Evidence for a sialic acid salvaging pathway in lepidopteran insect cells

Jason Hollister2, Harald Conradt3 and Donald L. Jarvis1,2

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
We previously described a transgenic insect cell line, Sfß4GalT/ST6, that expresses mammalian ß-1,4-galactosyltransferase and {alpha}2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP–sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfß4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfß4GalT/ST6 cells is relatively large—it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfß4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfß4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfß4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfß4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP–sialic acid.

Key words: baculovirus expression system / glycoprotein biosynthesis / insect cells / sialic acids / sialylation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
N-glycosylation is a common and extremely important protein modification. Insect expression systems are often used to produce recombinant glycoproteins for various biomedical applications because they can N-glycosylate newly synthesized glycoproteins (reviewed by Luckow and Summers, 1988Go; O'Reilly et al., 1992Go; Jarvis, 1993Go, 1997Go; Pfeifer, 1998Go). However, these systems generally fail to produce recombinant mammalian glycoproteins with authentic N-glycans. In fact, the major processed N-glycans linked to recombinant glycoproteins produced by insect-based expression systems is Man3GlcNAc2. The structure of this glycan reflects the dominant N-glycosylation pathway in the insect cell lines, such as Sf9 (Summers and Smith, 1987Go), typically used to produce recombinant glycoproteins (reviewed by Marz et al., 1995Go; Altmann et al., 1999Go; Marchal et al., 2001Go). Several years ago, we began to address this problem by creating transgenic Sf9 cell derivatives with extended N-glycan processing capabilities. One of these cell lines, designated Sfß4GalT/ST6, was engineered to constitutively express mammalian ß1,4-galactosyltransferase and {alpha}2,6-sialyltransferase genes (Hollister and Jarvis 2001Go). Extensive structural characterization of the N-glycans isolated from a recombinant glycoprotein produced by these cells revealed that the {alpha}1,3 arm of the trimannosyl core was elongated by the addition of N-acetylglucosamine, galactose, and sialic acid (Hollister and Jarvis, 2001Go; Hollister et al., 2002Go). The presence of significant amounts of terminal sialic acids on these insect-derived glycoproteins was somewhat surprising because previous studies had shown that Sf9 cells have only extremely low amounts of sialic acid (Lawrence et al., 2000Go) and no detectable CMP–sialic acid (Hooker et al., 1999Go; Tomiya et al., 2001Go). Furthermore, Sfß4GalT/ST6 cells had not been engineered in any way to produce sialic acid or CMP–sialic acid. This raised an important and interesting question: How do Sfß4GalT/ST6 cells acquire the sialic acids needed for glycoprotein sialylation?

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, 1982Go). This pathway involves phosphorylation of N-acetylmannosamine by N-acetylmannosamine kinase to produce N-acetylmannosamine-6-phosphate (Ghosh and Roseman, 1961Go). This intermediate is subsequently condensed with phosphoenolpyruvate by N-acetylneuraminyl-9-phosphate synthase to produce N-acetylneuraminyl-9-phosphate (Roseman et al., 1961Go; Watson et al., 1966Go). This intermediate is then dephosphorylated by a specific phosphatase to produce N-acetylneuraminic acid, the most common form of sialic acid (Warren and Felsenfeld, 1961Go). Finally, this compound is activated by CMP-N-acetylneuraminic acid synthetase to produce CMP-N-acetylneuraminic acid (Kean, 1970Go). 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., 1981Go; Mendla et al., 1988Go; Chigorno et al., 1996Go). 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 CMP–sialic acid and transport into the Golgi apparatus, where it serves as the only known donor substrate for the sialyltransferases (Comb, 1966Go).

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, 2000Go; Kim et al., 2002Go), the low levels of sialic acid (Lawrence et al., 2000Go) and absence of CMP–sialic acid in Sf9 cells (Hooker et al., 1999Go; Tomiya et al., 2001Go), together with their general inability to produce sialylated N-glycans (reviewed by Marchal et al., 2001Go), 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 hypothesis—we 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 CMP–sialic acid.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sfß4GalT/ST6 cells require fetal bovine serum for glycoprotein sialylation
Initially, we examined fetal bovine serum as a potential source of sialic acids for de novo glycoprotein sialylation by Sfß4GalT/ST6 cells. A recombinant baculovirus was used to express a soluble, glutathione-S-transferase-tagged form of a Spodoptera frugiperda (Sf) class I {alpha}-mannosidase (GST-SfManI) (Kawar et al., 2000Go) in Sfß4GalT/ST6 cells cultured in the presence or absence of serum. The GST-SfManI was affinity-purified, and its purity was verified by gel electrophoresis with Coomassie blue staining (data not shown). Subsequently, the N-glycans were released, recovered, and analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The results showed that the GST-SfManI produced in the presence of serum contained two major processed N-glycans (peaks 1 and 2 in Figure 1A). Based on previous HPAEC-PAD and mass spectrometry analyses, we knew that peak 1 represented a monogalactosylated N-glycan, whereas peak 2 represented this same structure with a terminal sialic acid (Hollister et al., 2002Go). To confirm this, the N-glycans in peaks 1 and 2 were isolated by preparative HPAEC-PAD, digested with either ß-galactosidase or neuraminidase, and then rechromatographed. Digestion of the N-glycan from peak 1 with ß-galactosidase eliminated this peak and produced a new peak (peak 3) that eluted earlier in the gradient, as expected from removal of a terminal galactose residue (Figure 1B). Neuraminidase digestion of the N-glycan from peak 2 eliminated this peak and produced two new peaks, one coeluting with peak 1 and the other coeluting with standard N-acetylneuraminic acid (peak 4 in Figure 1C). These results confirmed our previous finding that Sfß4GalT/ST6 cells grown in the presence of serum can produce terminally sialylated N-glycans. In contrast, the only processed N-glycan detected using GST-SfManI produced by Sfß4GalT/ST6 cells cultured in the absence of serum was the terminally galactosylated structure (peak 1 in Figure 1D). In addition, the GST-SfManI produced under these latter conditions failed to react with Sambucus nigra agglutinin (SNA) in lectin blotting assays (data not shown), indicating that it contained no detectable terminal sialic acid. These results revealed that Sfß4GalT/ST6 cells could produce terminally sialylated N-glycans in the presence but not in the absence of fetal bovine serum.



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Fig. 1. Fetal bovine serum supports glycoprotein sialylation by Sfß4GalT/ST6 cells. This figure shows the HPAEC-PAD profiles of the N-glycans isolated from GST-SfManI produced by Sfß4GalT/ST6 cells cultured in the presence (AC) or absence (D) of fetal bovine serum. B and C show the profiles obtained when the N-glycans in peak 1 (A) were treated with ß-galactosidase and when those in peak 2 (A) were treated with neuraminidase, respectively. The arrows and symbols identify the N-glycan species and free sialic acid observed in these analyses.

 
The serum factor(s) supporting glycoprotein sialylation is large
Among other possibilities, the serum factor(s) supporting glycoprotein sialylation by Sfß4GalT/ST6 cells could have been a low-molecular-weight compound, such as a sialic acid precursor or sialic acid itself, or a high-molecular-weight compound, such as a glycoconjugate with covalently bound sialic acids. Thus, we used dialysis to examine the relative size of the serum component(s) supporting glycoprotein sialylation. Sfß4GalT/ST6 cells were cultured for several passages in medium supplemented with serum that had been extensively dialyzed in either low- (1000) or high- (50,000) molecular-weight cutoff membranes. These cells were then infected with the baculovirus encoding GST-SfManI, the recombinant glycoprotein was affinity-purified, and the N-glycans were released and analyzed by HPAEC-PAD. The results showed that sialylated N-glycans were released from the GST-SfManI produced by baculovirus-infected Sfß4GalT/ST6 cells cultured in either medium (data not shown). Thus, the serum factor(s) supporting glycoprotein sialylation by Sfß4GalT/ST6 cells was not removed by extensive dialysis in low- or high- molecular-weight cutoff membranes, indicating that this factor(s) has a relatively high molecular weight.

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 CMP–sialic 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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Fig. 2. Purified sialoglycoproteins support glycoprotein sialylation by Sfß4GalT/ST6 cells. This figure shows the HPAEC-PAD profiles of the N-glycans isolated from GST-SfManI produced by Sfß4GalT/ST6 cells cultured in the presence of a serum-free medium supplemented with purified fetuin (A) or asialofetuin (B). The arrows and symbols identify the N-glycan species observed in these analyses.

 
Sialylated N-glycans support glycoprotein sialylation by Sfß4GalT/ST6 cells
The ability of purified fetuin, but not asialofetuin, to support glycoprotein sialylation by Sfß4GalT/ST6 cells suggested that these cells have a sialic acid salvaging pathway that can specifically utilize at least one extracellular, terminally sialylated glycoprotein. An alternative possibility was that fetuin induced glycoprotein sialylation by functioning as a growth factor that activated otherwise silent functions in these cells. To distinguish between these possibilities, we isolated the terminally sialylated N-glycans from fetuin and examined their ability to support glycoprotein sialylation by Sfß4GalT/ST6 cells in the absence of the polypeptide backbone. These disialylated biantennary and trisialylated triantennary N-glycans were added to serum-free medium in concentrations equal to those associated with 1 mg/ml fetuin. As before, the Sfß4GalT/ST6 cells were cultured in these media and infected with the recombinant baculovirus, and GST-SfManI was used as the reporter glycoprotein. However, because we could prepare only limited amounts of the purified glycans for these studies, these experiments were scaled down and lectin blotting was used instead of HPAEC-PAD for the N-glycan analyses. The recombinant glycoprotein produced under various culture conditions was affinity-purified, resolved by gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and then probed with either anti-GST, to show that equal amounts were loaded into each lane, or SNA, to determine if the recombinant glycoprotein contained terminally sialylated N-glycans (Figure 3).



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Fig. 3. N-glycans from fetuin support glycoprotein sialylation by Sfß4GalT/ST6 cells. This figure shows immunoblotting (A) and SNA lectin blotting (B) analyses of the GST-SfManI produced by Sfß4GalT/ST6 cells cultured in a serum-free medium supplemented with disialylated biantennary (lane 3) or trisialylated triantennary (lane 4) N-glycans isolated from fetuin. The GST-SfManI from Sfß4GalT/ST6 cells cultured in serum-free medium supplemented with nothing (lane 2) or fetuin (lane 1) were included as positive and negative controls. The position of the 97-kDa protein standard in each blot is indicated by the arrows.

 
The results of these analyses showed that SNA bound to the GST-SfManI produced by cells cultured in the presence of either N-glycan (lanes 3–4). But significantly more SNA bound to an equivalent amount of GST-SfManI from Sfß4GalT/ST6 cells cultured in serum-free medium supplemented with fetuin (lane 1). Thus, the complex N-glycans isolated from fetuin supported sialylation of GST-SfManI by these cells but to a much lower degree than the intact sialoglycoprotein. The absence of any SNA binding to GST-SfManI from Sfß4GalT/ST6 cells cultured in serum-free medium alone indicated that SNA binding was specific for terminal sialic acids. Extensive additional evidence for the specificity of our lectin blotting assays has been presented in previous studies (Jarvis and Finn, 1995Go, 1996Go; Hollister et al., 1998Go, 2002Go; Hollister and Jarvis, 2001Go; Jarvis et al., 2001Go; Seo et al., 2001Go) and is also provided in the accompanying study (Aumiller et al., 2003).

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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Fig. 4. Sialic acid or N-acetylmannosamine supports glycoprotein sialylation by Sfß4GalT/ST6 cells. This figure shows the HPAEC-PAD profiles of the N-glycans isolated from GST-SfManI produced by Sfß4GalT/ST6 cells cultured in the presence of a serum-free medium supplemented with N-acetylneuraminic acid (A) or N-acetylmannosamine (B). The arrows and symbols identify the N-glycan species observed in these analyses.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Sf9 is a widely utilized lepidopteran insect cell line that contains only low levels of sialic acid (Lawrence et al., 2000Go), no detectable CMP–sialic acid (Hooker et al., 1999Go; Tomiya et al., 2001Go), and no detectable sialyltransferase activity (Stollar et al., 1976Go; Butters et al., 1981Go; Hooker et al., 1999Go; Hollister and Jarvis, 2001Go). Accordingly, it does not usually produce sialylated N-glycans. Historically, the inability of these cells to produce sialylated glycoproteins limited their utility as hosts for baculovirus-mediated recombinant glycoprotein production (reviewed by Jarvis, 1997Go; Altmann et al., 1999Go; Marchal et al., 2001Go). Therefore, several years ago, we initiated efforts to extend the N-glycosylation pathway in these cells by genetic transformation. The incorporation of mammalian glycosyltransferase genes into these cells yielded transgenic derivatives, Sfß4GalT/ST6 and SfSWT-1, that can constitutively express these genes and produce sialylated recombinant glycoproteins (Hollister and Jarvis, 2001Go; Hollister et al., 2002Go). However, these results were somewhat unexpected because we made no attempt to increase sialic acid or CMP–sialic acid levels in these cells. This led us to consider that Sf9 cells might have a salvage pathway that would allow them to acquire sialic acids from extracellular glycoconjugates, as previously described in mammalian systems (Ferwerda et al., 1981Go; Mendla et al., 1988Go; Chigorno et al., 1996Go). The purpose of the present study was to begin to test this hypothesis.

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., 2002Go). 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., 1981Go; Mendla et al., 1988Go; Chigorno et al., 1996Go). 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., 1993Go), 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 CMP–sialic acid because this nucleotide sugar is the only known donor substrate for the rat {alpha}2,6-sialyltransferase catalyzing N-glycan sialylation in these cells (Comb, 1966Go; Weinstein et al., 1987Go). During the course of this study, we tried to detect CMP–sialic 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 CMP–sialic 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., 2003Go). We concluded that the intracellular pool of CMP–sialic acid is below the detection limits of this method. Assuming that Sf9 cells can indeed convert free sialic acid to CMP–sialic acid for de novo glycoprotein sialylation, they also must have a nucleotide sugar transporter that can transport CMP–sialic acid into the Golgi apparatus. Two putative CMP–sialic acid transporter genes have been identified in the Drosophila genome (Ferraz et al., 1999Go; Kim et al., 2002Go), but one was experimentally shown to be a UDP-galactose/UDP-N-acetylgalactosamine transporter (Aumiller and Jarvis, 2002Go; Segawa et al., 2002Go) 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 CMP–sialic 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, 1961Go; Roseman et al., 1961Go; Watson et al., 1966Go). This conclusion is consistent with the finding that expression of D. melanogaster (Kim et al., 2002Go) and human (Lawrence et al., 2000Go) 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., 1999Go) 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, 2000Go; Kim et al., 2002Go).

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 CMP–sialic acid synthetase genes, because this led to high levels CMP–sialic 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
N-acetylmannosamine and N-acetylneuraminic acid were purchased from Pfanstiehl Laboratories (Waukegan, IL). Bovine fetuin and asialofetuin were purchased from Sigma (St. Louis, MO). HyQSFX-INSECT serum-free insect cell culture medium and fetal bovine serum were purchased from HyClone (Logan, UT). Dialyzed fetal bovine serum was prepared by placing 50-ml aliquots of the commercial serum into sterile 1000 or 50,000 molecular weight cutoff membranes (Pierce, Rockford, IL) and dialyzing for 24 h at 4°C against 2 L SFX-INSECT medium. The dialysate was then replaced with 2 L fresh medium, dialysis was continued for another 24 h, and the serum was harvested under aseptic conditions. GSH-Sepharose was purchased from Amersham-Pharmacia Biotech (Piscataway, NJ). Graphitized carbon cartridges were purchased from Alltech (Deerfield, IL). The oligosaccharide standards (NA2F and A1F) used for HPAEC-PAD analyses were purchased from Glyko (Novato, CA).

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, 2001Go). 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., 2000Go).

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 Tris–HCl, 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 sulfate–polyacrylamide gel electrophoresis (Laemmli, 1970Go) 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, 2001Go). For the HPAEC-PAD analyses, N-glycans were prepared by treating 250–500 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., 1998Go) 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, 1988Go; Hermentin et al., 1992Go). 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 3.3–20% solvent B, a 10-min linear gradient of 20–30% 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.


    Acknowledgements
 
This work was supported by NIH Grant no. GM49734.

1 To whom correspondence should be addressed; e-mail: dljarvis{at}uwyo.edu Back


    Abbreviations
 
GST, glutathione-S-transferase; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; PNGase-F, peptide:N-glycosidase F; SNA, Sambucus nigra agglutinin


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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