2 Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA
3 Protein Glycosylation, Gesellschaft Fur Biotechnologische Forschung mbH, Braunschweig, Germany
Received on December 26, 2002; revised on January 27, 2003; accepted on February 5, 2003
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Abstract |
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Key words: baculovirus expression system / glycoprotein biosynthesis / insect cells / sialic acids / sialylation
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Introduction |
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The mammalian pathway for de novo biosynthesis of sialic acid and CMP-NeuAc was elucidated in pioneering studies that were published some time ago (reviewed by Schauer and Corfield, 1982). This pathway involves phosphorylation of N-acetylmannosamine by N-acetylmannosamine kinase to produce N-acetylmannosamine-6-phosphate (Ghosh and Roseman, 1961
). This intermediate is subsequently condensed with phosphoenolpyruvate by N-acetylneuraminyl-9-phosphate synthase to produce N-acetylneuraminyl-9-phosphate (Roseman et al., 1961
; Watson et al., 1966
). This intermediate is then dephosphorylated by a specific phosphatase to produce N-acetylneuraminic acid, the most common form of sialic acid (Warren and Felsenfeld, 1961
). Finally, this compound is activated by CMP-N-acetylneuraminic acid synthetase to produce CMP-N-acetylneuraminic acid (Kean, 1970
). Mammalian cells also appear to have a salvage pathway for the acquisition of sialic acids from either free or covalently bound sources in the extracellular milieu (Ferwerda et al., 1981
; Mendla et al., 1988
; Chigorno et al., 1996
). The acquisition of covalently bound sialic acids requires internalization of sialoglycoconjugates, their transport to the lysosomes, removal of the terminal sialic acid residues by lysosomal sialidases, and the movement of free sialic acids into the cytoplasm. Reincorporation of the free sialic acid into a newly synthesized glycoprotein requires its conversion to CMPsialic acid and transport into the Golgi apparatus, where it serves as the only known donor substrate for the sialyltransferases (Comb, 1966
).
Although there is evidence to suggest that some insect cells encode at least some of the enzymes involved in de novo sialic acid biosynthesis (Angata and Varki, 2000; Kim et al., 2002
), the low levels of sialic acid (Lawrence et al., 2000
) and absence of CMPsialic acid in Sf9 cells (Hooker et al., 1999
; Tomiya et al., 2001
), together with their general inability to produce sialylated N-glycans (reviewed by Marchal et al., 2001
), suggest that de novo sialic acid biosynthesis is not a major metabolic pathway in these cells or their transgenic derivatives. Thus, our working hypothesis has been that these cells have a sialic acid salvaging pathway, which supports N-glycan sialylation by its transgenic derivatives. The results of the present study supported this hypothesiswe found that Sfß4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence of fetal bovine serum or in serum-free medium supplemented with a purified bovine sialoglycoprotein, fetuin. Conversely, these cells did not produce sialylated N-glycans when cultured in serum-free medium or serum-free medium supplemented with asialofetuin. We also found that prolonged exposure of Sfß4GalT/ST6 cells to N-acetylneuraminic acid or N-acetylmannosamine supported N-glycan sialylation but only at very low levels. These data not only provide the first evidence that at least some insect cells have a sialic acid salvaging pathway but also begin to explain how Sfß4GalT/ST6 and other transgenic insect cells can produce sialylated glycoproteins in the absence of a more obvious source of CMPsialic acid.
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Results |
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Purified sialoglycoproteins support glycoprotein sialylation by Sfß4GalT/ST6 cells
One class of high-molecular-weight fetal bovine serum components that could potentially support N-glycan sialylation by Sfß4GalT/ST6 cells is the glycoproteins. By analogy to mammalian cells, Sfß4GalT/ST6 cells could salvage sialic acids by internalizing extracellular sialoglycoproteins, removing their terminal sialic acids with lysosomal sialidases, and reutilizing the free sialic acids for CMPsialic acid biosynthesis. If this hypothesis is correct, Sfß4GalT/ST6 cells cultured in serum-free media supplemented with a highly purified sialoglycoprotein should be able to produce terminally sialylated N-glycans. Thus, we analyzed the N-glycans from GST-SfManI produced by Sfß4GalT/ST6 cells cultured in a serum-free medium supplemented with a commercial bovine fetuin preparation that had been extensively purified by ammonium sulfate precipitation and gel filtration. HPAEC-PAD analyses showed that the GST-SfManI produced by these cells contained sialylated N-glycans (peak 2 in Figure 2A). In direct contrast, Sfß4GalT/ST6 cells cultured in serum-free media supplemented with bovine asialofetuin produced GST-SfManI with no detectable sialylated N-glycans (Figure 2B).
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N-acetylneuraminic acid and N-acetylmannosamine support glycoprotein sialylation by Sfß4GalT/ST6 cells
We also examined the ability of a free sialic acid and N-acetylmannosamine, a sialic acid precursor, to support glycoprotein sialylation by Sfß4GalT/ST6 cells. N-glycans were isolated from the GST-SfManI produced by Sfß4GalT/ST6 cells cultured in serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine and analyzed by HPAEC-PAD. The major processed N-glycans produced under both conditions were terminally galactosylated structures (peak 1 in Figures 4A,B). However, these cells also produced an extremely minor subpopulation of sialylated N-glycans under both culture conditions (peak 2 in Figures 4A,B). In each case, the presence of terminal sialic acids on the N-glycans in peak 2 was confirmed by neuraminidase treatment, which eliminated this peak and produced two new peaks, one coeluting with peak 1 and the other coeluting with standard N-acetylneuraminic acid (data not shown). The presence of sialic acids on the GST-SfManI produced under both culture conditions was also confirmed by SNA lectin blotting assays (data not shown). Together, these results indicated that N-acetylneuraminic acid and N-acetylmannosamine can support glycoprotein sialylation by Sfß4GalT/ST6 cells but to a much lower degree than fetal bovine serum or fetuin.
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Discussion |
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Initially, we found that Sfß4GalT/ST6 cells produced sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. We also found that the serum factor(s) supporting N-glycoprotein sialylation by these cells has a relatively high molecular weight. One specific high-molecular-weight serum factor that supported N-glycoprotein sialylation was the sialoglycoprotein fetuin, and the relevant moiety was found to be its complex, terminally sialylated N-glycans. Together, these results provided the first evidence that Sfß4GalT/ST6 cells have a salvage pathway for the acquisition of sialic acids from extracellular sialoglycoconjugates, such as fetuin. It is possible that fetuin is preferentially utilized by the salvage pathway in these cells. However, our results did not eliminate the possibility that these cells could also acquire sialic acids from other glycoproteins and/or other classes of large glycoconjugates, such as glycolipids. Finally, the results presented were not unique to Sfß4GalT/ST6 cells because many of these same results were obtained in parallel studies with SfSWT-1 cells (data not shown) (Hollister et al., 2002). This was no surprise because both of these transgenic insect cell lines were derived from the same parental cell line, Sf9.
It is reasonable to speculate that the Sf9 cell sialic acid salvaging pathway involves endocytosis of sialoglycoconjugates from the extracellular milieu, their desialylation by lysosomal sialidases, and the movement of free sialic acids into the cytoplasm, by analogy to the mammalian pathway (Ferwerda et al., 1981; Mendla et al., 1988
; Chigorno et al., 1996
). The present study provided no direct evidence to support these specific details. But we have previously documented the presence of sialidase activity in Sf9 cells (Licari et al., 1993
), and the present study justifies and directs future studies designed to more directly examine the specific steps in this pathway.
The present study also showed that N-acetylneuraminic acid supported glycoprotein sialylation by Sfß4GalT/ST6 cells but at significantly lower levels than serum or fetuin. This finding indicated that Sf9 cells can convert free sialic acids from extracellular sources to CMPsialic acid because this nucleotide sugar is the only known donor substrate for the rat 2,6-sialyltransferase catalyzing N-glycan sialylation in these cells (Comb, 1966
; Weinstein et al., 1987
). During the course of this study, we tried to detect CMPsialic acid in Sf9 cells cultured in the presence of fetal bovine serum using a standard extraction method and HPAEC-PAD analysis. However, we were unable to detect any CMPsialic acid in these cells, despite the fact that we could detect large amounts of this nucleotide sugar in positive controls (data not shown; see accompanying article, Aumiller et al., 2003
). We concluded that the intracellular pool of CMPsialic acid is below the detection limits of this method. Assuming that Sf9 cells can indeed convert free sialic acid to CMPsialic acid for de novo glycoprotein sialylation, they also must have a nucleotide sugar transporter that can transport CMPsialic acid into the Golgi apparatus. Two putative CMPsialic acid transporter genes have been identified in the Drosophila genome (Ferraz et al., 1999
; Kim et al., 2002
), but one was experimentally shown to be a UDP-galactose/UDP-N-acetylgalactosamine transporter (Aumiller and Jarvis, 2002
; Segawa et al., 2002
) and the other has not yet been tested. Thus, although the ability of transgenic Sf9 cell derivatives to produce sialylated glycoproteins indicates that they must be able to produce and transport CMPsialic acid, there remains no direct evidence to date for either capability in any insect cell system.
The present study also showed that N-acetylmannosamine supported extremely low levels of glycoprotein sialylation by Sfß4GalT/ST6 cells. This finding suggests that Sf9 cells have endogenous N-acetylmannosamine kinase and sialic acid synthase activities, which are required to convert this precursor to N-acetylneuraminic acid (Ghosh and Roseman, 1961; Roseman et al., 1961
; Watson et al., 1966
). This conclusion is consistent with the finding that expression of D. melanogaster (Kim et al., 2002
) and human (Lawrence et al., 2000
) sialic acid synthases in Sf9 cells led to the production of N-acetylneuraminic acid when these cells were cultured in the presence of N-acetylmannosamine. This conclusion is also consistent with the detection of N-acetylmannosamine kinase activity in uninfected Sf9 cell lysates (Effertz et al., 1999
) and the presence of a gene in Drosophila with similarity to the kinase domain of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (Angata and Varki, 2000
; Kim et al., 2002
).
In the context of a putative sialic acid salvaging pathway, there are plausible reasons why N-acetylneuraminic acid and N-acetylmannosamine supported de novo glycoprotein sialylation by Sfß4GalT/ST6 cells to significantly lower degrees than serum, dialyzed serum, or fetuin. The negative charge on free N-acetylneuraminic acid would be expected to prevent it from efficiently crossing the plasma membrane of Sf9 cells, and it might only be taken up inefficiently by a nonspecific pinocytotic mechanism. N-acetylmannosamine should enter Sf9 cells more readily, but this compound might be inefficiently converted to N-acetylneuraminic acid by limited amounts of sialic acid synthase activity in these cells. This latter possibility is supported by the results of the accompanying study, in which SfSWT-1 cells were engineered to express mammalian sialic acid synthase and CMPsialic acid synthetase genes, because this led to high levels CMPsialic acid production and more efficient sialylation of newly synthesized recombinant glycoproteins. Despite the technological success of this latter endeavor, it will be interesting and important in the future to elucidate the details of the sialic acid salvaging pathway in Sf9 cells and their transgenic derivatives.
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Materials and methods |
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Disialylated biantennary and trisialylated triantennary N-glycans were prepared from a commercial source of bovine fetuin. Briefly, bovine fetuin was deglycosylated and the N-glycans were isolated by preparative HPAEC-PAD. Eluted products were desalted using graphitized carbon cartridges, and their purity was verified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Streptococcus pneumoniae ß-galactosidase and Arthrobacter urefaciens neuraminidase were purchased from Calbiochem (La Jolla, CA).
Cells and viruses
Sfß4GalT/ST6 is a transgenic insect cell line that was described previously (Hollister and Jarvis, 2001). Sfß4GalT/ST6 cells were routinely maintained as shake flask cultures in HyQ SFX-INSECT protein-free medium. AcGST-SfManI is a previously described recombinant baculovirus that was used to express GST-SfManI under the control of the viral polyhedrin promoter (Kawar et al., 2000
).
Expression and purification of recombinant GST-SfManI
A shake flask culture of Sfß4GalT/ST6 cells maintained in serum-free culture medium was used to seed fresh 50 ml cultures in the same serum-free medium supplemented with nothing, 10% fetal bovine serum (undialyzed or dialyzed in 1000 or 50,000 molecular weight cutoff membranes, as described), 1 mg/ml fetuin, 1 mg/ml asialofetuin, 10 mM N-acetylmannosamine, or 5 mM N-acetylneuraminic acid. The cells were passaged twice in the appropriate medium and then seeded into fresh flasks, allowed to reach middle log phase, and infected with AcGST-SfManI at a multiplicity of 5 plaque-forming units per cell. After a 2 h adsorption period, the virus was removed by low-speed centrifugation and the cells were washed twice with serum-free medium before being resuspended in their original culture media and returned to their flasks.
At 72 h postinfection, each culture was harvested, and cell-free supernatants were prepared by low-speed centrifugation. The progeny virus particles produced during infection were removed by ultracentrifugation at 20,000 x g for 30 min at 4°C in a Beckman Model XL-100 ultracentrifuge and a Beckman Ti-45 rotor. These cell and virus-free supernatants were harvested, diluted 10-fold with glutathione column binding buffer [25 mM TrisHCl, pH 8.0; 250 mM NaCl; 1.5% (v/v) Triton X-100], and then 2 ml of a 75% (w/v) slurry of GSH-Sepharose were added to each diluted supernatant. The soluble GST-SfManI was allowed to bind to the resin for 2 h at room temperature with gentle agitation; then the bound GST-SfManI was collected by gravity flow through 10-ml disposable columns fitted with a plastic frit. The GSH-Sepharose beads retained in the columns were washed with 50 ml glutathione column binding buffer, then 50 ml glycosidase buffer (5 mM NaH2PO4, pH 7.5). Finally, the bound GST-SfManI was eluted using glycosidase buffer supplemented with 10 mM glutathione. The eluted material was dialyzed against glycosidase buffer with no glutathione, and then total protein concentrations were determined using a commercial modified Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. The purity of each GST-SfManI preparation was verified by analyzing 5-µg samples by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Laemmli, 1970) with Coomassie blue staining.
For the production of GST-SfManI in the presence of the N-glycans isolated from fetuin, 75-cm2 tissue culture flasks (Corning Glass, Corning, NY) were seeded with Sfß4GalT/ST6 cells that had been maintained in serum-free medium. After the cells had attached, the growth medium was removed and replaced with fresh serum-free medium supplemented with nothing, 1 mg/ml fetuin, 125 µg/ml of the disialylated biantennary glycan, or 125 µg/ml of the trisialylated triantennary glycan. The cells were incubated for another 24 h, then the media were removed and the cells were infected with AcGST-SfManI at multiplicity of infection of 5 pfu/cell. After a 2-h adsorption period, the cells were washed three times with serum-free medium, the original media were returned to each flask, and GST-SfManI was affinity purified from the cell and virus-free supernatants at 72 h postinfection, as described.
N-glycan analyses
Lectin blotting and immunoblotting assays were performed on equivalent amounts of GST-SfManI isolated from Sfß4GalT/ST6 cells cultured under various conditions, as previously described (Hollister and Jarvis, 2001). For the HPAEC-PAD analyses, N-glycans were prepared by treating 250500 µg of each purified GST-SfManI preparation with peptide:N-glycosidase F for 12 h at 37°C in glycosidase buffer. The released N-glycans were isolated on graphitized carbon cartridges, as previously described (Packer et al., 1998
) then analyzed using a Dionex Bio LC system (Dionex, Sunnyvale, CA) equipped with a 0.4 x 25 cm CarboPac PA 100 column and a pulsed amperometric detector (Hardy and Townsend, 1988
; Hermentin et al., 1992
). The PA100 column was equilibrated in 96.7% solvent A (0.15 M NaOH) and 3.3% solvent B (0.15 M NaOH; 0.6 M NaOAc) prior to sample injections. The elution profile included an initial 10-min isocratic run with the equilibration solvent, a 30-min linear gradient of 3.320% solvent B, a 10-min linear gradient of 2030% solvent B, and then a 5-min isocratic run with 100% solvent B, all at a flow rate of 1 ml/min. To digest individual N-glycan species with exoglycosidases, the relevant glycans were resolved by HPAEC-PAD, as described, collected as they eluted from the column, and desalted using graphitized carbon. The glycans were then resuspended in glycosidase buffer and treated with no enzyme, S. pneumoniae ß-galactosidase or A. urefaciens neuraminidase, using conditions recommended by the manufacturer. The reaction products were then directly analyzed by HPAEC-PAD using the same conditions described.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: dljarvis{at}uwyo.edu
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Abbreviations |
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References |
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