Expression of a membrane-bound form of Trypanosoma cruzi trans-sialidase in baculovirus-infected insect cells: a potential tool for sialylation of glycoproteins produced in the baculovirus–insect cells system

Ingrid Marchal2, Martine Cerutti3, Anne-Marie Mir2, Sylvie Juliant3, Gérard Devauchelle3, René Cacan1,2 and André Verbert2

2Laboratoire de glycobiologie structurale et fonctionnelle, Unité Mixte de Recherche du CNRS no. 8576, Université des Sciences et Technologies de Lille I, 59655 Villeneuve d’Ascq cedex, France, and 3Station de Pathologie Comparée INRA/Unité Mixte de Recherche du CNRS no. 5087/ Université de Montpellier II, 30380 Saint-Christol lez Alès, France.

Received on January 17, 2001; revised on March 20, 2001; accepted on March 21, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A chimeric protein containing the catalytic domain of Trypanosoma cruzi trans-sialidase, the transmembrane domain of the major envelope glycoprotein of the baculovirus (gp67), and the signal peptide of ecdysteroid glucosyltransferase of the baculovirus was expressed under the control of the very late promoter p10 in baculovirus-infected lepidopteran cells. The recombinant protein was found to be enzymatically active. Three days after infection, equal amounts of activity were found associated to the plasma membrane and in the infection medium, both forms having the same apparent molecular weight and being N-glycosylated. When exogenous galactosylated acceptors (lactose or asialo-{alpha}1-acid glycoprotein) were added in the culture medium of cells infected with the recombinant baculovirus in the presence of a sialylated donor, a sialylation could be observed. Therefore, we propose the use of trans-sialidase as a potential tool for sialylation of glycoconjugates in the baculovirus–insect cells system.

Key words: baculovirus/insect cells/recombinant glycoproteins/sialic acid/trans-sialidase


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Insect cells, in combination with baculovirus-based expression vectors, are widely used as hosts for the expression of heterologous eukaryotic gene products (reviewed by Miller, 1988Go; Fraser, 1992Go; Jarvis, 1997Go). The relative ease and speed with which a recombinant protein is produced and the high expression levels allowed by the use of strong promoters; in addition, the processing abilities of insect cells, essentially similar to those of vertebrates, have contributed to their development. In terms of safety, the baculovirus expression system is also promising because the cells can be cultured in serum-free and even protein-free media. However, for therapeutic purposes, a strictly controlled quality of the recombinant proteins is required, and this has stimulated the examination of the posttranslational processes that occur in insect cells. Lepidopteran insect cell lines, by far the most widely used, have been shown to perform most of the posttranslational events, including specific proteolytic cleavages, oligomerization of multimeric proteins, phosphorylation, acylation, and glycosylation.

However, it is now well known that N- and O-glycosylation are highly cell type–dependent modifications, and it has been extensively reviewed that insect cells glycosylation substantially differs from the mammalian-type glycosylation (März et al., 1995Go). As summarized by Altmann et al. (1999)Go, this constitutes a serious barrier for therapeutic use of baculovirus-expressed glycoproteins. Typically, the early events in glycosylation pathways are conserved from insects to mammals, and the divergences reside in the terminal reactions. The O-glycans produced by insect cells are generally reduced to a single GalNAc (Tn antigen) or the disaccharide Galß1,3GalNAc (T antigen) (Thomsen et al., 1990Go; Grabenhorst et al., 1993Go; Sugiyama et al., 1993Go; Lopez et al., 1999Go). The N-glycosylation potential has been investigated more, both on endogenous and baculovirus-expressed glycoproteins. Most studies showed that the major structures are truncated N-glycans, highly fucosylated (Grabenhorst et al., 1993Go; Manneberg et al., 1994Go; Lopez et al., 1997Go). These short species are explained by a trimming reaction catalyzed by a Golgi-located ß-N-acetylglucosaminidase (Altmann et al., 1995Go; Wagner et al., 1996aGo; Marchal et al., 1999Go). However, in some cell lines showing low levels of this enzyme, such as Ea4 from Estigmene acrea or in High Five cells (TN-5B1-4) derived from Trichoplusia ni, the glycosylation capacities can be extended to N-glycans containing low amounts of terminal galactose (Ogonah et al., 1996Go; Hsu et al., 1997Go; Hooker et al., 1999Go; Rudd et al., 2000Go). The presence of sialic acids (reviewed by Marchal et al., 2001Go) has been reported in some cases; however, it must be recognized that the sialylation in insect cells occurs with extreme low frequency and seems to be restricted to particular glycoproteins (see, for example, Davidson et al., 1990Go) or to be tissue-specific. Sialic acids were detected during the development of Drosophila embryos (Roth et al., 1992Go) and, more recently, in larval tissues of Galleria mellonella (Lepidoptera) (Karaçali et al., 1997Go, 1999) and of the cicada Philaenus spumarius (Malykh et al., 1999Go). Thus, it can be speculated that complex N-glycosylation might be essential at some developmental stages in insects and that it is repressed at other stages. Accordingly, Karaçali et al. (1999)Go observed a reduction of 16 to 1 of sialic acids levels from larvae to adults.

When looking at the enzyme levels, several authors have shown that cultured insect cells lack terminal transferases like ß1,4-galactosyl- and sialyltransferases (Lopez et al., 1999Go; Hooker et al., 1999Go). Furthermore, Hooker et al. (1999)Go were unable to detect any cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) among the nucleotide pool of uninfected cells from Spodoptera frugiperda (Sf9 and Sf21) and E. acrea (Ea4) cells, as well as in baculovirus-infected Sf21 cells.

Accordingly, insect cells are a promising model for engineering the glycosylation pathways (reviewed by Jarvis et al., 1998Go). The first attempts to correct the glycan structures by adding mammalian glycosyltransferases were promising: the overexpression of human N-acetylglucosaminyltransferase I (GNTI) was found to lead to a fourfold increase in terminal GlcNAc residues on a baculovirus-expressed fowl plague hemagglutinin (Wagner et al., 1996bGo). Similarly, the expression of bovine ß1,4-galactosyltransferase under the control of a viral immediate early promoter, either by the way of a baculovirus expression vector (Jarvis and Finn, 1996Go) or in stably transfected cells, enabled the cells to galactosylate the N-glycans on a viral glycoprotein (Hollister et al., 1998Go) or on recombinant human transferrin (Ailor et al., 2000Go). The elongation of N-glycans can be achieved because insect cells contain the proper sugar donors, namely, uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphogalactose (UDP-Gal). However, the availability of the sialic acid donor CMP-Neu5Ac is not likely to be sufficient in cultured lepidopteran cells (Hooker et al., 1999Go), therefore, engineering the sialylation in these cells will probably require the addition of more than one gene.

The protozoan parasite Trypanosoma cruzi, the causing agent of Chagas disease, expresses a developmentally regulated trans-sialidase (TS) (reviewed by Schenkman et al., 1994Go). This enzyme allows the parasite, which does not synthesize sialic acids, to sialylate mucin-like molecules on its surface, at the expense of glycoconjugates from the host. This sialylation is thought to have a role in adhesion of T. cruzi trypomastigotes to red blood cells, in the invasion (Schenkman et al., 1991Go), and in preventing recognition by the immune system (Pereira-Chioccola and Schenkman, 1999Go). The enzyme has the unique property to transfer {alpha}2,3-linked sialic acids linked to ß-galactosides from various donors to an acceptor ß-galactoside (Scudder et al., 1993Go).

In this study, we developed an approach for sialylation in the baculovirus-insect cells system using a membrane-bound TS. As a first approach, T. cruzi TS was expressed under the control of the strong baculovirus promoter p10. We report the expression of a chimeric protein consisting in the catalytic domain of T. cruzi TS fused to the C-terminally located transmembrane domain of the major envelope glycoprotein gp67 (also called gp64) of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). The recombinant enzyme was found to be active and membrane-bound, although partially soluble, and was able to sialylate ß-galactosides using fetuin or sialyllactose as sialic acid donor.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Construction of a recombinant baculovirus containing a TS cDNA
The construction is schematized in Figure 1. A cDNA encoding a truncated and soluble 6-His-tagged form of T. cruzi TS (Buscaglia et al., 1998Go) was fused to a sequence encoding the last 30 amino acids of the major envelope glycoprotein of the baculovirus AcMNPV, gp67 (Whitford et al., 1989Go), encompassing the transmembrane anchor sequence and the short cytoplasmic tail. To avoid any recombination inside the baculovirus genome, a degenerated sequence was constructed using 10 overlapping oligonucleotides. The TS sequence was inserted downstream of a sequence encoding a signal peptide from another baculovirus gene (the gene for ecdysteroid glucosyltransferase of AcMNPV, O'Reilly and Miller, 1989Go). The final construction was inserted into the p10 locus of a baculovirus expression vector, AcSLP10 (Chaabihi et al., 1993Go). Following the procedures described in Materials and Methods, we obtained a recombinant baculovirus, AcP10TS, which was used for all subsequent experiments.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. Construction of the recombinant baculovirus. (A) The cDNA encoding a 6-His-tagged truncated form of T. cruzi TS was used as a template for PCR to generate modified cDNA ends containing additional restriction sites. The modified 5' end (307 bp) and the modified 3' end (315 bp) were sequenced and reinserted into the TS sequence using the indicated restriction sites. (B) The C-terminal sequence from gp67 gene containing the transmembrane domain and the cytoplasmic tail (CtGP67, 135 bp) was reconstituted using 10 overlapping oligonucleotides (indicated as arrows), inserted into a pUC vector containing the signal sequence from ecdysteroid glucosyltransferase (PS EGT) and sequenced. (C) The modified TS sequence was excised and inserted between PS EGT and CtGP67. After sequencing the construction was cloned into the p119 transfer vector.

 
Time course analysis of the recombinant TS expression
The expression of the TS was monitored during the course of infection by assaying its capacity to transfer Neu5Ac residues onto lactose. The amounts of TS activity were examined in lysates of cells infected with the recombinant baculovirus AcP10TS and in a suspension of intact, washed cells. Fetuin was used as the sialic acid donor because it is a nonpermeant glycoprotein, rich in {alpha}2,3-linked Neu5Ac, thus allowing to measure the plasma membrane–bound activity. This activity was found to represent one third to one half of the total activity found in the lysates (Figure 2). Both activities were detectable as early as 24 h after infection and were maximal and reached a plateau at 48 h postinfection (p.i.), which was consistent with an expression under the control of the late promoter p10 (Quant-Russell et al., 1987Go). An increasing TS activity was also detected in the medium after 24 h p.i. This activity was approximately as high as the activity in the lysates 3 days after infection. In contrast, no TS activity was detectable in AcSLP10-infected Sf9 cells.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Time-course analysis of TS expression. Sf9 cells (106 cells) were infected with AcP10TS. At various times p.i., the TS activity was assayed as described in Materials and methods in the lysates (filled triangles), in the infection medium (filled circles) or in a suspension of intact cells (open squares). The data are representative of two independent experiments and are given for 106 cells. Vertical bars indicate the standard deviation for the TS activity measurements performed in triplicate in one set of experiment.

 
Western blot analysis of the recombinant TS
A rabbit polyclonal antibody directed against the catalytic domain of TS was produced by immunization of a rabbit with a bacteria-expressed, 6-His-tagged TS. This antibody was used for detection of the TS protein in the cell lysates (Figure 3A). The antibody was found to be highly specific for the TS. The protein could be detected in the lysates of AcP10TS-infected Sf9 cells but not in Sf9 cells infected with the control baculovirus, AcSLP10. The predicted molecular weight of the protein after cleavage of the signal peptide was approximately 75 kDa. As expected, the TS migrated with an apparent molecular weight close to the migration of human apotransferrin (79 kDa). Because the TS sequence contained two potential N-glycosylation sites, peptide N-glycanase F (PNGase F) digestion was performed. This resulted in a shift down of the TS to a size of about 75 kDa, indicating that at least one N-glycosylation site was occupied and that the protein was correctly addressed to the plasma membrane via the secretion pathway.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3. Western blot analysis of the TS in lysates and in the medium. Sf9 cells adapted to EXTRA SB5 serum-free medium were infected by AcP10TS or by AcSLP10 (control). At 3 days p.i., the cells were lysed and the infection media were collected, clarified by ultracentrifugation at 50,000 x g for 20 min to eliminate the viral particles and concentrated. Both lysates (A) and supernatants (B) were analyzed by western blot using an anti-TS antibody and a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were revealed by chemiluminescence. The molecular mass standards are indicated on the left. The 79-kDa standard corresponds to human apotransferrin. The samples were treated with PNGase F (+) or with the buffer only (–) before SDS–PAGE.

 
Flow cytofluorimetry analysis of TS membrane expression
To obtain a plasma-membrane TS activity, the catalytic domain was fused with a transmembrane segment from the baculovirus envelope glycoprotein gp67. To check for the correct location of the enzyme, the expression of the TS construction was analyzed by fluorescence-assisted cell sorting. Cells infected by AcP10TS or by the control baculovirus AcSLP10 were analyzed using the polyclonal antibody. As shown in Figure 4, a clear shift of the fluorescence was observed after incubation of AcP10TS-infected cells with the anti-TS antibody, as compared to incubation with the preimmune serum, and this shift was not observed for AcSLP10-infected Sf9 cells. It has to be noted that for AcSLP10-infected cells, the presence of polyhedrin crystals dramatically change the physical properties of the cells, and this significantly alter the background fluorescence. The permeabilization of the AcP10TS-infected cells prior to incubation with the antibodies did not significantly modify the shift, suggesting that the majority of TS activity associated to the cells was bound to the plasma membrane.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4. Flow cytofluorimetry analysis of TS membrane expression. Sf9 cells were infected with AcP10TS or with the control virus AcSLP10. The TS was detected by flow cytofluorimetry on intact AcP10TS-infected cells (top panel), on ethanol-permeabilized AcP10TS-infected cells (middle panel), or on intact AcSLP10-infected cells (bottom panel). At 2 days p.i., the cells were harvested, washed, and incubated with a rabbit immune serum against TS (black open peaks) or with the preimmune serum (filled peaks) and with a secondary anti-rabbit antibody (FITC conjugated).

 
Analysis of the TS activity on extracellular viral particles
As observed in Figure 2, an increasing activity was detected in the infection medium. This infection medium contained the extracellular viral particles but also the proteins released by secretion or by cell lysis. As demonstrated by flow cytofluorimetry analyses, the TS was membrane-bound, thus we expected that it could be recovered on viral particles. To test this possibility, the extracellular viral particles were isolated from an infection medium at 3 days p.i. by ultracentrifugation. After centrifugation 20 min at 50,000 x g the viral pellet was found to contain TS activity, but a 200-fold higher activity was found as soluble material in the clarified medium. When the pelleted viral particles were analyzed by ultracentrifugation in a sucrose density gradient, a peak of TS activity coincided with the major envelope glycoprotein of the baculovirus, gp67, detected by western blot (Figure 5), indicating that TS was associated to the extracellular viral particles. But again, a part of the activity did not penetrate the gradient and remained in the top fractions, indicating that it was not incorporated into the viral envelopes.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5. Analysis of the TS activity in the infection medium. Sf9 cells were infected with AcP10TS, and the infection medium was collected at 3 days p.i. and submitted to an ultracentrifugation at 50,000 x g for 20 min to obtain a pellet of viral particles. AcP10TS viral particles were then submitted to an ultracentrifugation through a 25–56% sucrose gradient (w/v). Each fraction was assayed for TS activity. The data are expressed as percentage of the input activity, vertical bars indicate the standard deviation for triplicate determination of the TS activity. Each fraction was analyzed by western blot after SDS–PAGE with an antibody against gp67. As a control, an aliquot of the viral pellet was also analyzed by western blot (lane virus).

 
Analysis of the TS activity in the clarified infection medium
Because the major part of the TS activity measured in the infection medium was not associated with the viral particles, one hypothesis could be that part of the cellular TS activity was released into the medium after a proteolytic cleavage. However, as shown in Figure 3B, the western blot analysis after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of the clarified infection medium clearly indicated that the TS had the same apparent molecular weight as the cellular protein; in addition, its susceptibility to PNGase digestion showed that it was also N-glycosylated.

Another hypothesis to explain this soluble TS activity could be its incorporation into vesicular membranes or exosomes (Trams et al., 1981Go). To determine if this could be the case, the clarified infection medium was submitted to ultracentrifugations at increasing forces, and the TS activity remaining in the supernatant was assayed (Figure 6A). We observed that even after a 15 h centrifugation at 100,000 x g, a condition where all potential vesicles should be sedimented, 60% of the TS activity remained in the supernatant. Similarly, after filtration through a 0.22-µm filter, which should retain the vesicular material, 90% of the TS activity was recovered in the filtrates (Figure 6B). Taken together, these results strongly indicate that the TS activity in the infection medium was mainly present as a soluble protein and not integrated into exosomes. Because this protein possesses the hydrophobic tail from gp67, it could be able to form stable aggregates. To test this possibility, the proteins contained in the clarified infection medium were resolved on a nondenaturing PAGE and the TS was detected by western blot. Figure 6C shows that the immunoreactive material could penetrate into the gel, remaining in the upper part. This result shows that the TS present in the clarified infection medium was not associated to vesicles but rather was present as aggregates of very high molecular weight.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6. Analysis of the soluble TS activity. Sf9 cells were infected by AcSLP10. At 3 days p.i., the infection medium was collected, clarified by ultracentrifugation at 50,000 x g for 20 min to eliminate the viral particles and (A) submitted to increasing centrifugation forces (from 103 x g x h to 1.5 x 106 x g x h). After each centrifugation an aliquot of the supernatant was assayed for TS activity. The data are representative of two independent experiments and are expressed as the remaining TS activity in the supernatant after centrifugation. The vertical bars indicate the standard deviations of triplicate TS assays. (B) The clarified infection medium was filtered through 0.45- and 0.22-µm filters and the TS activity was assayed in the filtrates. The data are expressed as percentages of TS activity remaining in the filtrates (taking the unfiltered activity as 100%). The vertical bars indicate the standard deviations of triplicate TS assays. (C) Native gel electrophoresis of the soluble TS. Sf9 cells grown in EXTRA SB5 were infected with AcP10TS; at 3 days p.i. the infection medium was clarified and concentrated. An aliquot was analyzed by PAGE under native conditions and revealed by western blot.

 
Sialylation of galactosylated acceptors
The aim of this study was to evaluate the possibility to use T. cruzi TS as a tool for sialylation of glycoconjugates in the baculovirus-insect cells system. To test the efficiency of this enzyme and in a first approach, we used exogenous galactosylated acceptors added in the culture medium, with either fetuin or sialyl-{alpha}2,3-lactose as sialic acid donors.

In a first experiment, Sf9 cells were infected with AcP10TS, and 1 day after infection the medium was replaced by a fresh medium containing 3 mg/ml fetuin and 0.2 mg/ml lactose. Figure 7A shows the paper chromatography analysis of the medium after a 24-h incubation (i.e., 2 days p.i.). More than 30% of the lactose was converted to sialyllactose, that is, approximately 600 µg of sialyllactose was produced in 24 h by 5 x 106 infected cells.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 7. Sialylation of galactosylated exogenous acceptors. (A) 5 x 106 Sf9 cells were infected with AcP10TS and at 24 h p.i. the medium was replaced by a fresh medium containing 15 mg fetuin and 1 mg lactose (2 x 106 d.p.m. of [14C]lactose as marker). After a 24-h incubation an aliquot of the medium was analyzed by descending paper chromatography according to Materials and methods. The radioactivity was analyzed along the chromatographic path. The positions of authentic standards are indicated by arrows (SL, sialyllactose; Lac, lactose). (B) Sf9 cells grown in EXTRA SB5 serum free medium were infected with either AcSLP10 or AcP10TS. At 24 h p.i. the infection medium was replaced by fresh medium containg 1 mM sialyl-{alpha}2,3-lactose and 1 mg/ml asialo-{alpha}1AGP. Controls were performed without sialyllactose or without asialo-{alpha}1AGP. At various times p.i. aliquots of the medium were analyzed by SDS–PAGE and blotted. The proteins were visualized by Ponceau red staining (top panel) and {alpha}2,3 sialyl residues were revealed by digoxigenin-conjugated MAA (bottom panel). + indicates that the samples were treated with C. perfringens sialidase before electrophoresis.

 
Finally, we tested the possibility to sialylate N-glycans. For this purpose, Sf9 cultured in serum-free medium were infected with AcP10TS, and 1 day after infection the medium was replaced by a fresh medium containing 1 mM sialyl-{alpha}2,3-lactose and 1 mg/ml desialylated {alpha}1-acid glycoprotein ({alpha}1-AGP). Figure 7B shows the time course analysis of the sialylation of asialo-{alpha}1-AGP revealed by lectin blotting with Maackia amurensis agglutinin (MAA). It clearly appears that asialo-{alpha}1-AGP acquired {alpha}2,3-linked sialic acids. The specificity of the lectin is demonstrated by the absence of signal when the resialylated {alpha}1-AGP was treated with C. perfringens sialidase before SDS–PAGE. Controls were performed in the absence of asialo-{alpha}1-AGP, in the absence of sialyl-{alpha}2,3-lactose and with AcSLP10-infected cells incubated with both the donor and the acceptor. No sialylation was observed with any of these controls, demonstrating that the TS uses sialyllactose to sialylate the desialylated {alpha}1-AGP in the culture medium.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Recent reports, summarized by Jarvis et al. (1998)Go, have shown that engineering the N-glycosylation pathway in the baculovirus–insect cells expression system can be considered. The obtention of baculovirus-expressed glycoproteins carrying mammalian-type N-glycans up to the galactose can reasonably be hoped for in the near future. However, the problem of the sialylation is not resolved because cultured insect cells do not display significant amounts of the donor CMP-Neu5Ac (Hooker et al., 1999Go). This result predicts that the addition of more than one gene will be needed to allow the sialylation in insect cells. This point of view is still contradictory, since Hollister and Jarvis (2001) have recently obtained a stably transformed insect cell line, Sfß4GalT/ST6, possessing the mammalian cDNAs for a ß1,4-galactosyltransferase and an {alpha}2,6-sialyltransferase, which is able to transfer {alpha}2,6-linked Neu5Ac onto a baculoviral glycoprotein.

This study was aimed to explore another possibility for the sialylation in the baculovirus system using T. cruzi S. This enzyme, which has a unique catalytic activity, has been extensively studied (Schenkman et al., 1994Go), and several groups are concerned with the exploitation of this activity in vitro (Tomlinson et al., 1992Go; Ito and Paulson, 1993Go).

Due to the surface expression of this enzyme, T. cruzi is able to sialylate mucin-like acceptors using circulating or cellular donors. Our idea was to mimic this property of the parasite in insect cells. To achieve this purpose, the catalytic domain of the enzyme was fused to the transmembrane domain of gp67. The envelope fusion protein gp67 or gp64 has been shown to drive the viral budding (Oomens and Blissard, 1999Go) and to be the main insect cell receptor (Hefferon et al., 1999Go). An envelope fusion domain and a trimerization domain have been defined in the gp67 ectodomain, and a number of mutations in the ectodomain result in viruses with reduced infectivity (Monsma and Blissard, 1995Go). In contrast, the deletion of the C-terminal cytoplasmic tail did not significantly modify the infectivity or the virions production (Oomens and Blissard, 1999Go), suggesting that this sequence does not play any essential role in the virion assembly. Therefore, it was postulated in this study that the addition of only the transmembrane sequence and the cytoplasmic tail, that is, the last 30 amino acids of AcMNPV gp67 sequence (Whitford et al., 1989Go), would direct the chimeric protein to be integrated in the plasma membrane and not specifically in the envelope of the virus. Our strategy was different from the baculovirus display strategy (Boublik et al., 1995Go; Grabherr et al., 1997Go; Ernst et al., 2000Go), which takes advantage of the specific integration of gp67 into the viral envelope to express proteins on the surface of budded virions. However, because the envelope of budded virions is known to derive from the cytoplasmic membrane of infected cells, it was expected that the TS construction would also be present in the viral envelope. In fact, after sedimentation of the viral particles through a sucrose gradient, we were able to detect TS activity in the virus-containing fractions.

When the chimeric TS was overexpressed under the control of the p10 promoter in Sf9 cells, the TS activity recovered was mainly associated with the cells and in the infection medium. Because in the flow cytofluorimetry experiments the expression of the TS was comparable for intact and permeabilized AcP10TS-infected cells, we concluded that the majority of the cellular TS was bound to the plasma membrane. Only a minor part seemed to be associated with virus budding.

It was more surprising to observe that an equal amount of TS activity was found on the plasma membrane and as soluble material in the infection medium, even after high-speed centrifugations in the conditions used to pellet the viruses. It was first thought that the protein was shed from the cell surface by a proteolytic cleavage resulting in an active, truncated form of the enzyme. But after SDS–PAGE and western blot analysis we could not detect any difference in the size of the protein found in cell lysates and in the infection supernatants. Therefore, another possible explanation for this soluble activity could be the incorporation of the TS, through the hydrophobic segment from gp67, into vesicles exfoliated from the cellular membrane. This exfoliation process was first observed by Trams et al. (1981)Go who proposed the name of exosomes for these vesicles. However, taking their experimental conditions (ultracentrifugation and ultrafiltration), we were unable to characterize the association of soluble TS activity with exosomes. Furthermore, an electrophoresis under native conditions showed that the TS could penetrate into the gel and confirmed that the enzyme was not linked to vesicular membranes. Our interpretation is that the TS synthesized in the endoplasmic reticulum follows the secretion pathway, because both cellular and soluble forms are N-glycosylated. Due to the overexpression driven by the p10 promoter, a part of the glycoprotein remains plasma membrane–associated, while an other part is released as aggregates. Indeed, the time-course analysis shows that the TS associated to the membranes rapidly reaches a plateau, whereas the released activity continuously increases during the infection. The plasma membrane–bound activity was found to represent one third to one half of the total cellular activity as assayed in the lysates, and this appears not to be in agreement with the flow cytofluorimetry analyses, which indicated that the majority of the TS was bound to the plasma membrane, because no difference in the antibody fixation was observed for permeabilized cells as compared with intact cells. One possible explanation is that a part of the intracellular TS activity is soluble and hence is lost after permeabilization. But this discrepancy may also reflect a difference in activity or in substrate accessibility between the solubilized and membrane-bound forms of the enzyme.

More important is the observation that the expression of a TS in the baculovirus system enables the insect cells to sialylate exogenous galactosylated acceptors, such as lactose and asialo-N-glycans. But further efforts should concern the optimization of the model. The first point to consider is that in this study the TS was overexpressed under the control of a strong late promoter. Because this could affect the synthesis of a glycoprotein of interest, it could be useful to express the TS under the control of a moderate and early promoter (for review, see Jarvis et al., 1990Go). The second point is that we were only concerned to sialylate exogenous galactosylated acceptors, because Sf9 cells do not produce significant amounts of galactose-terminated glycoproteins. As discussed above, the expression of TS in insect cells engineered to extend the glycosylation pathway up to the galactose seems to be a key point. Future studies should concern the coexpression of a ß-galactosyltransferase in combination with the TS, together with a glycoprotein of interest. Alternatively, it could be interesting to express the TS in insect cell lines that are known to synthesize higher levels of galactosylated glycoproteins, such as Ea4 or High Five cells.

Another essential improvement concerns the efficiency of the TS reaction and the optimization of the sialylation levels. The choice of the most appropriate sialic acid donor seems to be crucial. Several requirements have to be adressed. First, the donor will have to allow an easy purification of the glycoprotein of interest. For example, a variety of low molecular weight or insolubilized sialic acid donors can be substrates for the TS. A good donor should also be cost-effective. Finally, the quality of the donor and the ratio donor versus acceptor have to be tested for optimal sialylation levels.

Though a major limitation for sialylation of recombinant glycoproteins using TS is that it transfers only {alpha}2,3-linked sialic acids, the main advantage is that a variety of acceptor motifs, including O- and N-glycans, can be sialylated. This is generally not the case with sialyltransferases, as these enzymes have a very high specificity toward the acceptor (for a review, see Harduin-Lepers et al., 1995Go). Moreover, the strategy, which uses in vitro sialylation of the purified glycoprotein by a sialyltransferase, is constrained by the cost of CMP-NeuAc, which is the exclusive donor for this family of enzymes.

Although the recent study of Hollister and Jarvis (2001) reports that the coexpression of a ß1,4-galactosyltransferase and an {alpha}2,6-sialyltransferase in a stably transformed cell line derived from Sf9 allows the sialylation of a recombinant glycoprotein, the way by which CMP-NeuAc is synthesized remains unclear. In addition, the sialylation levels reached in this system presumably are limited by the CMP-NeuAc availability. Therefore, the trans-sialidase appears to be a potential alternative for the production of {alpha}2,3-sialylated glycoproteins in the baculovirus-insect cells system.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cells and viruses
Spodoptera frugiperda Sf9 cells (ATCC CRL 1711) were maintained at 28°C in EXTRA 1x medium (Eurobio, Les Ulis, France) supplemented with 5% heat-inactivated fetal calf serum (Gibco BRL Life Technologies, France), and antibiotics: penicillin (3.5 mM) and streptomycin (35 µM) (both from Sigma, St. Louis, MO) or in a serum-free medium, EXTRA SB5 (Eurobio), supplemented with cholesterol (4 mg/l, Sigma) and antibiotics. The cells were cultured as monolayers and subcultured twice a week.

The viruses were propagated in Sf9 cells, and all procedures were done essentially as described by Summers and Smith (1987)Go. For infection, cells were inoculated with a viral suspension at multiplicities of infection (MOIs) of 5–10 PFU/cell. After a 1-h adsorption at room temperature, the viral inoculum was removed and fresh culture medium was added. Infected cells were further incubated at 28°C for various times. The viral titers were determined by plaque assay.

Construction of the recombinant baculovirus
The cDNA encoding a 6-His tagged truncated form of T. cruzi trans-sialidase (GenBank accession number L26499), cloned in the EcoRI site of the pTrcHisA vector (Invitrogen, The Netherlands) was a kind gift of Dr A.C.C. Frasch (see Buscaglia et al., 1998Go). This plasmid served as a template for polymerase chain reaction (PCR) to generate modified ends of the TS sequence (Figure 1), using primers from Eurogentec (Belgium) and the VENT polymerase (New England Biolabs, Beverly, MA). Oligonucleotides TST1 (5'-CGA-TTC-TAG-ACT-GGC-ACC-CGG-ATC-GAG-CCG-AGT-TGA-3') and TST2 (5'-CAC-TGT-GGG-ATC-CAC-CAC-ACG-AGA-AAC-AGA-3') were designed for generation of a modified 5' end containing an additional XbaI site and deleted of the 6-His tag. The amplified fragment (307 bp) was excised from the agarose gel and cloned into a pBlueScript plasmid for sequencing using the T7 polymerase kit from Pharmacia (Uppsala, Sweden) and d[35S]ATP from Amersham (UK). The obtained plasmid was then digested by XbaI and BamHI. The digestion fragment was reinserted into pTrcHisATS to generate pTrcHisATSt. Oligonucleotides TSQ1 (5'-GGG-TAA-GAG-GTA-CCA-CGT-CGT-TCT-3') and TSQ2 (5'-CTG-AGA-ATT-CCT-AGG-GCA-CTC-GTG-TCG-CTG-CTG-CTG-TC-3') were used for generation of a modified 3' end containing an additional AvrII site and deleted of the stop codon. The amplified 315-bp-long fragment was excised from the gel and cloned into a pUC19 plasmid for sequencing. The KpnI–EcoRI fragment was then reinserted into pTrcHisATSt to generate pTrcHisATStq.

A degenerated sequence encoding the C-terminal transmembrane domain from the baculovirus envelope glycoprotein gp67 (Whitford et al., 1989Go, GenBank accession number M25420) and containing unique restriction sites at both ends was constructed using a set of 10 overlapping oligonucleotides. The dephosphorylated oligonucleotides were denatured by heating, mixed, and allowed to renature overnight. The resulting fragment (135 bp long) was cloned into a HpaI/SacI-cut pUC vector carrying the signal sequence from the ecdysteroid glucosyltransferase gene of the baculovirus AcMNPV (O'Reilly and Miller, 1989Go, GenBank accession number M22619). Several independent clones obtained after ligation and transformation of Escherichia coli were sequenced, and one clone was found to contain the right sequence.

The modified TS sequence was excised by XbaI/AvrII digestion and inserted between the signal peptide and the transmembrane sequence. The final construction was then subcloned into the transfer vector p119, designed for insertion into the p10 locus of the baculovirus.

Sf9 cells were cotransfected by lipofection (Felgner and Ringold, 1989Go) using DOTAP (Boehringer Mannheim, Germany) with p119-TS and genomic DNA of the modified baculovirus AcSLP10. AcSLP10 is derived from wild-type AcMNPV and possesses only one strong promoter, p10, with the polyhedrin coding sequence inserted downstream of this promoter (Chaabihi et al., 1993Go). Thus, it has an occlusion body–positive phenotype, whereas the recombinant virus is occlusion body–negative. The screening and purification of the recombinant baculovirus AcP10TS were carried out as described by Summers and Smith (1987)Go.

Production of the antibodies
E. coli strain DH5{alpha} bacteria were transformed with pTrcHisATS, cultured in 200 ml LB medium with 50 µg/ml; ampicillin and induced by 1 mM isopropyl ß-D-thiogalactoside (Sigma) when the optical density reached 0.6. After a 4-h induction at 30°C, cells were harvested, washed in a cold Tris buffer (pH 7.5) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and were lysed using a French press at a pressure of 10,000 psi. After centrifugation and SDS–PAGE analysis, the pellet was found to contain the TS. The pellet was resuspended in denaturing buffer (urea 8 M, NaH2PO4 100 mM, Tris 10 mM, pH 8, PMSF 1 mM). Then the 6-His-tagged TS was batch-purified using an Ni-NTA-agarose resin (Qiagen GmbH, Germany) as described in the manufacturer’s protocol. Each fraction was analyzed by SDS–PAGE; the fractions containing the pure TS were pooled, dialyzed against water, and lyophilized. An approximate yield of 800 µg was obtained and used for immunization of a rabbit by three successive injections.

The antiserum was collected 8 weeks after the first injection, and the antibodies were precipitated with ammonium sulfate and resuspended in a half volume of phosphate buffered saline (PBS, pH 7.2) and dialyzed. The specificity of the antibodies was evaluated by western blot analysis (not shown).

TS assays
For TS assays, we used a protocol in which the sialic acid donor was fetuin (rich in {alpha}2,3-linked sialic acids) and the acceptor was [14C]-labeled lactose (Amersham, UK). Each assay contained 1 mM lactose (0.4 µCi) and 20 mg/ml fetuin (Sigma, Neu5Ac content approximately 5%) in a final volume of 100 µl in PBS pH 7.2, with or without 0.5% Triton X100. The incubations were performed at 28°C for 1 h, then the macromolecules were precipitated by the addition of 900 µl cold ethanol. Following centrifugation, the supernatants were dried under nitrogen flow and resuspended in 70% ethanol, and the labeled compounds were separated by paper electrophoresis as described by Leguizamon et al. (1994)Go. Sialyllactose and lactose could also be separated by descending paper chromatography in the following solvent: pyridine/ethyl acetate/acetic acid/water 5/5/1/3 (by volume).

For analysis of the TS activity in cell lysates, the infected cells were harvested, washed with cold PBS pH 7.2, and lysed in the lysis buffer (50mM Tris–HCl, pH 7.5, 1% Triton X100) and the TS assay was performed as described above.

For analysis of the membrane-bound activity, the infected cells were harvested, washed with cold PBS pH 7.2, and resuspended in PBS. The assay was carried out as described above with this cell suspension (without Triton X100). The cell viability was estimated by Trypan blue exclusion to be around 90%.

For analysis of the soluble activity, the infection medium was clarified by centrifugation 20 min at 50,000 x g in a SW41TI rotor at 4°C. The clarified infection medium was then centrifuged at various speeds and for various times in a SW41TI rotor at 4°C, and the supernatants were assayed for TS activity as above. The clarified infection medium was also filtered through 0.45- and 0.22-µm filters (Pall Gelman Sciences, Ann Arbor, MI) and the filtrates were assayed for TS activity.

When asialo-{alpha}1-AGP was used as sialic acid acceptor, the desialylation was carried out by incubation of human {alpha}1-AGP (Sigma) with 3 M acetic acid at 80°C for 3 h, and the desialylation was checked by gas-liquid chromatography. Pure sialyl-{alpha}2,3-lactose was the kind gift of Dr. Gérard Strecker.

Flow cytofluorimetry analysis
Sf9 cells grown in EXTRA 1x medium were infected with the recombinant baculovirus AcP10TS or with the control baculovirus AcSLP10. The cells were harvested at 48 h p.i. and washed three times in cold PBS, then incubated with the antibody or with the preimmune serum diluted in PBS containing 0.1% bovine serum albumin (BSA) for 1 h in ice. Cells were washed then incubated 1 h with the secondary antibody: fluorescein conjugated anti-rabbit (Sigma) diluted in PBS–BSA. Then the cells were analyzed in a FACScalibur (Beckton Dickinson, Sunnyvale, CA).

Permeabilization of the cells prior to incubation with the antibodies was performed by incubating the cells for 2 h in ice-cold 70% ethanol at a temperature of –20°C. Then the cells were washed three times with cold PBS and incubated with the antibodies as described above.

Preparation of viral particles and sedimentation on sucrose gradient
Sf9 cells cultured in EXTRA 1x medium were infected with AcP10TS in a 75-cm2 T-flask. Three days after infection, the medium was collected and the viral particles were pelleted by ultracentrifugation at 50,000 x g for 20 min at 4°C (according to Loisel et al., 1997Go). The supernatant was saved and assayed for TS activity. The pellet was resuspended in cold PBS and centrifuged at low speed to eliminate any cellular debris. This supernatant was then pelleted by another ultracentrifugation at 50,000 x g for 20 min at 4°C. The pellet was resuspended in 1.2 ml of TE buffer (Tris 10 mM, EDTA 1 mM, pH 7.4) containing 1 mM PMSF (Sigma), assayed for TS activity, and layered onto a linear continuous sucrose gradient (25–56% w/v in TE buffer). The gradient was centrifuged at 100,000 x g for 90 min in a SW41TI rotor at 4°C. Fractions were collected from the bottom to the top of the gradient, dialyzed against water, and assayed for TS activity as described. An aliquot of each fraction was analyzed by SDS–PAGE, and western blot analysis was performed using a mouse monoclonal antibody against gp67, the major envelope glycoprotein of the baculovirus. This anti-gp67 antibody (AcV5, Hohmann and Faulkner, 1983Go; Monsma and Blissard, 1995Go) was a kind gift of Dr. G.W. Blissard. The secondary antibody was a goat anti-mouse antibody conjugated to horseradish peroxydase (Sigma). The immunoreactive bands were detected by chemiluminescence.

Western blot analysis of the recombinant TS
To allow an easier detection, Sf9 cells adapted to a serum-free medium, EXTRA SB5 supplemented with cholesterol, were used for western blot analysis. Sf9 cells adapted to this medium were infected with AcP10TS or with the control AcSLP10 at MOIs of 10. At 3 days p.i., the cells were harvested and lysed in the lysis buffer (Tris–HCl, pH 7.5, 1% Triton X100) and assayed for TS activity. The infection supernatants were clarified by centrifugation at 50,000 x g for 20 min at 4°C and concentrated five times using a Microsep unit (Pall Gelman Sciences). PNGase F digestions were carried out according to the manufacturer’s protocol (New England Biolabs). The samples were first denatured 10 min at 100°C in denaturing buffer (0.5% SDS, 1% ß-mercaptoethanol), then the incubations were carried out for 1 h at 37°C with 15 mIU PNGase F in the incubation buffer (sodium phosphate 0.05 M, pH 7.5) with 1% NP40. Control samples were denatured similarly and treated with the buffer only. Finally, the samples were placed in Laemmli buffer, boiled, and ran on denaturing gel electrophoresis (Laemmli, 1970Go). Proteins were electrotransferred to nitrocellulose sheets (Schleicher & Schuell, Germany), and visualized by Ponceau red staining (Sigma). The nitrocellulose membranes were blocked in 2% gelatin (Sigma) in Tris buffered saline (TBS) buffer (Tris–HCl 15mM, pH 8, NaCl 140 mM, 0.05% Tween 20). The proteins were detected by using the polyclonal anti-TS antibody as the primary antibody (1/10,000 in TBS buffer) and a goat anti-rabbit IgG conjugated to horseradish peroxidase (Dako, Glostrup, Denmark, 1/1000 in TBS buffer). The immunoreactive bands were detected by chemiluminescence using an ECL kit from Pharmacia (Uppsala, Sweden).

Native gel electrophoresis
For nondenaturing gels, the samples were placed in a sample buffer (187 mM Tris–HCl, pH 8.8, 1% sucrose, 0.005% bromophenol blue) and ran in a 5–15% gradient gel without SDS in the electrophoresis buffer (Tris–HCl, 0.025 M, glycine 0.2 M, pH 8.5). The western blot was performed as above.

Lectin blotting
After SDS–PAGE and electrotransfer as described above, the nitrocellulose sheets were blocked in polyvinylpyrrolidone (2% in TBS buffer), then incubated with digoxigenin-conjugated MAA (specific for {alpha}2,3-linked sialic acids); blocked again in blocking reagent; incubated with Fab–anti-digoxigenin fragments, conjugated to alkaline phosphatase, and finally revealed with nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl-phosphate (all reagents from Boehringer Mannheim). Control samples were treated before SDS–PAGE with 50 mU/ml C. perfringens sialidase (Sigma) in 100 µl of incubation buffer (sodium citrate 50 mM, pH 6, NaCl 0.9%, CaCl2 0.1%) for 1 h at 37°C.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was initiated by Dr. André Verbert and we wish to dedicate it to his memory. André Verbert died on 20 May 2000. We miss his friendship and enthusiasm. We are very grateful to Dr. A.C.C. Frasch (University of Buenos Aires) for his kind gift of the trans-sialidase cDNA and to Dr. G.W. Blissard (Boyce Thompson Institute, Cornell University, Ithaca, NY) for the gift of the AcV5 antibody. We sincerely acknowledge Dr. Philippe Delannoy for helpful discussions. We are indebted in Dr. Joël Mazurier for the cytofluorimetry experiments, Jean-Pierre Decottignies for the immunization of a rabbit, Dr. Jean-Pierre Bohin for the bacterial cultures, and Dr. Gérard Strecker for gift of the sialyllactose. We also sincerely thank the members of the Station de Pathologie Comparée, Unité Mixte de Recherche INRA/CNRS/Université de Montpellier II no. 5087 and of the Unité Mixte de Recherche du CNRS no. 8576, particularly Dr. Sandrine Duvet, Dr. Frédéric Chirat, and François Foulquier for their help and support. This work has been supported by the Université des Sciences et Technologies de Lille, by the Ministère de la Recherche and by the Centre National de la Recherche Scientifique, "Programme Interdisciplinaire Physique et Chimie du Vivant, Réseau GT-rec."


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}1-AGP, {alpha}1-acid glycoprotein; AcMNPV, Autographa californica multicapsid nuclear polyhedrosis virus; BSA, bovine serum albumin; CMP-Neu5Ac, cytidine monophosphate N-acetylneuraminic acid; EGT, ecdysteroid glucosyltransferase; GNTI, ß-1,2-N-acetylglucosaminyltransferase I; MAA, Maackia amurensis agglutinin; MOI, mutiplicity of infection; PBS, phosphate buffered saline; PCR, polymerase chain reaction; p.i., post-infection; PMSF, phenylmethylsulfonyl fluoride; PNGase, peptide N-glycanase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris buffered saline; TS, trans-sialidase; UDP-Gal, uridine diphosphogalactose; UDP-GlcNAc, uridine diphospho-N-acetylglucosamine.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ailor, E., Takahashi, N., Tsukamoto, Y., Masuda, K., Rahman, B.A., Jarvis, D.L., Lee, Y.C., and Betenbaugh, M.J. (2000) N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase. Glycobiology, 10, 837–847.[Abstract/Free Full Text]

Altmann, F., Schwihla, H., Staudacher, E., Glossl, J., and März, L. (1995) Insect cells contain an unusual, membrane-bound beta-N-acetylglucosaminidase probably involved in the processing of protein N-glycans. J. Biol. Chem., 270, 17344–17349.[Abstract/Free Full Text]

Altmann, F., Staudacher, E., Wilson, I.B.H., and März, L. (1999) Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj. J., 16, 109–123.[ISI][Medline]

Boublik, Y., Di Bonito, P., and Jones, I.M. (1995) Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface. Biotechnology (NY), 10, 1079–1084.

Buscaglia, C.A., Campetella, O., Leguizamon, M.S., and Frasch, A.C. (1998) The repetitive domain of Trypanosoma cruzi trans-sialidase enhances the immune response against the catalytic domain. J. Infect. Dis., 177, 431–436.[ISI][Medline]

Chaabihi, H., Ogliastro, M.H., Martin, M., Giraud, C., Devauchelle, G., and Cerutti, M. (1993) Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells. J. Virol., 67, 2664–2671.[Abstract]

Davidson, D.J., Fraser, M.J., and Castellino, F.J. (1990) Oligosaccharide processing in the expression of human plasminogen cDNA by lepidopteran insect (Spodoptera frugiperda) cells. Biochemistry, 29, 5584–5590.[ISI][Medline]

Ernst, W.J., Spenger, A., Toellner, L., Katinger, H., and Grabherr, R.M. (2000) Expanding baculovirus surface display. Modification of the native coat protein gp64 of Autographa californica NPV. Eur. J. Biochem., 267, 4033–4039.[Abstract/Free Full Text]

Felgner, P.L., and Ringold, G.M. (1989) Cationic liposome-mediated transfection. Nature, 337, 387–388.[ISI][Medline]

Fraser, M.J. (1992) The baculovirus-infected insect cell as a eukaryotic gene expression system. Curr. Top. Microbiol. Immunol., 158, 131–172.[ISI][Medline]

Grabenhorst, E., Hofer, B., Nimtz, M., Jager, V., and Conradt, H.S. (1993) Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications. Eur. J. Biochem., 215, 189–197.[Abstract]

Grabherr, R., Ernst, W., Doblhoff-Dier, O., Sara, M., and Katinger, H. (1997) Expression of foreign proteins on the surface of Autographa californica nuclear polyhedrosis virus. Biotechniques, 22, 730–735.[ISI][Medline]

Harduin-Lepers, A., Recchi, M.A., and Delannoy, P. (1995) 1994, the year of sialyltransferases. Glycobiology 5, 741–758.[ISI][Medline]

Hefferon, K.L., Oomens, A.G., Monsma, S.A., Finnerty, C.M., and Blissard, G.W. (1999) Host cell receptor binding by baculovirus GP64 and kinetics of virion entry. Virology, 258, 455–468.[ISI][Medline]

Hohmann, A.W., and Faulkner, P. (1983) Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis. Virology, 125, 432–444.[ISI][Medline]

Hollister, J.R., and Jarvis, D.L. (2001) Engineering lepidopteran insect cells for sialoglycoprotein expression by genetic transformation with mammalian ß1, 4-galactosyltransferase and {alpha}2, 6-sialyltransferase genes. Glycobiology, 11, 1–9.[Abstract/Free Full Text]

Hollister, J.R., Shaper, J.H., and Jarvis, D.L. (1998) Stable expression of mammalian beta 1, 4-galactosyltransferase extends the N-glycosylation pathway in insect cells. Glycobiology, 8, 473–480.[Abstract/Free Full Text]

Hooker, A.D., Green, N.H., Baines, A.J., Bull, A.T., Jenkins, N., Strange, P.G., and James, D.C. (1999) Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells. Biotechnol. Bioeng., 63, 559–572.[ISI][Medline]

Hsu, T.A., Takahashi, N., Tsukamoto, Y., Kato, K., Shimada, I., Masuda, K., Whiteley, E.M., Fan, J.Q., Lee, Y.C., and Betenbaugh, M.J. (1997) Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J. Biol. Chem., 272, 9062–9070.[Abstract/Free Full Text]

Ito, Y., and Paulson, J.C. (1993) Combined use of trans-sialidase and sialyltransferase for enzymatic synthesis of {alpha}NeuAc2->3ßGal-OR. J. Am. Chem. Soc., 115, 7862–7863.[ISI]

Jarvis, D.L. (1997) Baculovirus expression vectors. In Miller, L.K., (eds), The Baculoviruses. Plenum Publishing Corporation, New York, pp. 389–431.

Jarvis, D.L., and Finn, E.E. (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat. Biotechnol., 14, 1288–1292.[ISI][Medline]

Jarvis, D.L., Fleming, J.G.W., Kovacs, G.R., Summers, M.D., and Guarino, L.E. (1990) Use of early baculovirus promoters for continuous expression and efficient processing of foreign gene products in stably transformed lepidopteran cells. Bio/Technology, 8, 950–955.[ISI][Medline]

Jarvis, D.L., Kawar, Z.S., and Hollister, J.R. (1998) Engineering N-glycosylation pathways in the baculovirus-insect cell system. Curr. Opin. Biotechnol., 9, 528–533.[ISI][Medline]

Karaçali, S., Kirmizigul, S., and Deveci, R. (1999) Sialic acids in developing testis of Galleria mellonella (Lepidoptera). Invert. Reprod. and Develop., 35, 225–229.

Karaçali, S., Kirmizigul, S., Deveci, R., Deveci, O., Onat, T., and Gurcu, B. (1997) Presence of sialic acid in prothoracic glands of Galleria mellonella (Lepidoptera). Tissue and Cell, 29, 315–321.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Leguizamon, M.S., Campetella, O.E., Gonzalez Cappa, S.M., and Frasch, A.C. (1994) Mice infected with Trypanosoma cruzi produce antibodies against the enzymatic domain of trans-sialidase that inhibit its activity. Infect. Immun., 62, 3441–3446.[Abstract]

Loisel, T.P., Ansanay, H., St-Onge, S., Gay, B., Boulanger, P., Strosberg, A.D., Marullo, S., and Bouvier, M. (1997) Recovery of homogeneous and functional beta 2-adrenergic receptors from extracellular baculovirus particles. Nat. Biotechnol., 15, 1300–1304.[Medline]

Lopez, M., Coddeville, B., Langridge, J., Plancke, Y., Sautiere, P., Chaabihi, H., Chirat, F., Harduin-Lepers, A., Cerutti, M., Verbert, A., and Delannoy, P. (1997) Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells. Glycobiology, 7, 635–651.[Abstract]

Lopez, M., Tetaert, D., Juliant, S., Gazon, M., Cerutti, M., Verbert, A., and Delannoy, P. (1999) O-glycosylation potential of lepidopteran insect cell lines. Biochim. Biophys. Acta, 1427, 49–61.[ISI][Medline]

Malykh, Y.N., Krisch, B., Gerardy-Schahn, R., Lapina, E.L., Shaw, L., and Schauer, R. (1999) The presence of N-acetylneuraminic acid in Malpighian tubules of larvae of the cicada Philaenus spumarius. Glycoconj. J., 16, 731–739.[ISI][Medline]

Manneberg, M., Friedlein, A., Kurth, H., Lahm, H.W., and Fountoulakis, M. (1994) Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells. Prot. Sci., 3, 30–38.[Abstract/Free Full Text]

Marchal, I., Mir, A.M., Kmiecik, D., Verbert, A., and Cacan, R. (1999) Use of inhibitors to characterize intermediates in the processing of N- glycans synthesized by insect cells: a metabolic study with Sf9 cell line. Glycobiology, 9, 645–654.[Abstract/Free Full Text]

Marchal, I., Jarvis, D.L., Cacan, R., and Verbert, A. (2001) Glycoproteins from insect cells: sialylated or not? Biol. Chem., 382, 151–159.[ISI][Medline]

März, L., Altmann, F., Staudacher, E., and Kubelka, V. (1995) Protein glycosylation in insects. In Montreuil J., Schachter H. and Vliegenthart, J.F.G., eds., Glycoproteins. Elsevier, Amsterdam, pp. 543–563.

Miller, L.K. (1988) Baculoviruses as gene expression vectors. Annu. Rev. Microbiol., 42, 177–199.[ISI][Medline]

Monsma, S.A., and Blissard, G.W. (1995) Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. J. Virol., 69, 2583–2595.[Abstract]

Ogonah, O.W., Freedman, R.B., Jenkins, N., Patel, K., and Rooney, B.C. (1996) Isolation and characterization of an insect cell line able to perform complex N-linked glycosylation on recombinant glycoproteins. Bio/Technology, 14, 197–202.[ISI]

Oomens, A.G., and Blissard, G.W. (1999) Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus. Virology, 254, 297–314.[ISI][Medline]

O’Reilly, D.R., and Miller, L.K. (1989) A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase. Science, 245, 1110–1112.[ISI][Medline]

Pereira-Chioccola, V.L., and Schenkman, S. (1999) Biological role of Trypanosoma cruzi trans-sialidase. Biochem. Soc. Trans, 27, 516–518.[ISI][Medline]

Quant-Russell, R.L., Pearson, M.N., Rohrmann, G.F., and Beaudreau, G.S. (1987) Characterization of baculovirus p10 synthesis using monoclonal antibodies. Virology, 160, 9–19.[ISI][Medline]

Roth, J., Kempf, A., Reuter, G., Schauer, R., and Gehring, W.J. (1992) Occurrence of sialic acids in Drosophila melanogaster. Science, 256, 673–675.[ISI][Medline]

Rudd, P.M., Downing, A.K., Cadene, M., Harvey, D.J., Wormald, M.R., Weir, I., Dwek, R.A., Rifkin, D.B., and Gleizes, P.E. (2000) Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells. Biochemistry, 39, 1596–1603.[ISI][Medline]

Schenkman, S., Eichinger, D., Pereira, M.E., and Nussenzweig, V. (1994) Structural and functional properties of Trypanosoma trans-sialidase. Annu. Rev. Microbiol., 48, 499–523.[ISI][Medline]

Schenkman, S., Jiang, M.S., Hart, G.W., and Nussenzweig, V. (1991) A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell, 65, 1117–1125.[ISI][Medline]

Scudder, P., Doom, J.P., Chuenkova, M., Manger, I.D., and Pereira, M.E. (1993) Enzymatic characterization of beta-D-galactoside alpha 2, 3-trans-sialidase from Trypanosoma cruzi. J. Biol. Chem., 268, 9886–9891.[Abstract/Free Full Text]

Sugiyama, K., Ahorn, H., Maurer-Fogy, I., and Voss, T. (1993) Expression of human interferon-alpha 2 in Sf9 cells. Characterization of O-linked glycosylation and protein heterogeneities. Eur. J. Biochem., 217, 921–927.[Abstract]

Summers, M.D., and Smith, G.E. (1987) A manual of methods for baculovirus and insect cell culture procedures. Texas Agric. Exp. State Bull., 1555, 1–5.

Thomsen, D.R., Post, L.E., and Elhammer, A.P. (1990) Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9. J. Cell. Biochem., 43, 67–79.[ISI][Medline]

Tomlinson, S., Pontes de Carvalho, L., Vandekerckhove, F., and Nüssenzweig, V. (1992) Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes. Glycobiology, 2, 549–551.[Abstract]

Trams, E.G., Lauter, C.J., Salem N.J.R., and Heine, U. (1981) Exfoliation of membrane ecto-enzymes in the form of micro-vesicles. Biochim. Biophys. Acta, 645, 63–70.[ISI][Medline]

Wagner, R., Geyer, H., Geyer, R., and Klenk, H.D. (1996a) N-acetyl-beta-glucosaminidase accounts for differences in glycosylation of influenza virus hemagglutinin expressed in insect cells from a baculovirus vector. J. Virol., 70, 4103–4109.[Abstract]

Wagner, R., Liedtke, S., Kretzschmar, E., Geyer, H., Geyer, R., and Klenk, H.D. (1996b) Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1, 2-N-acetylglucosaminyltransferase I. Glycobiology, 6, 165–175.[Abstract]

Whitford, M., Stewart, S., Kuzio, J., and Faulkner, P. (1989) Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J. Virol., 63, 1393–1399.[ISI][Medline]