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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Gangliosides are of importance as targets for cancer immunotherapy (Livingston, 1998Go) and for treatment of disorders of the nervous system including spinal cord injury (Geisler, 1998Go) and Parkinson’s disease (Schneider et al., 1998Go). Recent reports (Lannert et al., 1998Go; Giraudo et al., 1999Go) 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., 1992Go; Rother et al., 1992Go) and Golgi (Coste et al., 1986Go; Futerman and Pagano, 1991Go; Jeckel et al., 1992Go; Schweizer et al., 1994Go), 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., 1998Go). Although initial reports had described GM3 synthase as an early Golgi resident (Trinchera and Ghidoni, 1989Go; Trinchera et al., 1990Go; Iber et al., 1992Go), recently both LacCer synthase and GM3 synthase were localized to the late Golgi of rat liver (Lannert et al., 1998Go). Furthermore, GM2 synthase was localized to the trans Golgi network (TGN) (Lannert et al., 1998Go; Giraudo et al., 1999Go). Thus, according to the current model (Lannert et al., 1998Go), 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, 1985Go). In the CHO cells we used in the present study, GM3 is the only ganglioside endogenously produced (Yogeeswaren et al., 1974Go; Briles et al., 1977Go), but following transfection with GM2 synthase, GM2, GM1, and GD1a are produced (Rosales Fritz et al., 1997Go).

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., 1996bGo). 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 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., 1997Go). 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., 1993Go) to the cis Golgi (Roth et al., 1994Go). The results of this study support a late Golgi location for LacCer to GM3 conversion in CHO cells.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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., 1995Go), the myc epitope, and the ppGaNTase transmembrane domain and flanking regions which include one N-linked chain (Homa et al., 1993Go). 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., 1996aGo; Zhu et al., 1997Go). 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.


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Table I. Polypeptide domains used for the construction of chimeric GM2 synthase variants
 


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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.

 
We previously described the release of a soluble form of wild type GM2 synthase/myc as a result of proteolytic cleavage near the border between the transmembrane and lumenal domains (Jaskiewicz et al., 1996aGo). 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., 1996aGo). 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 anti-myc 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 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.



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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 {alpha}-glucosidase II (ER marker enzyme; open circles) and galactosyl transferase (Golgi marker enzyme; solid circles).

 
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., 1996bGo). Giraudo et al. (1999)Go 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., 1994Go) 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.


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Table II. GM2 synthase activities and ganglioside profiles of CHO cells expressing chimeric GM2 synthase
 


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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).

 
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., 1996bGo). 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, 1998Go), 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., 1998Go). Similarly, although we detected GnTI/GM2 synthase by immunoelectronmicroscopy in clone B5 cells only in the medial Golgi (Jaskiewicz et al., 1996bGo), 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., 1993Go; Rabouille et al., 1995Go). 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 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 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., 1996bGo). 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, 1996Go). 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 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., 1996bGo), 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 (Figure 4); i.e., in these cells GM2 functioned primarily as a metabolic intermediate and did not accumulate significantly (see Discussion).



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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 FITC-conjugated 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.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
GM3 synthase has been cloned (Ishii et al., 1998Go; Kono et al., 1998Go; Fukumoto et al., 1999Go; Kapitonov et al., 1999Go), 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, 1989Go; Trinchera et al., 1990Go) but to the trans Golgi more recently (Lannert et al., 1998Go). 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., 1992Go). 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., 1998Go). 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)Go that GM3 synthase is located in the late Golgi and not the cis Golgi as originally proposed (Trinchera and Ghidoni, 1989Go; Trinchera et al., 1990Go). In addition our results extend the findings of Lannert et al., 1998Go, 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, 1998Go), 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)Go 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, 1990Go; Riboni et al., 1997Go; Gillard et al., 1998Go). In contrast, here we analyzed the chemical patterns of ganglioside 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., 1998Go). 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, 1999Go). 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 {alpha}1,3-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, 1999Go). 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., 1994Go). 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., 1998Go). 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 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, 1984Go; Piller et al., 1990Go). A precedent for another GalNAc transferase being able to function in the ER was provided by Rottger et al., 1998Go, 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., 1998Go).

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)Go 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., 1988Go) 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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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., 1996aGo,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 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., 1996aGo,b).

Flow cytometry
Staining of transfected CHO cells with monoclonal IgM anti-GM2 10–11 (Zhu et al., 1998Go). 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 PBS-BSA, 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-ß-N-acetylglucosaminidase H (endo H) and Western blotting with anti-myc were described previously (Jaskiewicz et al., 1996aGo,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., 1996aGo). 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 {alpha}-glucosidase II, and 0.055 ml for GalT. The in vitro assay for GM2 synthase activity was performed as described elsewhere (Jaskiewicz et al., 1996bGo) 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., 1986Go) 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)Go. Lipids were extracted, Folch partitioned, and upper phase lipids desalted on Bond Elut C18 columns (Analytichem, Harbor City, CA; Schnaar, 1994Go). 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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Cer, ceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; CHO, Chinese hamster ovary; PBS-BSA, phosphate buffered saline pH 7.4 containing 1% bovine serum albumin.


    Footnotes
 
1 Present address: NIH, Building 10 Room 9-D16, 9000 Rockville Pike, Bethesda, MD 20892 Back

2 These authors contributed equally to this work Back

3 To whom correspondence should be addressed at: Dental School, University of Louisville, 501 S. Preston St., Louisville, KY 40292 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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