©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Coupling of Glycosyl Transfer Steps for Synthesis of Gangliosides in Golgi Membranes from Neural Retina Cells (*)

(Received for publication, June 2, 1995; and in revised form, June 19, 1995)

Mariana K. Maxzúd (§) José L. Daniotti Hugo J. F. Maccioni (¶)

From the Centro de Investigaciones en Qu&ıacute;mica Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Qu&ıacute;mica Biológica, Facultad de Ciencias Qu&ıacute;micas, Universidad Nacional de Córdoba, CC61, 5016 Córdoba, Argentina

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-[^3H]NeuAc or UDP-[^3H]GalNAc or UDP-[^3H]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-[^3H]Gal, in which most of the [^3H]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.


INTRODUCTION

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 (^1)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.


EXPERIMENTAL PROCEDURES

Preparation of Retina Golgi Membranes

Retinas dissected from 14-day-old chicken embryo were suspended in 10 mM Tris-HCl buffer (pH 7.2) containing 0.25 M sucrose (buffer A) and washed twice in the same buffer solution. The tissue was homogenized and the homogenate centrifuged at 600 g for 5 min to obtain a post-nuclear supernatant fraction that was used as the starting material to obtain a Golgi fraction essentially as described (Landa et al., 1981; Iber et al., 1992): it was overlaid onto a discontinuous sucrose gradient made of 9 ml each of 0.8, 1.0, and 1.2 M-sucrose, and centrifuged 2.5 h at 54,000 g in the SW 25 rotor of the Spinco Ultracentrifuge. The Golgi membrane enriched fraction was collected from the interphase 0.8-1.2 M sucrose and pooled. It showed an enrichment of Sial-T2 and GalNAc-T about 5-fold relative to the total homogenate. These activities were determined following already published procedures (for references, see Daniotti et al., 1994).

Incubation Conditions

Two step protocols were usually run. In the first step the incubation system contained, in a final volume of 20 µl, 15 mM MnCl(2), 10 µM of UDP-[^3H]Gal, or CMP-[^3H]NeuAc or UDP-[^3H]GalNAc, 25 mM HEPES-KOH (pH 7.0), 25 mM KCl, 2.5 mM magnesium acetate, and 15 µg of protein of Golgi membranes. Unless otherwise indicated, the system was incubated for 90 min at 37 °C. In experiments in which incorporation was determined at 1, 10, 50, 100, and 200 µM of sugar nucleotide, it was found that 10-20 µM produced about 60% of the maximal incorporation obtained at 50-100 µM (not shown). Ten µM was within the range of the apparent K values of the sugar nucleotide transporters (Hirschberg and Snider, 1987). For each condition, the labeled nucleotide was added to give a specific radioactivity of 7,500 cpm/pmol. After the first step, membranes were freed from incubation medium ingredients by layering onto 0.6 M sucrose and centrifugation at 13,000 g for 6 min at 4 °C. The washed membrane pellet was resuspended to the original incubation volume with the ingredients of the first step but lacking the radioactive nucleotide. To the washed membranes, unlabeled UDP-Gal, UDP-GalNAc, or CMP-NeuAc, alone or in mixtures of two or three and at concentrations that will be indicated in each case, were added and the second incubation step was carried out during 2 h.

Determination of Radioactivity and Chromatographic Analysis of Radioactive Gangliosides

After incubation, the system was cooled in ice, diluted to 1 ml with distilled water, and centrifuged at 13,000 g for 30 min in a refrigerated centrifuge. The pellet was supplemented with appropriate amounts of lactosylceramide (from human erythrocytes), GM3 (from canine spleen), GM2 (from Tay Sachs brain), and bovine brain gangliosides as internal standards and extracted by sonication in 2 ml of chloroform/methanol/water (60:30:4.5 by volume). Water-soluble contaminants were removed by passing the extract through a Sephadex G-25 column equilibrated in the same solvent mixture (Maccioni et al., 1974). For determination of total incorporation, an aliquot of the lipid extract was transferred to a vial, evaporated, resuspended in 50 µl of 10% sodium dodecyl sulfate, mixed with a scintillation fluid containing 10% naphthalene and 2,5-diphenyloxazole, and counted in a liquid scintillation spectrometer. For chromatographic analysis, the lipid extract was evaporated under N(2) and run on HPTLC in a developing tank as described by Nores et al.(1994) using chloroform/methanol, 0.25% CaCl(2) (60:35:8 by volume) as solvent for samples labeled from UDP-[^3H]Gal or CMP-[^3H]NeuAc and chloroform/methanol, 0.25% CaCl(2) (45:45:10 by volume) for samples labeled from UDP-[^3H]GalNAc. In two-dimensional HPTLC separations, the solvent in the second dimension was chloroform/methanol, 7% NH(4)OH (60:35:8 by volume). Positions of the internal standard gangliosides were determined by exposure of the plate to iodine vapors. Fluorography was carried out by dipping the plate in 2-methyl-naphthalene containing 0.4% 2,5-diphenyloxazole and further exposure of the plate to an x-ray film for 4-8 days at -70 °C. The relative contribution of individual radioactive gangliosides was determined by densitometric scanning of the x-ray film in a CS 930 Shimadzu UV/vis scanner.


RESULTS

Labeling of Endogenous Gangliosides

Fig. 1A shows that the incorporation of radioactivity from UDP-[^3H]Gal or UDP-[^3H]GalNAc or CMP-[^3H]NeuAc into total endogenous ganglioside acceptors of the Golgi membrane-enriched fraction was linear as a function of the protein concentration in the range of 0.25-1.5 mg protein/ml. Fig. 1B shows that the incorporation as a function of the incubation time was linear up to 30 min and then decreased and tend to plateau after 2 h. Results of Table 1show that no inactivation of Gal-T2, GalNAc-T, and Sial-T4 occurred by preincubation of the membranes during 2 h before measuring their activities. This indicates that the tendency to plateau in the labeling of the endogenous acceptors observed in Fig. 1B was due to saturation of the endogenous acceptor capacity of the membranes which, if no dilution effect by endogenous sugar nucleotides had occurred, was about 90 pmol/mg of protein for NeuAc and GalNAc and about 270 pmol/mg of protein for Gal.


Figure 1: Incorporation of [^3H]GalNAc, [^3H]NeuAc, and [^3H]Gal into endogenous ganglioside acceptors of Golgi membranes from 14-day-old chicken embryos. Golgi membranes were incubated with 10 µM of UDP-[^3H]GalNAc (), or CMP-[^3H]NeuAc (bullet), or UDP-[^3H]Gal (up triangle, filled) 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-[^3H]GalNAc or CMP-[^3H]NeuAc or UDP-[^3H]Gal during 1.5 and 4 h. Radioactive GM2 and GD2 were the major [^3H]GalNAc-labeled products, radioactive GM3, GD3, GD1a, GT1b the major [^3H]NeuAc-labeled products, and radioactive LacCer, GM1, GD1b the major [^3H]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): [^3H]GalNAc was incorporated into endogenous GM3 and GD3, [^3H]NeuAc was incorporated into endogenous LacCer, GM3, GM1, and GD1b, and [^3H]Gal was incorporated into endogenous GlcCer, GM2, and GD2. Formation of small amounts of [^3H]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(2) (60:36:8 by volume) and the second dimension (2nd D) in chloroform/methanol, 0.25% NH(4)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).



Effect of Incubation of [^3H]GalNAc-labeled Golgi Membranes with Unlabeled UDP-Gal or UDP-Gal Plus CMP-NeuAc

Except for a small amount of radioactive GM3 and GT1b observed in the labeling from UDP-[^3H]Gal, the experiments of Fig. 2evidenced a failure of the system to sustain further glycosylations of the immediate radioactive products formed. To examine the possibility that this was due to a lack of the necessary endogenous sugar nucleotides, membranes were incubated with one radioactive nucleotide as in Fig. 2, washed, and then incubated in a second step for an additional 2-h period in the presence of the non-radioactive sugar nucleotide necessary for the transfer step that would use the radioactive endogenous glycolipid formed in the first step.

Fig. 4, A and B, shows that if membranes that were labeled from UDP-[^3H]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 [^3H]GalNAc-labeled GM1 into GD1a and of [^3H]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 ([^3H]GalNAc-labeled GM2 and GD2, respectively) as substrate for synthesis of GM1 and GD1b; Sial-T4 was, in turn, able to convert [^3H]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-[^3H]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-[^3H]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.



Effect of Incubation of [^3H]NeuAc-labeled Golgi Membranes with Unlabeled UDP-GalNAc or UDP-GalNAc Plus UDP-Gal

To assess whether transfer steps catalyzed by Sial-T1 and/or Sial-T2 colocalize with those catalyzed by GalNAc-T, Gal-T2, and Sial-T4, CMP-[^3H]NeuAc was used in the first incubation step, and increasing concentrations of UDP-GalNAc from 0 to 50 µM in the second. Fig. 5, A and B, shows that about 60% of [^3H]NeuAc-labeled GM3 and GD3 formed in the first incubation step were converted into GM2 and GD2, respectively, indicating that a considerable fraction of the [^3H]NeuAc-labeled GM3 and GD3 was synthesized in a compartment that also bears GalNAc-T. Fig. 5, C and D, shows that incubation of membranes with CMP-[^3H]NeuAc in the first step and 50 µM of UDP-GalNAc plus increasing concentrations of UDP-Gal, from 0 to 50 µM in the second step, led to a decrease of [^3H]NeuAc-labeled GM2 and GD2 and to an increase of [^3H]NeuAc-labeled GM1 and GD1b. This result is consistent with an increasing galactosylation of the products of GalNAc addition to [^3H]NeuAc-labeled GM3 and [^3H]NeuAc-labeled GD3 (GM2 and GD2, respectively), as UDP-Gal concentration increases and indicates that transfer steps catalyzed by Sial-T1, Sial-T2, GalNAc-T, and Gal-T2 colocalize and are functionally coupled in the same compartment.


Figure 5: Radioactive labeling pattern of gangliosides from Golgi membranes incubated with CMP-[^3H]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-[^3H]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.



Effect of Incubation of [^3H]Gal-labeled Golgi Membranes with Unlabeled CMP-NeuAc or CMP-NeuAc Plus UDP-GalNAc

To examine if the synthesis of lactosylceramide occurs in the compartment supporting the transfer steps catalyzed by Sial-T1, Sial-T2, GalNAc-T, Gal-T2, and Sial-T4, membranes were incubated with UDP-[^3H]Gal in the first step to obtain [^3H]Gal-labeled lactosylceramide. Incubation of these membranes with increasing concentrations of CMP-NeuAc from 0 to 50 µM in a second step (Fig. 6, A and B) led to a gradual decrease of radioactive lactosylceramide and to an increase of GM3 and GD3. About 80% of [^3H]Gal-labeled lactosylceramide was sialylated to GM3 and then to GD3. Formation of GM3 was maximal at 10 µM while that of GD3 reached plateau at 25 µM. This indicates that most radioactive lactosylceramide synthesized in the first step was accessible to Sial-T1 and that about half of the formed GM3 was in turn used as substrate for GD3 synthesis by Sial-T2 at about 25 µM CMP-NeuAc concentration. GM1 and GD1b were not converted into labeled GD1a and GT1b at any CMP-NeuAc concentration (except for an increase in GD1a from 2.5 to 5%), indicating that not all intermediates formed in the first step were substrates for a next transfer step.


Figure 6: Radioactive labeling pattern of gangliosides from Golgi membranes incubated with UDP-[^3H]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-[^3H]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-[^3H]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 [^3H]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.

Effect of Incubation of Radioactive Golgi Membranes with Three Unlabeled Sugar Nucleotides: Time Dependence of Conversion

According to the results of Fig. 4-6, a precursor product relationship between the immediate acceptor of [^3H]Gal labeled in the first step (lactosylceramide) and the end product GD1a should be obtained if the three unlabeled nucleotides were present in the second step. Fig. 7shows the results of an experiment similar to the one described in Fig. 6, C and D, in which UDP-[^3H]Gal was in the first step and in addition to 50 µM of CMP-NeuAc and UDP-GalNAc, 50 µM of UDP-Gal was also present in the second step; the experiment was run for different periods of time from 1 min to 2 h. It is clear that with the three nucleotides in excess in the second step, labeled lactosylceramide was progressively converted to GD1a in a time-dependent fashion, being practically exhausted after 30 min; transient formation of labeled GM3, GM2, and GM1 (this last one superimposed to the GM1 fraction not convertible into more complex gangliosides, see Fig. 6) was also noticeable. Dilution of the incubation system (up to 20-fold) in the second step or extensive washing of the membranes with buffer A containing 1 M KCl before incubation to remove possible attached cytosolic proteins was without effect on the pattern of progression of radioactivity to more glycosylated species (not shown).


Figure 7: Time dependence of the conversion of LacCer to GD1a. Golgi membranes were labeled with UDP-[^3H]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.



Labeling from UDP-[^3H]Glc

Fig. 8shows the pattern of labeling of the endogenous acceptors after incubation of the membranes with UDP-[^3H]Glc in the first step and unlabeled UDP-Gal, UDP-GalNAc, and CMP-NeuAc in the second. Although the membranes synthesize an important amount of [^3H]Glc-labeled glucosylceramide, only 10% of it was converted to [^3H]Glc-labeled lactosylceramide. This amount did not increase after a second incubation step in the presence of the three unlabeled sugar nucleotides, suggesting that this is the fraction of GlcCer accessible to the luminal Gal-T1 (Deutscher and Hirschberg, 1986). Of the [^3H]Glc-labeled lactosylceramide, no more than 10% was converted to GD1a, which contrasts with the near 80% conversion of [^3H]Gal-labeled lactosylceramide formed by addition of [^3H]Gal to glucosylceramide shown in Fig. 7. The simplest explanation for the contrast between the experiments of Fig. 7and Fig. 8is that, at difference with [^3H]Gal-labeled lactosylceramide, the [^3H]Glc-labeled lactosylceramide formed in the compartment in which Glc-T synthesizes glucosylceramide does not largely colocalize with distal Golgi GalNAc-T, Gal-T2, and Sial-T4.


Figure 8: Radioactive labeling pattern of gangliosides from Golgi membranes incubated with UDP-[^3H]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-[^3H]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.



Artifactual Coupling between Compartments Is Not a Major Event in the Conditions of the Assay

The possibility was considered that the different transfer steps were located in distinct compartments but that these compartments became mixed due to promiscuous fusion among them under the conditions of the in vitro assay. This possibility was considered since the experiments were carried out in presence of 15 mM Mn , which has been suggested to be fusogenic at concentrations of about 30 mM (Paiement et al., 1982). To check this possibility we took advantage of the fact that the activities of Sial-T2 and GalNAc-T are relatively high and low, respectively, in 7 day chick embryo retina, and that the converse occurs in 14 day embryos (Panzetta et al., 1980; Daniotti et al., 1994); an indication of this is the pattern of labeling from CMP-[^3H]NeuAc of the endogenous gangliosides of microsomal membranes from retinas at early (Fig. 9, lane 1) and late (Fig. 9, lane 3) developmental stages, which differ mostly in the lower labeling of GD3 and presence of labeled GD1a in 14 day retinas. Lane 2 is the pattern of labeling of the 7 day preparation that after the incubation with CMP-[^3H]NeuAc in a first step was washed, mixed with membranes from 14 day embryonic retina, and incubated in the standard conditions in presence of the three unlabeled nucleotides. It was expected that, if mixing of vesicles from the two embryonic stages had occurred, the GalNAc-T activity of 14 day membranes would convert labeled GD3 molecules from the 7 day membranes to labeled GD2 and/or GD1b. Results from Fig. 9, lane 2, show that no modification of the pattern of labeling of the 7 day preparation had occurred after 2 h of incubation and hence that the two membrane preparations did not behave as if they were promiscuously mixed.


Figure 9: Lack of unspecific fusion among [^3H]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-[^3H]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-[^3H]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).

A Fraction of [^3H]Gal-labeled GM1 and GD1b Molecules Is Not Sialylated to GD1a and GT1b

The experiments of Fig. 6and 7 showed that most of the endogenous GM2 and GD2 molecules completed with [^3H]Gal ([^3H]Gal-GM1 and [^3H]Gal-GD1b) were not sialylated to GD1a and GT1b in a second step. This is in contradiction with the results of Fig. 4, in which about 60% of the GM1 formed by addition of Gal to [^3H]GalNAc-labeled GM2 was converted to GD1a and with those of Fig. 7, in which the decay of radioactivity of [^3H]Gal-lactosylceramide corresponded fairly well with the increase of [^3H]Gal radioactivity in GD1a. We were able to isolate [^3H]Gal-labeled lactosylceramide and [^3H]Gal-labeled GA2 after mild acid hydrolysis (1 N sulfuric acid, 90 °C, 2 h) of the radioactive GD1a band isolated from the HPTLC plate of the experiment of Fig. 7, an indication that part of the ^3H-Gal incorporated was in the position proximal to ceramide. On the contrary, no radioactive lactosylceramide or GA2 were detected after mild acid hydrolysis of the radioactive GM1 band isolated from the same HPTLC plate, indicating that in this ganglioside most of the incorporated [^3H]Gal was terminal in the oligosaccharide. This indicates that some endogenous intermediates do not couple with the corresponding next transfer step; these molecules may be in Golgi vesicles that either because they belong to non-GD1a expressing neuronal cell types or because they represent a particular Golgi membrane domain, or because they depend on regulatory conditions absent from the in vitro assay, are devoid of Sial-T4 activity.


DISCUSSION

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-[^3H]Gal or UDP-[^3H]GalNAc or CMP-[^3H]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 [^3H]Gal-labeled lactosylceramide formed from UDP-[^3H]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 [^3H]Gal ([^3H]Gal-labeled lactosylceramide) were more efficiently transformed into GD1a than [^3H]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, (^2)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 [^3H]Gal, [^3H]GalNAc, and [^3H]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.


FOOTNOTES

*
This work was supported by Grants PID 3-105700/88 from Consejo Nacional de Investigaciones Cient&ıacute;ficas y Técnicas (CONICET), 2436/92 from Consejo de Investigaciones Cient&ıacute;ficas y Tecnológicas de la Provincia de Córdoba (CONICOR), 117 from Secretar&ıacute;a de Ciencia y Tecnolog&ıacute;a de la Universidad Nacional de Córdoba, and 4218 from The Council for Tobacco Research, U.S.A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a CONICET Fellowship. This research is part of a Ph. D. thesis.

To whom correspondence should be addressed. Tel.: 054-51-604955; 054-51-608813; Fax: 054-51-680833; hmaccion{at}uncbqb.edu.ar.

(^1)
The abbreviations used are: GlcCer, glucosylceramide; HPTLC, high-performance thin-layer chromatography; LacCer, lactosylceramide; cpm, counts/min; BFA, brefeldin A. The nomenclature used for gangliosides is that recommended by Svennerholm (1963).

(^2)
V. Rosales Fritz and H. J. F. Maccioni, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. V. Rosales Fritz, J. A. Martina, and G. A. Nores for useful discussions and B. L. Caputto for critical reading of the manuscript.


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