Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase genes

Jason R. Hollister and Donald L. Jarvis1

Department of Molecular Biology, University of Wyoming, P.O. Box 3944, Laramie, WY 82071–3944, USA

Received on August 31, 1999; revised on September 1, 2000; accepted on September 6, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Recombinant mammalian glycoproteins produced by the baculovirus–insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.

Key words: N-glycosylation/insect cells/baculovirus expression system/genetic engineering/cell transformation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Many higher eukaryotic glycoproteins have glycans with terminal sialic acids, which directly or indirectly influence their functions (Jenkins and Curling, 1994Go; Weigel, 1994Go). In higher eukaryotes, sialylation of newly synthesized N-linked glycoproteins is the last step in an elaborate biosynthetic pathway (Kornfeld and Kornfeld, 1985Go). This pathway begins with the cotranslational transfer of oligosaccharide precursors to nascent proteins. The precursors are subsequently trimmed and elongated by enzymes localized in the endoplasmic reticulum and Golgi apparatus. A key intermediate is GlcNAcMan3GlcNAc2-N-Asn, which can be sequentially elongated by various glycosyltransferases to produce complex N-glycans containing terminal sialic acids.

Insect cells have an analogous N-glycosylation pathway, which also can transfer oligosaccharide precursors to nascent proteins and convert them to GlcNAcMan3GlcNAc2-N-Asn (Marz et al., 1995Go; Jarvis, 1997Go). However, insect cells generally appear to have extremely low levels of N-acetylglucosaminyltransferase II and galactosyltransferase activities and no detectable sialyltransferase activities (Stollar et al., 1976Go; Butters et al., 1981Go; Altmann et al., 1993Go; van Die et al., 1996Go; Hooker et al., 1999Go). Furthermore, some insect cells have an N-acetylglucosaminidase, which removes the terminal GlcNAc residue from GlcNAcMan3GlcNAc2-N-Asn and eliminates the intermediate required for complex N-glycan production (Licari et al., 1993Go; Altmann et al., 1995Go; Wagner et al., 1996Go; Marchal et al., 1999Go). Finally, it has been reported that there is no detectable CMP-sialic acid, which is the donor substrate required for sialoglycoprotein synthesis, in one insect cell line (Hooker et al., 1999Go). Consequently, the major processed N-glycan typically produced by insect cells is the paucimannose structure, Man3GlcNAc2(±Fuc)-N-Asn.

This fundamental difference between the N-glycosylation pathways of insects and higher eukaryotes has important implications for biotechnology, as insect cells are widely used as hosts for baculovirus-mediated production of recombinant glycoproteins (Summers and Smith, 1987Go; O'Reilly et al., 1992Go; Jarvis, 1997Go). Recombinant mammalian glycoproteins produced using the baculovirus-insect cell expression system usually have Man3GlcNAc2(±Fuc)-N-Asn in place of complex, terminally sialylated glycans found on the native product. Depending upon the recombinant glycoprotein being produced and its intended application, this structural difference can be a serious problem (Jenkins and Curling, 1994Go; Weigel, 1994Go).

In this study, we engineered the baculovirus-insect cell expression system for sialoglycoprotein production by genetically transforming an insect cell line with mammalian genes encoding {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase. These modified insect cells had high levels of both transferase activities and served well as hosts for baculovirus infection. Surprisingly, these cells also produced sialylated foreign N-glycoproteins, despite the fact that we made no effort to engineer pathways for CMP-sialic acid production or transport.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Incorporation of mammalian glycosyltransferase genes into the baculovirus expression system
A bovine {beta}1,4-galactosyltransferase cDNA (Shaper et al., 1986Go) was subcloned into an immediate early expression plasmid (Jarvis et al., 1996Go), and the resulting construct was used to isolate a stably-transformed Sf9 cell variant designated Sf{beta}4GalT (Hollister et al., 1998Go). These cells constitutively expressed integrated copies of the {beta}1,4-galactosyltransferase cDNA and produced high levels of {beta}1,4-galactosyltransferase activity (Figure 1A). Sf{beta}4GalT cells had higher levels of {beta}1,4-galactosyltransferase activity than COS cells (Figure 1A; Gluzman, 1981Go) or a hybridoma cell line (data not shown). Sf{beta}4GalT cells also produced foreign glycoproteins with terminally galactosylated N-glycans (Hollister et al., 1998Go), which are acceptor substrates for sialyltransferases (Hollis et al., 1989Go). Thus, these cells were an excellent target for the subsequent incorporation of an {alpha}2,6-sialyltransferase gene.



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Fig. 1. Glycosyltransferase activities in parental and stably-transformed insect cells. Cell lysates were prepared from Sf9, Sf{beta}4GalT, Sf{beta}4GalT/ST6, or COS cells and assayed for the presence of galactosyltransferase (A) and sialyltransferase (B) activities as described in Materials and methods. Activities were reported as the average cpm of tritiated galactose or sialic acid transferred to the acceptor substrate/1 x 106 cells/h.

 
This was accomplished by subcloning a rat {alpha}2,6-sialyltransferase cDNA (Weinstein et al., 1987Go) into an immediate early expression plasmid and using this construct to isolate a stably-transformed variant of Sf{beta}4GalT cells, designated Sf{beta}4GalT/ST6. These cells constitutively expressed integrated copies of the {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase cDNAs and produced higher levels of both activities than COS cells (Figure 1A,B) or a hybridoma cell line (data not shown). In contrast, untransformed Sf9 cells produced neither activity, as there was no significant difference between the amounts of radioactive sugars transferred by unboiled or boiled Sf9 cell extracts (Figure 1). The glycosyltransferase activity levels in both Sf{beta}4GalT and Sf{beta}4GalT/ST6 cells fluctuated during routine passage in the laboratory, which is typical of stably-transformed Sf9 cell lines (Jarvis et al., 1990Go). However, we have observed no significant loss of either activity during routine passage of these cells since they were first isolated in December of 1998 (data not shown). Thus, Sf{beta}4GalT/ST6 is a stably transformed derivative of Sf9 cells.

Sf{beta}4GalT/ST6 cells as hosts for baculovirus infection
The baculovirus–insect cell expression system is a binary system consisting of a recombinant baculovirus vector and its host, an insect cell. The virus delivers the gene encoding a glycoprotein of interest to the cell, then the gene is expressed by virus-encoded transcription factors and the protein is translated and glycosylated by host cell machinery during viral infection. Accordingly, Sf{beta}4GalT/ST6 cells would be useful for foreign sialoglycoprotein production only if they could support baculovirus infection while continuing to provide galactosyltransferase and sialyltransferase activities. One-step viral growth curves showed that there were no significant differences in baculovirus replication when Sf9, Sf{beta}4GalT, or Sf{beta}4GalT/ST6 cells were used as the host (Figure 2). Furthermore, Sf{beta}4GalT/ST6 cells continued to provide both galactosyltransferase (Figure 3A) and sialyltransferase (Figure 3B) activities throughout the course of infection. Both activities were stimulated early in infection and gradually declined, but both were higher in infected than uninfected cells, even at 72 h after infection. Sialyltransferase activity declined with increasing cell density in uninfected Sf{beta}4GalT/ST6 cells, for reasons that remain to be determined.



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Fig. 2. Baculovirus growth in Sf{beta}4GalT/ST6 cells. Sf9 (circles), Sf{beta}4GalT (squares), and Sf{beta}4GalT/ST6 (triangles) cells were infected with AcMNPV and samples were harvested at various times after infection. Infectious progeny were measured using a standard AcMNPV plaque assay, as described in Materials and methods.

 


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Fig. 3. Glycosyltransferase activities in baculovirus-infected Sf{beta}4GalT/ST6 cells. Sf9 (circles) and Sf{beta}4GalT/ST6 (squares) cells were either mock-infected (open symbols) or AcMNPV-infected (solid symbols) and samples containing 2 x 106 cells were harvested at various times after infection. Cell extracts were prepared from each sample and normalized aliquots were assayed for galactosyltransferase (A) or sialyltransferase (B) activities as described in Materials and methods. Activities were reported as the average cpm of tritiated galactose or sialic acid transferred to the acceptor substrate/1 x 106 cells/h.

 
Production of foreign sialoglycoproteins by baculovirus-infected Sfb4GalT/ST6 cells
The major baculovirus structural glycoprotein, gp64, is expressed during the late phase of infection. When produced by various insect cell lines, gp64 acquires N-glycans with no detectable galactose or sialic acid. However, when produced by COS cells, which have the requisite glycosyltransferase activities, gp64 acquires terminally sialylated N-glycans (Jarvis and Finn, 1995Go). Thus, we used gp64 as a reporter to determine if Sf{beta}4GalT/ST6 cells could produce a sialylated foreign glycoprotein during baculovirus infection. gp64 was isolated from the budded virus progeny produced by baculovirus-infected Sf9, Sf{beta}4GalT, or Sf{beta}4GalT/ST6 cells and analyzed by lectin blotting, as described in Materials and methods. The results showed that Sambucus nigra agglutinin (SNA), which specifically recognizes terminal sialic acids, bound only to the gp64 produced by Sf{beta}4GalT/ST6 cells (Figure 4A). Preincubation with a competing sugar blocked SNA binding, suggesting that SNA binding to gp64 was carbohydrate-specific (Figure 4B). Furthermore, pretreatment of gp64 with either peptide:N-glycosidase F (PNGase-F) or neuraminidase precluded SNA binding (Figure 5). These results strongly suggested that baculovirus-infected Sf{beta}4GalT/ST6 cells can produce a foreign sialoglycoprotein with terminally sialylated N-glycans.



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Fig. 4. Lectin blotting analysis of gp64 produced by parental or stably-transformed insect cells. gp64 was isolated from the progeny budded virus produced by AcMNPV-infected Sf9 (lanes 1), Sf{beta}4GalT (lanes 2), and Sf{beta}4GalT/ST6 (lanes 3) cells as described in Materials and methods. Samples were resolved by SDS-PAGE and transferred to Immobilon filters, which were then cut into three panels and probed with rabbit anti-gp64 (Ab), RCA, or SNA. The lectin blots were done either in the absence (A) or presence (B) of competing sugars. The arrows on the right indicate the positions of gp64 (gp64) and IgG heavy chain (IgG).

 


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Fig. 5. Glycosidase analysis of gp64 produced by parental or stably-transformed insect cells. gp64 was isolated from the progeny budded virus produced by baculovirus-infected Sf{beta}4GalT/ST6 cells as described in Materials and methods. gp64 was then treated with nothing (S), buffer alone (B), PNGase-F (P), or neuraminidase (N), as described in Materials and methods. Samples were resolved by SDS-PAGE, transferred to Immobilon filters, and strips were probed with rabbit anti-gp64 (Ab), RCA, or SNA. The arrows on the right indicate the positions of glycosylated gp64 (gp64) and deglycosylated gp64 (p64).

 
This interpretation was supported by structural analyses of the N-glycans isolated from recombinant GST-SfManI, a glutathione-S-transferase-tagged Sf9 cell class I {alpha}-mannosidase (Kawar et al., 1997Go, 2000), produced by Sf{beta}4GalT/ST6 cells. GST-SfManI was used instead of gp64 for these analyses because it was produced at higher levels, it could be effectively purified by glutathione affinity chromatography, it had only one N-glycan as compared to four on gp64 (Jarvis et al., 1998Go), and it could be efficiently deglycosylated under native conditions. Sf{beta}4GalT/ST6 cells were infected with the recombinant baculovirus, AcGST-SfManI, and GST-SfManI was isolated from the cell-free medium by glutathione affinity chromatography, as described in Materials and methods. SDS-PAGE and Coomassie blue staining showed that GST-SfManI was the only detectable protein eluted from the affinity column (Figure 6A). This protein was clearly identified by immunoblotting with an anti-GST antibody and reacted specifically with SNA, indicating that it had been sialylated in Sf{beta}4GalT/ST6 cells. Subsequently, samples of the purified GST-SfManI preparation were treated with PNGase-F alone or PNGase-F plus neuraminidase, and the N-glycans were recovered and analyzed by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). PNGase-F treatment produced two major peaks (peaks 1 and 2; Figure 6B), the second of which eluted late in the gradient, as expected of a sialylated N-glycan. Treatment with both PNGase-F and neuraminidase produced a larger peak 1, no peak 2, and a new peak (peak 3; Figure 6C). Standard Neu5Ac eluted at the same position as peak 3, relative to peak 1, when added to a GST-SfManI/PNGase-F/neuraminidase reaction mixture (data not shown). These results indicated that peak 2 represents a sialylated N-glycan, which yields a desialylated N-glycan (peak 1) plus a sialic acid (peak 3) upon neuraminidase treatment.




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Fig. 6. Lectin blotting and HPAEC-PAD analysis of GST-SfManI produced by stably-transformed insect cells. Sf{beta}4GalT/ST6 cells were infected with AcGST-SfManI and GST-SfManI was isolated by glutathione affinity chromatography, as described in Materials and methods. In (A), purified GST-SfManI was treated with buffer alone (lanes C) or neuraminidase (lanes N), the digests were resolved by SDS-PAGE, and proteins were transferred to nylon filters. The filters were stained with Coomassie blue (CB), anti-SfManI (Ab) (Kawar et al., 2000), or SNA that had been preincubated with buffer alone (SNA-) or a competing sugar (SNA+), as described in Materials and methods. In (B) and (C), purified GST-SfManI was treated with PNGase-F alone or with PNGase-F and neuraminidase, respectively, and the glycans were recovered and analyzed by HPAEC-PAD, as described in Materials and methods.

 
These data supported and extended our lectin blotting results and were consistent with the idea that the GST-SfManI produced by Sf{beta}4GalT/ST6 cells had acquired a sialylated N-glycan, but there also was an alternative interpretation. It was possible that the sialyltransferase produced by Sf{beta}4GalT/ST6 cells was cleaved, secreted, and the soluble form of this enzyme was able to sialylate GST-SfManI in the extracellular growth medium using CMP-sialic acid from the fetal bovine serum as the donor substrate. To address this possibility, GST-SfManI was isolated from baculovirus-infected Sf{beta}4GalT/ST6 cells cultured in growth medium supplemented with fetal bovine serum that had been extensively dialyzed to remove nucleotide sugars. This GST-SfManI preparation was then treated with PNGase-F or PNGase-F and neuraminidase and the glycans were recovered and analyzed by HPAEC-PAD. Treatment with PNGase-F alone produced essentially the same glycan profile (Figure 7A) as was obtained with GST-SfManI isolated from cells grown in nondialyzed serum (Figure 6B). In fact, the relative amount of sialylated glycan (peak 2) was larger with the GST-SfManI from cells grown in dialyzed serum. Again, neuraminidase eliminated peak 2 and produced a new peak (peak 3 in Figure 7B), which was located at the same position as peak 3 in Figure 6B and standard Neu5Ac in Figure 7C. In addition, two other new peaks (peaks 4 and 5), which were not prominent in the experiment shown in Figure 6, were observed between peaks 1 and 3 (Figure 7B). The origin of these two peaks is unclear. They might be nonsialylated glycans, as both were observed with PNGase-F alone (Figure 7A). Alternatively, peak 4 might represent an acetylated version of the sialic acid represented by peak 3, as it was eliminated and peak 3 was larger after treatment with 0.1 N NaOH for 30 min at 37°C (data not shown). In any case, the results of these analyses showed that GST-SfManI acquired a sialylated N-glycan whether Sf{beta}4GalT/ST6 cells were grown in nondialyzed or extensively dialyzed fetal bovine serum. This indicates that sialylation occurred intracellularly, within the confines of the modified N-glycan processing pathway of Sf{beta}4GalT/ST6 cells, not by the action of a soluble sialyltransferase and CMP-sialic acid in the extracellular medium.



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Fig. 7. HPAEC-PAD analysis of GST-SfManI produced by stably-transformed insect cells grown in dialyzed serum. Sf{beta}4GalT/ST6 cells cultured in HyQ Sfx-Insect medium containing extensively dialyzed fetal bovine serum were infected with AcGST-SfManI and GST-SfManI was isolated by glutathione affinity chromatography, as described in Materials and methods. The purified GST-SfManI preparation was treated with PNGase-F alone (A) or with PNGase-F and neuraminidase (B) and the glycans were recovered and analyzed by HPAEC-PAD, as described in Materials and methods. (C) shows the elution profiled for standard Neu5Ac under identical conditions.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The baculovirus–insect cell expression system is widely used for recombinant glycoprotein production because it is a eukaryotic system that can provide protein modifications, including glycosylation. But, a major limitation of this system is that it cannot produce structurally authentic sialoglycoproteins. One fundamental basis for this limitation is that insect cells lack adequate levels of the sialyltransferases and CMP-sialic acids needed for routine sialoglycoprotein production (Stollar et al., 1976Go; Butters et al., 1981Go; Hooker et al., 1999Go). Consequently, insect cells typically fail to convert N-glycan processing intermediates to complex, terminally sialylated end products and convert these intermediates to paucimannose structures, instead. This results in a structural difference between the N-glycans of recombinant glycoproteins and their native counterparts, which can adversely affect their secretion (Nagayama et al., 1998Go; McFarlane et al., 1999Go), enzymatic activities (Fast et al., 1993Go), ligand interactions (Janosi et al., 1999Go), and in vivo circulatory half-lives (Chitlaru et al., 1998Go). Another problem is that insect cells can add {alpha}1,3-linked fucosyl residues to the N-glycan core of recombinant glycoproteins, which can stimulate an allergic response in humans (Marz et al., 1995Go).

We addressed the first of these problems by genetically engineering an established insect cell line to create a new cell line with constitutively expressible mammalian glycosyltransferase genes. This new line, designated Sf{beta}4GalT/ST6, supported normal levels of baculovirus replication and produced both {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase activities during infection. Furthermore, unlike the unmodified parental insect cell line, Sf{beta}4GalT/ST6 cells produced foreign glycoproteins containing complex, terminally sialylated N-glycans. These results demonstrate that the baculovirus–insect cell expression system can be engineered for sialoglycoprotein production. Surprisingly, this was accomplished simply by increasing {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase activities, with no further genetic modifications. This finding provides new information on the fundamental nature of the N-glycosylation pathway in Sf9 cells. It shows that these insect cells can meet all the requirements for the action of these enzymes, including proper spatial and temporal positioning of both glycosyltransferases along the secretory pathway, production of nucleotide sugar donors, including both UDP-galactose and CMP-sialic acid, and transport of these donors to the appropriate subcellular locations (Kornfeld and Kornfeld, 1985Go). The presence of this infrastructure was unexpected from most structural data on insect cell-derived glycoproteins, which indicate that these cells cannot produce terminally sialylated N-glycans. Indeed, upon finding no CMP-sialic acid in Sf9 cells, Hooker and coworkers concluded that "genetic modification of N-glycan processing in these cells will be constrained by substrate availability to terminal galactosylation" (Hooker et al., 1999Go). Our results oppose this conclusion and were predictable from the previous observation that baculovirus-infected insect cells can produce one authentic recombinant sialoglycoprotein (Davidson et al., 1990Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Construction of immediate early expression plasmids
pIE1Hygro and pIE1ST6 are immediate early expression plasmids, which encode E. coli hygromycin phosphotransferase (Yates et al., 1985Go) or rat {alpha}2,6-sialyltransferase (Weinstein et al., 1987Go) under the control of a baculovirus ie1 promoter. The ie1 promoter provides constitutive foreign gene expression in uninfected insect cells and, therefore, immediate early expression plasmids can be used for stable transformation of established insect cell lines (Jarvis et al., 1990Go). The rat {alpha}2,6-sialyltransferase cDNA originally had a cysteine codon at position 123, but we used site-directed mutagenesis to change it to a tyrosine codon, as this form of the protein has higher activity (Ma et al., 1997Go). The immediate early expression plasmids used for this study were constructed by inserting the modified {alpha}2,6-sialyltransferase or hygromycin phosphotransferase genes into pIE1HR3 (Jarvis et al., 1996Go).

Cells and viruses
Sf9 (Vaughn et al., 1977Go), Sf{beta}4GalT (Hollister et al., 1998Go), and Sf{beta}4GalT/ST6 cells were routinely maintained as shake-flask cultures in TNM-FH medium containing 10% heat-inactivated fetal bovine serum (HyClone; Logan, UT) and 0.1% (w/v) pluronic F68, as described previously (Summers and Smith, 1987Go). This medium was designated complete TNM-FH. In addition, all three cell lines were maintained as shake-flask cultures in HyQ Sfx-Insect protein free medium (Hyclone). Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) strain E2 (Smith and Summers, 1978Go) was used as wild-type baculovirus, and AcGST-SfManI (Kawar et al., 2000Go) was a recombinant baculovirus used to express a soluble GST-SfManI fusion protein under the control of the polyhedrin promoter.

Sf{beta}4GalT/ST6 is a stably transformed Sf9 cell derivative that was produced by using a modification of an established procedure (Jarvis et al., 1990Go). Sf{beta}4GalT cells were cotransfected with a mixture of pIE1Hygro and pIE1ST6 using a calcium phosphate method (Summers and Smith, 1987Go), then the cells were allowed to recover for 1 day, treated with 1 mg of hygromycin (Sigma) per milliliter of TNM-FH medium, and hygromycin-resistant clones were isolated by limiting dilution in 96-well plates. After stepwise amplification into larger cultures, lysates were prepared from each hygromycin-resistant clone and assayed for both {beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase activities, as described below. The clone with the highest levels of both activities was designated Sf{beta}4GalT/ST6.

One-step growth curves
Sf9, Sf{beta}4GalT, and Sf{beta}4GalT/ST6 cells were pelleted by low-speed centrifugation, gently resuspended in complete TNM-FH, and infected with AcMNPV at a multiplicity of about 10 plaque forming units (pfu)/cell. The virus and cells were incubated on a rocking platform for 1 h at 28°C, then the cells were repelleted and washed three times with complete TNM-FH. The cells were resuspended at a density of 0.5 million cells/ml in a total of 100 ml of complete TNM-FH, transferred to 250 ml shake flasks, and samples of the growth media were harvested at various times post infection. Progeny virus production was measured by plaque assays on Sf9 cells as described previously (Summers and Smith, 1987Go).

{beta}1,4-galactosyltransferase and {alpha}2,6-sialyltransferase assays
Sf9, Sf{beta}4GalT, and Sf{beta}4GalT/ST6 cells were seeded at a density of 0.5 x 106 cells/ml into 250 ml shake flasks containing a total of 100 ml of complete TNM-FH. Once the cells reached mid-log phase, they were mock- or AcMNPV-infected at a multiplicity of 5 pfu/cell. After a 1 h adsorption period at 28°C, the cells were washed three times with complete TNM-FH and, at various times after infection, duplicate samples containing 2 x 106 cells were taken from each culture. The cells were pelleted by low speed centrifugation, washed with TBS, and lysed by freeze–thaw in sialyltransferase (50 mM sodium cacodylate, pH 6.0; 100 mM NaCl; 30 mM MnCl2; and 1.5% (v/v) Triton CF54) or galactosyltransferase (10 mM HEPES, pH 7.4; 140 mM NaCl; 5 mM MnCl2; and 0.5% (v/v) Nonidet-P40) assay buffers. The lysates were clarified in a microcentrifuge, then samples of each representing 0.25 x 106 cells were used for galactosyltransferase assays, as described previously (Jarvis and Finn, 1996Go). Similarly normalized samples were used for sialyltransferase assays except these extracts were mixed with sialyltransferase buffer containing 0.3 µCi of CMP[9-3H]NeuAc (20 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO), 50 µg of asialofetuin (Sigma), and 50 µg of bovine serum albumin, then adjusted to a total volume of 60 µl. All assays were incubated for 1 h at 37° C, then trichloroacetic acid–insoluble radioactivity was measured as described previously (Hollister et al., 1998Go). Activities were reported as cpm of tritiated galactose or sialic acid transferred per million cells per hour to control for differences in total protein content of uninfected and baculovirus-infected cells.

Lectin blotting and glycosidase analysis of gp64
Sf9, Sf{beta}4GalT, and Sf{beta}4GalT/ST6 cells were infected with AcMNPV at a multiplicity of 5 pfu/cell, then washed and resuspended in complete TNM-FH, as described above. One day later, cells and debris were removed by low speed centrifugation and progeny budded virus particles were concentrated and partially purified from the supernatants by centrifugation through 25% (w/v) sucrose cushions. gp64 was extracted from each virus, immunoprecipitated with a monoclonal antibody (Hohmann and Faulkner, 1983Go), and samples were quantified by Western blotting. Equivalent amounts of gp64 from each virus were then used for lectin blotting assays, as described previously (Jarvis and Finn, 1996Go). Briefly, the membranes were cut into strips corresponding to individual lanes, blocked, and probed with rabbit anti-gp64 or either digoxigenylated (Boehringer-Mannheim Corporation, Indianapolis, IN) or biotinylated (Vector Laboratories, Inc., Burlingame, CA) lectins. The lectins used in this study were Ricinus communis agglutinin (RCA), which binds {beta}-linked galactose, and Sambucus nigra agglutinin (SNA), which binds terminal {alpha}2,6-linked sialic acid. Each lectin was preincubated in buffer alone or in buffer containing excess competing sugar to control for the specificity of lectin binding. The IgG introduced during immunoprecipitation served as an internal positive control for the lectin blotting assays, as it has complex, terminally sialylated N-glycans. Bound lectins or antibodies were detected by secondary reactions with alkaline phosphatase–conjugated anti-digoxigenin (Boehringer-Mannheim), streptavidin (Vector Laboratories), or goat anti-rabbit IgG (Sigma), followed by a standard color reaction (Blake et al., 1984Go).

Prior to some lectin blotting assays, gp64 was eluted from immune complexes and pretreated with buffer alone, 20,000 U/ml of PNGase F (New England Biolabs, Beverly, MA), or 2,000 U/ml of Arthobacter urefaciens neuraminidase (Calbiochem; La Jolla, California) for 6 h at 37°C, as described previously (Jarvis and Finn, 1996Go). After the incubation period, an equal volume of 2x Laemmli sample buffer was added to the samples, then they were heated for 10 min at 65°C and analyzed by SDS-PAGE and lectin blotting, as described above.

Production, purification, and analysis of GST-SfManI
Sf{beta}4GalT/ST6 cells were seeded at a density of 0.5 x 106 cells/ml in 500 ml shake flasks containing a total of 200 ml of complete TNM-FH, grown to mid-log phase, infected with AcGST-SfManI at a multiplicity of 2 pfu/cell, washed, and resuspended in complete TNM-FH, as described above. At 72 h post-infection, the cultures were harvested and cell-free media were prepared by low speed centrifugation. The budded virus was removed by ultracentrifugation and polyethylene glycol 8000 was added to the supernatant at a final concentration of 15% (w/v). The precipitate was harvested by centrifugation, dissolved in glutathione column binding buffer (25 mM Tris-HCl pH 8.0, 250 mM NaCl, and 1.5% (v/v) Triton X-100), dialyzed against the same buffer, and GST-SfManI was isolated by glutathione affinity chromatography, as described previously (Kawar et al., 2000Go). The affinity-purified GST-SfManI samples were redialyzed against glycosidase buffer (5 mM Na2HPO4, pH 7.5) and total protein concentrations were determined using a commercial Bradford assay (Bio-Rad, Hercules, CA) with BSA as the standard. Three microgram samples of the purified protein were used for SDS-PAGE and lectin blotting analyses, with or without neuraminidase pretreatment, as described above. Three hundred microgram samples were used for direct glycan analyses. One sample was incubated in glycosidase buffer alone for 8 h at 37°C, then treated with 2000 mU/ml of PNGase-F for 4 h. Another sample was incubated for 8 hrs at 37°C in glycosidase buffer containing 80 mU/ml of Arthobacter urefaciens neuraminidase, then treated with 2000 mU/ml of PNGase-F for 4 h. Subsequently, each sample was ethanol precipitated and the supernatants were recovered, evaporated, and redissolved in water. The samples were then analyzed using a HPAEC-PAD system equipped with a Carbo-Pac PA100 column (Townsend and Hardy, 1991Go). After equilibrating the column in 50 mM NaOH, the samples were injected, eluted for 10 min with 50 mM NaOH at a flow rate of 1 ml/min, and then eluted with a linear gradient of 0–125 mM sodium acetate over 45 min at the same flow rate. A sialic acid standard (Neu5Ac; Sigma, St. Louis, MO) also was analyzed under the same conditions after being added to a GST-SfManI/PNGase-F/neuraminidase reaction mixture and incubated and processed as described above.

Similar experiments were performed using affinity-purified GST-SfManI from Sf{beta}4GalT/ST6 cells grown in medium containing fetal bovine serum that had been dialyzed to remove nucleotide sugars. Forty milliliters of serum were dialyzed in a 10,000 molecular weight cutoff membrane (Pierce, Rockford, IL) for 24 h at 4°C against 2 l of HyQ Sfx-Insect cell culture medium, then the dialysate was replaced with 2 l of fresh medium and dialysis was continued for another 24 h. The dialyzed serum was then added to HyQ Sfx-Insect medium at a concentration of 10% (v/v), and this medium was used to set up a 200 ml Sf{beta}4GalT/ST6 cell culture, as described above. The cells were infected, washed, and fed with this same medium, then GST-SfManI was harvested at 72 h post-infection and affinity-purified, as described above. The protein was then treated with PNGase-F alone or PNGase-F and neuraminidase using the same treatment conditions described above. But, in this case, the protein was initially deglycosylated with PNGase-F, then the released glycans were harvested using a graphitized carbon cartridge (Packer et al., 1998Go; Carbograph; Alltech, Deerfield, IL). After being eluted from the cartridge, the glycans were treated with buffer alone or neuraminidase and analyzed by HPAEC-PAD, as described above.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. Joel and Nancy Shaper of Johns Hopkins University School of Medicine for providing the bovine {beta}1,4-galactosyltransferase cDNA and for encouraging discussions. We also thank Dr. James C. Paulson of the Scripps Research Institute for providing the rat {alpha}2,6-sialyltransferase cDNA. This work was supported by NIH Grant GM49734 and NSF Grant BES-9814157.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
AcMNPV, Autographa californica nuclear polyhedrosis virus; GST, glutathione-S-transferase; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; pfu, plaque-forming units; PNGase-F, peptide:N-glycosidase F; RCA, Ricinus communis agglutinin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SNA, Sambucus nigra agglutinin.


    Footnotes
 
1 To whom correspondence should be addressed Back


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