleotide donor to the proper glycolipid acceptor by specific membrane bound glycosyltransferases (for reviews, see Roseman, 1970; Caputto et al., 1974; Van Echten and Sandhoff, 1993).
Current
evidence indicates that within the Golgi complex ganglioside synthesis
starts in a proximal compartment and ends in a more distal compartment.
Thus, cytosolic addition of Glc to ceramide, Gal addition to GlcCer ()to form luminal LacCer and NeuAc addition to LacCer and
GM3 to form GM3 and GD3, respectively, seem to occur in cis-Golgi compartments (Futerman and Pagano, 1991; Trinchera
and Ghidoni, 1989; Jeckel et al., 1992). It has been recently
shown that NeuAc addition to GD3 to form GT3 also occurs in a proximal
Golgi compartment (Rosales Fritz and Maccioni, 1995). Addition of
subsequent sugars (GalNAc, Gal, and NeuAc) to give ganglio series
gangliosides is carried out by glycosyltransferases (GalNAc-T, Gal-T2,
and Sial-T4, see Fig. SI) which on subfractionation on isopycnic
sucrose gradients colocalize with medial/trans-Golgi markers
and peak at slightly different positions; these results were taken as
an indication that these enzymes were arranged along the Golgi complex
in the order in which they act (Trinchera and Ghidoni, 1989; Trinchera et al., 1990).
Figure SI: Scheme IThe pathways of biosynthesis of gangliosides of the a and b series.
In spite of the evidences indicating a compartmental organization of ganglioside biosynthesis along the Golgi complex, the issue of how the product from one transfer step couples as substrate for the next transfer step in the building up of the oligosaccharide is still unclear. Uncovering the natural relationships between the different transfer steps is of importance because in addition to the control of the glycosyltransferase activities (for references see Daniotti et al., 1994) and of the sugar nucleotide availability (Martina et al., 1995), the control of the compartmental organization of the synthetic machinery could also be of relevance in regulating the expression of gangliosides by cells. To approach this issue, we study here the in vitro labeling of the endogenous gangliosides of Golgi complex preparations from chick embryo retina cells at 14 days of incubation. At this stage retina tissue actively synthesizes GD1a (Panzetta et al., 1980, 1987) and is appropriate for examining the coupling between the N-acetylgalactosaminyl-, galactosyl-, and sialyl- transfer steps required for the synthesis of ganglio series gangliosides.
Figure 1:
Incorporation of
[H]GalNAc, [
H]NeuAc, and
[
H]Gal into endogenous ganglioside acceptors of
Golgi membranes from 14-day-old chicken embryos. Golgi membranes were
incubated with 10 µM of
UDP-[
H]GalNAc (
), or
CMP-[
H]NeuAc (
), or
UDP-[
H]Gal (
) during 90 min with the
indicated amount of protein (A), or during the indicated times
with 15 µg of Golgi membrane protein (B). The incubation
system and procedures for determination of radioactivity incorporated
into endogenous glycolipids were as indicated under ``Experimental
Procedures.'' Values are duplicates of a typical
experiment.
Fig. 2shows a
fluorogram of a HPTLC of radioactive endogenous gangliosides from Golgi
membranes incubated with 10 µM of
UDP-[H]GalNAc or
CMP-[
H]NeuAc or UDP-[
H]Gal
during 1.5 and 4 h. Radioactive GM2 and GD2 were the major
[
H]GalNAc-labeled products, radioactive GM3, GD3,
GD1a, GT1b the major [
H]NeuAc-labeled products,
and radioactive LacCer, GM1, GD1b the major
[
H]Gal-labeled products. Although the total
incorporation from each sugar nucleotide increased slightly from 1.5 to
4 h (Fig. 1B), the pattern of labeling from any of the
radioactive nucleotides did not substantially change. Actually, in
experiments not shown it was found that the pattern observed was
already established after 30 min of incubation. The results of Fig. 2indicate that with each sugar donor most of the
incorporated radioactivity was in the immediate acceptor of the
corresponding labeled sugar (see Fig. SI):
[
H]GalNAc was incorporated into endogenous GM3
and GD3, [
H]NeuAc was incorporated into
endogenous LacCer, GM3, GM1, and GD1b, and [
H]Gal
was incorporated into endogenous GlcCer, GM2, and GD2. Formation of
small amounts of [
H]Gal-labeled GM3 and GT1b was
also noticeable, suggesting that an endogenous pool of CMP-NeuAc was
present in the membrane vesicles.
Figure 2: HPTLC fluorography of the labeled endogenous ganglioside acceptors from Golgi membranes incubated with radioactive sugar nucleotides. Golgi membranes were incubated with 10 µM of the indicated radioactive sugar nucleotides during 1.5 and 4 h. Incubation conditions, chromatographic separation of radioactive gangliosides, and fluorography were as indicated under ``Experimental Procedures.'' Radioactive bands were named according to their chromatographic mobilities as compared with co-chromatographed authentic glycolipid standards visualized by exposure of the HPTLC plates to iodine vapors.
Fig. 3is a fluorogram of a bidimensional HPTLC of a mixture of the radioactive gangliosides of experiments as the ones described in Fig. 2, showing that each compound separated in the first dimension in a neutral solvent system behaved as a single entity after a second run in an alkaline solvent system.
Figure 3:
Two-dimensional HPTLC analysis of labeled
products. Radioactive gangliosides isolated from incubates as in Fig. 2were mixed and analyzed by bidimensional thin layer
chromatography. The first dimension (1st D) was developed in
chloroform/methanol, 0.25% CaCl (60:36:8 by volume) and the
second dimension (2nd D) in chloroform/methanol, 0.25%
NH
OH (60:36:8 by volume). A, fluorography of the
bidimensional HPTLC of the radioactive ganglioside mixture. B,
orcinol staining of chromatographed glycolipid standards (only shown is
the first dimension).
Fig. 4, A and B, shows that if
membranes that were labeled from UDP-[H]GalNAc in
the first incubation step were incubated with increasing concentrations
of non-radioactive UDP-Gal from 0 to 50 µM in the second,
about 70% of radioactive GM2 and GD2, the major products formed in the
first step, were converted into GM1 and GD1b, respectively. Conversion
was maximal at 25 µM of UDP-Gal. This indicates that an
important fraction of synthesized GM2 and GD2 molecules were positioned
as substrates for the Gal-T2 that acts at the next transfer step to
synthesize GM1 and GD1b. Fig. 4, C and D,
shows that incubation of the labeled membranes in the second step with
50 µM UDP-Gal and increasing CMP-NeuAc concentrations from
0 to 50 µM led to a substantial decrease of GM1 and GD1b
and to a concomitant increase of GD1a and GT1b. The simplest
interpretation of results of Fig. 4, according to Fig. SIof biosynthesis, is that conversion of
[
H]GalNAc-labeled GM1 into GD1a and of
[
H]GalNAc-labeled GD1b into GT1b had occurred and
was maximal at 25 µM CMP-NeuAc. This indicates that the
transfer steps catalyzed by GalNAc-T, Gal-T2, and Sial-T4 colocalize
and are functionally coupled in the same compartment; Gal-T2 was able
to use the product of GalNAc addition to GM3 and GD3
([
H]GalNAc-labeled GM2 and GD2, respectively) as
substrate for synthesis of GM1 and GD1b; Sial-T4 was, in turn, able to
convert [
H]GalNAc-labeled GM1 and GD1b into GD1a
and GT1b, respectively, by successive transfer reactions.
Figure 4:
Radioactive labeling pattern of
gangliosides from Golgi membranes incubated with
UDP-[H]GalNAc in the first step and with
unlabeled UDP-Gal (A and B) or UDP-Gal plus CMP-NeuAc (C and D) in the second step. Golgi membranes were
labeled with10 µM UDP-[
H]GalNAc
during 90 min (first step), washed, and incubated with the indicated
increasing concentrations of UDP-Gal (A and B) or
with the indicated increasing concentrations of CMP-NeuAc in the
presence of 50 µM of UDP-Gal (Cand D) for 120 min (second step). Incubation conditions, washing
of membranes, lipid extraction, determination of total incorporation,
and HPTLC fluorography were as described under ``Experimental
Procedures.'' The figure shows the fluorograms (top half) and their respective densitometric quantifications (bottom
half) of a representative experiment out of two with similar
results. Total incorporation value at the end of the first step was
7,500 cpm. Total incorporation values at the end of the second step for
0, 10, 25, and 50 µM UDP-Gal (A and B)
were 8,500, 9,400, 8,900, and 8,300 cpm, respectively while those for
0, 10, 25, and 50 µM CMP-NeuAc and 50 µM
UDP-Gal (C and D) were 8,500, 8,000, 8,000, and 8,100
cpm, respectively.
Figure 5:
Radioactive labeling pattern of
gangliosides from Golgi membranes incubated with
CMP-[H]NeuAc in the first step and with unlabeled
UDP-GalNAc (A and B) or UDP-GalNAc plus UDP-Gal (C and D) in the second step. Golgi membranes were
labeled with 10 µM CMP-[
H]NeuAc
during 90 min (first step), and with the indicated increasing
concentrations of UDP-GalNAc (A and B) or 50
µM UDP-GalNAc plus the indicated increasing concentrations
of UDP-Gal (C and D) for 120 min (second step). Other
details were as in Fig. 4. Percents of GD1a, GT3, and GT1b,
remaining essentially constant in C, were not plotted in D. Total incorporation value at the end of the first step was
10,000 cpm. Total incorporation values at the end of the second step
for 0, 10, 25, and 50 µM UDP-GalNAc were 13,500, 13,000,
15,000, and 15,000 cpm, respectively, while those for 50 µM UDP-GalNAc and 0, 10, 25, and 50 µM UDP-Gal were
13,500, 14,000, 14,700, and 14,300 cpm,
respectively.
Figure 6:
Radioactive labeling pattern of
gangliosides from Golgi membranes incubated with
UDP-[H]Gal in the first step and with unlabeled
CMP-NeuAc (A and B) or CMP-NeuAc plus UDP-GalNAc (C and D) in the second step. Golgi membranes were
labeled with 10 µM UDP-[
H]Gal during
90 min (first step), and with the indicated increasing concentrations
of CMP-NeuAc (A and B) or 50 µM CMP-NeuAc plus the indicated increasing concentrations of
UDP-GalNAc (C and D) for 120 min (second step). Other
details were as indicated in Fig. 4. The incorporation value at
the end of the first step was 22,500 cpm. Total incorporation values at
the end of the second step for 0, 10, 25, and 50 µM of
CMP-NeuAc were 26,800, 27,200, 25,900, and 26,000 cpm, while those for
50 µM CMP-NeuAc and 0, 10, 25, and 50 µM UDP-GalNAc were 26,800, 25,500, 24,900, and 25,000 cpm,
respectively.
Fig. 6, C and D, shows that incubation of membranes with
UDP-[H]Gal in the first step and with 50
µM of CMP-NeuAc plus increasing concentrations of
UDP-GalNAc, from 0 to 50 µM in the second step, led to a
decrease in the percent contribution of GM3 and GD3, noticeable at
10-25 µM UDP-GalNAc, and to an increase of labeled
GM2. This contrasts with the result of Fig. 6, A and B, in which most radioactive LacCer was converted to GM3 and
half of GM3 to GD3 when CMP-NeuAc was the sole nucleotide. According to Fig. SIof biosynthesis, the simplest interpretation of these
results is that a competition between SialT-2 and GalNAc-T for the
common GM3 acceptor had ocurred when their corresponding sugar donors
were both present in the assay; radioactive GM3 was increasingly used
for GM2 synthesis, rather than for GD3 synthesis, as UDP-GalNAc
concentration increased. This was particularly evident in this system
because at 14 days of development retina GalNAc-T activity exceeds
Sial-T2 activity in about 20-fold (Panzetta et al., 1980;
Daniotti et al., 1991). In addition, the results of Fig. 6indicate that addition of [
H]Gal to
endogenous GlcCer to form the radioactive lactosylceramide moiety of
GM3 and GD3 occurred in a compartment bearing not only Gal-T1 but also
Sial-T1, Sial-T2, GalNAc-T, and Gal-T2.
Figure 7:
Time dependence of the conversion of
LacCer to GD1a. Golgi membranes were labeled with
UDP-[H]Gal during 90 min (first step), washed,
and incubated with 50 µM each of unlabeled UDP-Gal,
UDP-GalNAc and CMP-NeuAc (second step) for the times indicated. Other
details were as in Fig. 6.
Figure 8:
Radioactive labeling pattern of
gangliosides from Golgi membranes incubated with
UDP-[H]Glc in the first step and with unlabeled
UDP-Gal, UDP-GalNAc, and CMP-NeuAc in the second step. Golgi membranes
were labeled with UDP-[
H]Glc during 90 min (first
step), washed, and incubated with (+) or without(-) 10
µM of UDP-Gal, UDP-GalNAc and CMP-NeuAc for 120 min
(second step). Other details were as in Fig. 4. Total
incorporation value at the end of the first step was 14,000 cpm. Total
incorporation values at the end of the second step were 15,500 and
16,000 cpm for membranes incubated without or with added unlabeled
nucleotides, respectively.
Figure 9:
Lack of unspecific fusion among
[H]NeuAc-labeled microsomal membranes from
7-day-old chick embryo retinas with unlabeled microsomal membranes from
14-day-old chick embryo retinas. Microsomal membranes from 7-day-old
chick embryo retinas (100 µg) were labeled with 10 µM CMP-[
H]NeuAc during 90 min, washed, and
incubated in a second step during 120 min with 10 µM unlabeled UDP-Gal, UDP-GalNAc, and CMP-NeuAc (lane 1) or
as in lane 1 but with the addition of 100 µg of protein of
14-day-old membranes (lane 2). Lane 3 is as lane
1 except that membranes were from 14-day-old embryos. Incubates
were processed as in Fig. 4.
It was also considered possible
that vesicles were in an inverted topography, so that acceptors from
one inside out vesicle could interact with transferases from another
inside out vesicle. This possibility was discarded for two reasons: (i)
because even at the end of the two-step assay, which implies additional
pelleting and homogenization of the membranes, at least 60% of the
endogenous gangliosides labeled from CMP-[H]NeuAc
were protected from the action of neuraminidase, as it was already
shown for rat brain Golgi membrane gangliosides (Maccioni et
al., 1974; Landa et al., 1981); and (ii) because the
protein concentration curve shown in Fig. 1A was
linear. If enzyme-substrate interactions between inside out particles
were occurring, an exponential curve should have been obtained
evidencing the compound effect of increasing the concentration of both
the enzyme and the substrate in a non-saturated, freely interacting
system (Reiner, 1959).
The labeling of the endogenous ganglioside acceptors of neural cell Golgi membranes was studied to gain information on the functional relationship among the different glycosyl transfer steps of the synthesis of gangliosides. This approach, used previously for similar studies in brain microsomal membranes (Arce et al., 1971), is based on the ability of Golgi membranes of incorporate sugars into intermediates in the pathway of completion upon incubation of the organelle with the appropriate sugar nucleotide donors. It provides a picture of a dynamic process of synthesis carried out by membrane-bound enzymes, glycolipid acceptors, and sugar transporters, which was interrupted at the moment of membrane isolation, and was resumed in vitro under controlled conditions.
Because they synthesize
mainly GD1a (Panzetta et al., 1980), retina cells from
14-day-old chick embryo are useful for examination of the functional
relationships among the various glycosyl transfer steps involved in
ganglio series ganglioside synthesis. Incubation of Golgi
membrane-enriched fractions from these cells with
UDP-[H]Gal or UDP-[
H]GalNAc
or CMP-[
H]NeuAc up to completion of their
endogenous acceptor capacity resulted in the radioactive labeling of
most ganglioside intermediates. The incorporated sugar remained mainly
in the immediate product of the transfer step marked by the radioactive
sugar nucleotide used. These labeled intermediates were substrates for
further glycosylations when the membranes were incubated in a second
step with the required unlabeled sugar nucleotides, and two, three, or
four transfer reactions were evidenced, depending on whether one, two,
or the three required unlabeled sugar nucleotides were present. A
summarizing experiment illustrating this functional coupling is the one
depicted in Fig. 7, in which
[
H]Gal-labeled lactosylceramide formed from
UDP-[
H]Gal in the first incubation step was
almost completely converted to labeled GD1a upon incubation in a second
step with unlabeled UDP-Gal, UDP-GalNAc, and CMP-NeuAc. Since factors
known to be necessary for vesicular traffic among compartments
(Rothman, 1994) were not present in the conditions of the assay, the
results indicate that ganglioside Gal-T1, Sial-T1, Sial-T2, GalNAcT,
Gal-T2, and Sial-T4 colocalize in a common compartment of the Golgi
apparatus of chick embryo retina cells, and that the products of the
corresponding transfer steps are able to couple with the next transfer
step of the pathway.
The above mentioned compartment could be in the
distal or proximal Golgi, with either proximal Golgi enzymes (Gal-T1,
Sial-T1, and Sial-T2) and acceptors (glucosylceramide,
lactosylceramide) extending their presence to the distal compartment,
or vice versa, with elements of distal transfer steps (GalNAc-T,
Gal-T2, and Sial-T4) present in the proximal compartment. The first
possibility is supported by the results of Fig. 7and Fig. 8, showing that acceptor GlcCer molecules completed with
[H]Gal ([
H]Gal-labeled
lactosylceramide) were more efficiently transformed into GD1a than
[
H]Glc-labeled lactosylceramide. In addition, the
first possibility is supported by the results of experiments with BFA.
BFA, which blocks anterograde vesicular transport and redistributes the cis/medial and trans-Golgi (but not TGN) back into the ER (Klausner et al., 1992) inhibits in vivo ganglioside biosynthesis with accumulation of LacCer, GM3, GD3 (van Echten et al., 1990; Young et al., 1990), and GT3 (Rosales Fritz and Maccioni, 1995) and depletion of more complex gangliosides. Although not proved, the effect of BFA strongly suggest that synthesis of LacCer, GM3, GD3, and GT3 occur in cis/medial/trans-Golgi compartments, and that these intermediates are transported in vesicles to a compartment beyond the trans-Golgi, most probably the TGN, for further glycosylation. Evidence for vesicular transport of glycolipids have been provided (Miller-Podraza and Fishman, 1984; Wattenberg, 1990; Young et al., 1992). Thus, the lack of supply of acceptor glycolipid (i.e. GlcCer) to the distally located Gal-T1, Sial-T1, and Sial-T2 and the absence of GalNAc-T in the proximal compartment would be concurrent factors leading to the observed effect of BFA on ganglioside synthesis in cells.
Apart from the possibilities of different strategies of subcompartmentation used for different cell types (Roth et al., 1986; Sjoberg and Varki, 1993), our results are in line with the reported immunoelectron microscopy data showing that antibody labeling of the trans-Golgi glycoprotein Gal-T and of the medial Golgi GlcNAc-T1 is highly overlapped in HeLa cells (Nilsson et al., 1993). Also, they agree with the reported overlapped distributions of ganglioside glycosyltransferases in subfractions of rat liver Golgi (Trinchera and Ghidoni, 1989; Trinchera et al., 1990) and with the cis/trans decreasing gradient distribution of ganglioside Sial-T1 and cis/trans increasing gradient distribution of Sial-T4 activities in Golgi preparations obtained by density gradient centrifugation from cultured cerebellar neurons (Iber et al., 1992). Using the endogenous labeling approach, compartmental overlapping was also deduced for some N-linked oligosaccharide biosynthetic activities in Golgi preparations from rat liver (Hayes and Varki, 1993; Hayes et al., 1993) and for GD3 synthesis and O-acetylation in Golgi membranes from melanoma cells (Sjoberg and Varki, 1993).
A key question about when coupling relationships
are being measured is whether they are a representative or just a minor
phenomenon. Considering the concentration of gangliosides in chicken
retina of about 3.5 nmol/mg protein (Panzetta et al., 1980)
and the half-life of about 48 h measured in mouse neuroblastoma N18 and
rat glioma C6 (Miller-Podraza and Fishman, 1982) and in chicken retina
cells, ()it follows that a synthesis rate of 36 pmol/mg of
protein/h would be necessary to keep the ganglioside pool constant. The
rate of completion of endogenous acceptors in the period from 0 to 30
min in Fig. 1B gives minimum values of 58 pmol/mg of
protein/h for the sum of [
H]Gal,
[
H]GalNAc, and [
H]NeuAc in
a 5-fold enriched Golgi preparation. This figure is about 30% of the
rate needed for a cell to maintain its ganglioside pool and suggests
that we are not dealing with a minor phenomenon.