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.
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Abstract |
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Key words: ganglioside/glycosyltransferase/GM2 synthase/Golgi
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Introduction |
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In the present study we tested if the location for LacCer to GM3 conversion is the early or late Golgi in CHO cells. Our experimental approach was to target GM2 synthase to the medial Golgi, the cis Golgi, and the ER and then to determine the relative ability of the enzyme in each of these locations to convert GM3 to GM2. If LacCer to GM3 conversion occurred in the cis Golgi, then GM3 produced in that early compartment could be utilized as a substrate to produce GM2 if GM2 synthase were targeted to any Golgi cisternae. In contrast if LacCer to GM3 conversion occurred in the trans Golgi and if we assume minimal retrograde trafficking of gangliosides to the early Golgi (see Discussion), then GM2 could be produced efficiently by GM2 synthase targeted to the trans Golgi or TGN, but GM2 synthesis would be progressively less efficient as GM2 synthase was targeted to the medial Golgi, cis Golgi, and ER, respectively. Previously, we reported that conversion of GM3 to GM2 occurred when GM2 synthase was targeted to the medial Golgi but not when it was targeted to the ER (Jaskiewicz et al., 1996b). However, our earlier analysis of ganglioside synthesis was performed on lipids metabolically labeled with [3H] palmitate for 4 h, and it did not reveal that the product GM2 could be further converted to GM1 and GD1a by endogenous enzymes as was subsequently shown by metabolic labeling with [3H] galactose (Rosales Fritz et al., 1997
). Therefore, in the present report we examined the chemical quantities of gangliosides produced in cells expressing these GM2 synthase chimeras in order to analyze the entire ganglioside profile. In addition we tested a new construct which targeted GM2 synthase to the ER and cis Golgi. This targeting approach was based on the localization of endogenously expressed UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGaNTase) (Homa et al., 1993
) to the cis Golgi (Roth et al., 1994
). The results of this study support a late Golgi location for LacCer to GM3 conversion in CHO cells.
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Results |
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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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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., 1996b). In clone E6 only 1.6% of GM3 was converted to higher gangliosides (Table II; Figure 3, lane 1). This minimal capability to produce GM2 cannot be explained simply by there being insufficient enzyme in clone E6 cells because the specific activity in these cells was more than 4-fold greater than that of wild type clone R12 (Table II). The Iip33 motif has been shown to be a signal for retrieval of proteins bearing the motif from the early Golgi to the ER (Teasdale and Jackson, 1996
). If the distribution of GM3 synthase were centered over the cis Golgi, then Iip33/GM2 synthase/myc and GM3 synthase should coexist in the same compartments at least transiently and produce GM2. The low amount of GM2 produced in clone E6 cells thus provides additional evidence against a cis Golgi location for GM3 synthase and, therefore, supports a late Golgi location for GM3 synthase in CHO cells.
Ganglioside patterns produced by chimeric enzymes
In clones C5 and R12 which expressed high and low levels of wild type enzyme, respectively, and in clone B5 expressing GnTI/GM2 synthase, GM2 was the major ganglioside (Figure 3, Table II). In contrast, we found previously that GM2 was not detectable on the surface of clone E6 cells expressing Iip33/GM2 synthase (Jaskiewicz et al., 1996b), and the same was true for clone E5 expressing ppGaNTase/GM2 synthase (Figure 4A). However, flow cytometry using fluoresceinated cholera toxin (Figure 4B) revealed the presence of ganglioside GM1 on the surface not only of clone E5 cells expressing ER and cis Golgi targeted ppGaNTase/GM2 synthase but also on clone E6 cells expressing Iip33/GM2 synthase. Thus, for clone E5 expressing the ER and cis Golgi targeted ppGaNTase/GM2 synthase and for clone E6 expressing the ER targeted Iip33/GM2 synthase, the small amount of GM2 that was produced was utilized to a large extent to produce GM1 and GD1a as judged by both chemical analysis (Table II) and flow cytometry (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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Discussion |
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An alternative explanation for the limited GM2 production by our chimeric enzymes would be that GM2 synthesis occurs following retrograde traffic of GM3. The subcellular fractionation performed by Lannert et al. (1998) provided elegant data about glycosyltransferase distribution but by its very nature could not be informative about the dynamics of ganglioside synthesis which includes not only de novo biosynthesis but also ganglioside synthesis via recycling pathways (Schwarzmann and Sandhoff, 1990
; Riboni et al., 1997
; Gillard et al., 1998
). In contrast, here we analyzed the chemical patterns of ganglioside profiles which are the net result of all synthetic pathways. In many cell lines recycling pathways produce the majority of gangliosides, particularly the more complex structures (Gillard et al., 1998
). The recycling pathways which have been analyzed consist of: (1) recycling of gangliosides from the plasma membrane via endosomes to the Golgi where they are subsequently utilized for the synthesis of more complex gangliosides; and (2) transfer of gangliosides from the plasma membrane to the lysosomes where the gangliosides are degraded to their sphingosine or sphinganine moiety which is then reutilized in the ER for new sphingolipid synthesis. In addition the Golgi cisternal maturation model offers the untested possibility of retrograde trafficking of gangliosides within the Golgi. At present it is not known if these retrograde vesicles are targeted only to the adjacent "younger" cisterna or whether they may reach all earlier cisternae. If GM3 produced in the trans Golgi were carried by these retrograde vesicles to all earlier cisternae, then the outcome of our present analysis would have been the production of high levels of GM2, regardless of the location of GM2 synthase. Similarly, the endocytotic recycling pathway potentially could transport GM3 from the cell surface to the early Golgi or even to the ER because it is clear from studies of cholera toxin, shiga toxin, and ricin that endocytosis can deliver material to the ER (Sandvig and Van Deurs, 1999
). If high amounts of cell surface to ER transport of GM3 had occurred in our cells, we would have detected high levels of GM2 regardless of the location of GM2 synthase. Clearly our results do not support either of these extreme cases because we found a progressive decrease in GM2 production as GM2 synthase was targeted to earlier compartments. However, the limited amount of GM2 produced by our chimeras could have been the result of either intra-Golgi retrograde trafficking of GM3 only to the adjacent cisterna or endocytotic recycling of GM3 from the cell surface which was most efficient for delivery to the late Golgi and progressively less efficient to earlier compartments.
Recently Grabenhorst and Conradt constructed chimeras of 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, 1999
). Here we have adopted a similar approach with GM2 synthase. The experimental design we employed included cis Golgi targeting of GM2 synthase via the transmembrane domain and flanking regions of ppGaNTase I. Previously endogenous ppGaNTase I was localized to the cis Golgi (Roth et al., 1994
). More recently, however, epitope tagged ppGaNTase I in transfected HeLa cells was found to be distributed throughout the Golgi stack rather than being only in the cis Golgi (Rottger et al., 1998
). The apparent discrepancy between these results may be due to cell type differences, differences due to overexpression of the transfected enzyme and/or epitope tagging, or the possibility that the antiserum used to detect endogenous ppGaNTase I may have recognized other members of the ppGaNTase family. Regardless of the explanation for this point of controversy, our data indicate that ppGaNTase/GM2 synthase hybrid molecules were targeted to the ER and cis Golgi as revealed by subcellular fractionation (Figure 2), which showed the presence of membrane-bound enzyme in the ER and Golgi, combined with endo H sensitivity (Figure 1).
An assumption in our experimental design is that the compartments to which our chimeras were targeted would allow GM2 synthesis if substrate GM3 were present. Clearly, the cis Golgi can provide the UDP-GalNAc required for GM2 synthesis because it has been identified as the site for GalNAc addition that initiates protein O-glycosylation (Roth, 1984; Piller et al., 1990
). A precedent for another GalNAc transferase being able to function in the ER was provided by Rottger et al., 1998
, who reported that O-linked specific GalNAc transferase 1 and 2 were capable of initiating protein O-glycosylation when they were relocated to the ER (Rottger et al., 1998
).
In the present study GM2 accumulated in cells in which GM2 synthase was targeted to the medial and late Golgi (clones B5, C5, and R12). However, in cells expressing GM2 synthase targeted to the ER and cis Golgi or to the ER alone (clones E5 and E6, respectively; Figures 3 and 4; Table II) GM2 did not accumulate but instead functioned primarily as a biosynthetic intermediate. Ruan et al. (1999) found that neuroblastoma cell lines which expressed moderate to high levels of GM2 synthase activity (which also catalyzes the conversion of GD3 to GD2 (Pohlentz et al., 1988
) and low levels of GD3 synthase activity contained high levels of GD2 but surprisingly low levels of GD3, i.e., a very high percentage of the intermediate GD3 was converted to GD2. The situation with our cis Golgi- and ER-targeted clones is similar in that the intermediate GM2 does not accumulate; however, in our case there is no correlation between the level of GM2 synthase activity and the accumulation of GM2. The lack of accumulation of GM2 in cells expressing GM2 synthase targeted to the early Golgi may simply be due to the amount of GM2 being below the level at which GM1 synthase is saturated. In the case of wild type clone R12 which expresses an even lower level of wild type enzyme (Table II) but accumulates GM2, one explanation for GM2 accumulation could be saturation of GM1 synthase. Alternatively, a portion of the GM2 produced in the TGN by wild type enzyme might proceed to the cell surface without being acted upon by GM1 synthase and remain there without significant endocytosis and utilization for GM1 production. In the case of clone B5 expressing medial Golgi-targeted GnTI/GM2 synthase/myc, saturation of GM1 synthase seems the likely explanation for GM2 accumulation because all GM2 produced in the medial Golgi should encounter GM1 synthase in the TGN on its way to the cell surface.
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Material and methods |
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Flow cytometry
Staining of transfected CHO cells with monoclonal IgM anti-GM2 1011 (Zhu et al., 1998). Briefly, cells were removed from monolayer culture with trypsin-EDTA and incubated for 30 min at 4°C with the primary antibody in PBS-BSA. The cells were then washed in cold PBS-BSA and incubated for 30 min at 4°C in PBS-BSA containing fluorescein-conjugated goat anti-mouse Ig. For staining with FITC-cholera toxin, transfected CHO cells were trypsinized and incubated for 30 min on ice with 10 µg/ml FITC-cholera toxin diluted in PBS-BSA. After washing, the cells were resuspended in 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., 1996a,b). For subcellular fractionation cells from 15 150 cm2 flasks were homogenized, and Golgi and ER fractions were separated on sucrose gradients as described previously (Jaskiewicz et al., 1996a
). Fractions (1.3 ml per fraction) were collected from the sucrose gradients and the following aliquots taken for each assay: 0.008 ml for anti-myc Western blotting, 0.02 ml for GM2 synthase activity, 0.1 ml for
-glucosidase II, and 0.055 ml for GalT. The in vitro assay for GM2 synthase activity was performed as described elsewhere (Jaskiewicz et al., 1996b
) except that the assay mixture contained 0.1 mM UDP-[3H] GalNAc and 10 mM CDP-choline and the product GM2 was isolated by butanol-water partitioning. Details of glycolipid analysis were described previously (Nichols et al., 1986
) with the modification that large scale cell culture was achieved in roller bottles using the serum-free medium CHO-S-SFM II (Life Technologies) according to Kolhekar et al. (1997)
. Lipids were extracted, Folch partitioned, and upper phase lipids desalted on Bond Elut C18 columns (Analytichem, Harbor City, CA; Schnaar, 1994
). Samples were separated on HPTLC plates in CHCl3:CH3OH:0.25% KCl in H2O, 50:40:10, v/v, and ganglioside bands were visualized by resorcinol.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 These authors contributed equally to this work
3 To whom correspondence should be addressed at: Dental School, University of Louisville, 501 S. Preston St., Louisville, KY 40292
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References |
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