Glycobiology, 2000, Vol. 10, No. 2 177-183
© 2000 Oxford University Press
An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection
Med. Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität Marburg, Robert-Koch-Strasse 17, D-35037 Marburg, Germany and 3Institut de Biologie de Lille-Institut Pasteur, Lille, France
Received on May 25, 1999; revised on August 26, 1999; accepted on August 30, 1999.
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Abstract |
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The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T.gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH2-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.
Key words: baculovirus expression system/GPI biosynthesis/Toxoplasma gondii surface antigen (SAG1)
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentAbbreviationsReferences |
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The baculovirus system is one of the most widely used eukaryotic expression systems, for production of high levels of protein for detailed structural and functional studies (Luckow and Summers, 1988a
). The ability of insect cells to perform many of the post-translational modifications of higher eukaryotes, including phosphorylation, glycosylation, correct signal peptide cleavage and the removal of introns by proper splicing, makes it a valuable tool for in vitro production of biologically active proteins (Luckow and Summers, 1988b). N-Glycosylation and GPI-anchor biosynthetic pathways play a particularly interesting role in the biology of parasites, since their surface antigens and their carbohydrate structures have to interact with the host’s immune system. In case of GPI-anchored proteins, it has been shown that substantial, biological and immunological properties are linked to this modification (Schofield and Tachado, 1996). There is a growing body of evidence that GPI-anchors exhibit a variety of functions other than the mere anchoring of membrane proteins, e.g., the sequestration of GPI-proteins in specialized membrane microdomains, the targeting of GPI-proteins to the apical membrane in polarized cells or participation in signaling mechanisms (Eckert et al., 1997). Such important functions of posttranslational modifications have to be taken into account when expressing surface antigens from parasitic protozoa in a heterologous system, such as, the baculovirus system for the purpose of vaccine development. The N-glycosylation capability of the baculovirus system has been elucidated in detail by several groups (Grünewald et al., 1996; Wagner et al., 1996a,b; Hsu et al., 1997). The expression of GPI-proteins in their anchored form in the baculovirus system has been described (Murphy et al., 1990; Davies and Morgan, 1993; Richardson et al., 1993; Chaudhri et al., 1994; Longacre et al., 1994). However, these earlier reports of GPI-anchored recombinant proteins provided only indirect evidence for GPI-anchoring, such as surface fluorescence, incorporation of radioactivity after metabolic labeling with GPI components or reactivity towards the GPI cross-reacting determinant (CRD)-antibody. We expressed the Toxoplasma gondii SAG1 surface antigen (Burg et al., 1988; Soldati and Boothroyd, 1993) in this system in order, to elucidate the structure of its GPI-anchor and for functional comparisons with the endogenous SAG1-anchor. However, the use of well established and highly sensitive techniques for the detection of GPI-anchors did not provide any evidence for a SAG1-linked GPI-anchor. Therefore, a cell-free system from a commonly used insect cell line (H5) was established for the analysis of GPI-biosynthesis in this expression system. Results obtained with this cell-free system demonstrate that the synthesis of GPI-anchors is reduced to almost background level following baculovirus infection by blocking the N-deacetylation of GlcNAc-PI that leads to the formation of GlcNH2-PI which is essential for the subsequent mannosylation steps. |
Results |
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Expression of SAG1
We investigated the capacity of the baculovirus system to modify the recombinant Toxoplasma gondii SAG1 surface antigen with a GPI-anchor. High expression levels of the recombinant protein were confirmed by Western blot analysis (Figure 1A, lane 3). Insect cells expressing the recombinant protein in vivo were labeled at 48 h post-infection (p.i.) with either tritiated mannose (Figure 1B) or tritiated glucosamine (Figure 1C) in the presence or absence of tunicamycin, a N-glycosylation inhibitor (Elbein, 1987
). SAG1 could only be labeled in the absence of tunicamycin (Figure 1B, lane 1; Figure 1C, lane 1) indicating that only N-glycans but no GPI-anchors could be labeled on the recombinant protein. None of the other well established and highly sensitive methods for the detection of GPI-anchors such as radiolabeling with specific components (ethanolamine and myoinositol), Triton X-114 phase-separation, generation of labeled neutral core-glycan by dephosphorylation, deamination, and reduction with tritiated sodium borohydride (Ferguson, 1993) provided any evidence for a GPI-anchor linked to the recombinant SAG1 (data not shown). Therefore to determine the capacity of the lepidopteran insect cell line High Five (H5, Trichoplusia ni) to perform GPI-anchor biosynthesis per se, we established a cell-free system for in vitro labeling of GPI-glycolipids.
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1Bac; lane 3). Each lane contains the equivalent of 2 x 105 cells. (B) and (C) Fluorography of SDS–PAGE-resolved cell extracts from SAG1In vitro GPIs biosynthesis
We first determined the ability of uninfected cells to produce GPI glycolipids precursors. Figure 2A shows the thin layer chromatography (TLC) profile of in vitro, mannose-labeled glycolipids. To demonstrate that this TLC-spectrum, indeed, represents GPI-glycolipids, PI-PLC digestion was performed followed by TLC-analysis (Figure 2B). This experiment showed that most of these peaks were sensitive towards this treatment, a strong indication of their GPI-nature. The fastest migrating peak still present after PI-PLC treatment represents Dol-P-Man, since its formation was blocked in the presence of the Dol-P-Man synthesis inhibitor amphomycin (Elbein, 1987
) (Figure 2C). To obtain structural information about the GPI core glycan generated from GPI glycolipids produced in vitro, we released the evolutionary conserved GPI-core glycan from bulk glycolipid preparations by dephosphorylation, deamination and reduction as described in the experimental protocol, followed by HPAEC analysis. This treatment released four oligosaccharides, which comigrated with Man
1–4anhydromannitol, Man1–6Man1–4anhydromannitol, Man1–2Man1–6Man1–4anhydromannitol, and Man1–2Man1–2Man1–6Man1–4anhydromannitol standards derived from P.falciparum GPIs (Gerold et al., 1994) (Figure 3A). The large injection peak present in these analyses resulted from the use of bulk glycolipids (Schmidt et al., 1998). Cleavage with Aspergillus saitoi 1–2 mannosidase (Figure 3B) generated two fragments. One fragment coeluted on HPAEC with Man2-AHM and the second with mannose coeluting with Man1-AHM standard indicating the removal of two terminal 1–2-linked mannose residues. Jack bean -mannosidase treatment produced a single mannose peak coeluting with Man1-AHM standard (Figure 3C). These data indicate that the released GPI-core-glycan represents the conserved trimannosyl core-structure (Man1–2Man1–6Man1–4anhydromannitol), modified by an additional mannose in 1–2 linkage.
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Analysis of protein bound GPI–anchors in uninfected cells
We investigated the capability of insect cells to transfer GPI-precursors to nascent proteins by performing in vivo labeling with tritiated glucosamine in the presence of tunicamycin. After cell lysis, total protein was delipidated, acetone-precipitated, followed by liberation of the GPI core-glycan. The released core-glycan was then analyzed by HPAEC. The resulting labeled glycan comigrates with Man4-anhydromannitol standard (Figure 4). These data clearly show, that uninfected cells not only are able to produce GPI-precursors but also to transfer them to nascent GPI-proteins.
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In vitro GPIs biosynthesis following infection
In order to solve the discrepancy between GPI-biosynthesis in uninfected cells and lack of anchoring for the recombinant SAG1, we therefore, investigated the formation of free GPI-glycolipids in a cell-free system derived from baculovirus-infected cells. Figure 5A shows the TLC profile of in vitro, mannose-labeled glycolipids recovered in chloroform/methanol/water (one-step extraction) from uninfected cells. Figure 5B shows the glycolipid spectrum from insect cells infected with the recombinant baculovirus 24 h p.i. Most of GPI glycolipids disappeared to a nondetectable level in the case of infected cells. Identical results were obtained with cells infected with wild-type baculovirus, Autografa californica mononuclear polyhedrosis virus (AcMNPV) (data not shown). These data strongly indicate that GPIs biosynthesis is repressed in insect cell lines following infection with either wild-type or recombinant baculovirus. In all cases, Dol-P-Man was shown to be the only persistent glycolipid, whose synthesis was unaffected in infected cells. This indicated that the suppression occurred either at one of the early steps in GPI-biosynthesis or affected the utilization of Dol-P-Man, the donor of mannose residues in GPI-anchor biosynthesis (Menon et al., 1990
). To determine the time course of this suppression during infection, in vitro-labeling with GDP-[3H]Man was performed at various time points after infection with recombinant type virus and the recovered glycolipids were resolved by TLC. Incorporation of mannose residues into the PI-PLC-sensitive glycolipids was determined by integrating these peaks. As shown in Figure 5C, incorporation of mannose residues into GPI-glycolipids decreased between 8 and 10 h p.i. to almost background levels.
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Analysis of the early GPI intermediates
To determine if this drastic reduction in GPI-biosynthesis activity is due to a block in the early steps of the GPI-biosynthetic pathway or in mannose utilization, we investigated the synthesis of the early GPI intermediates GlcNAc-PI and GlcNH2-PI. Figure 6 shows the TLC profile of in vitro glucosamine labeled glycolipids recovered in chloroform/methanol extracts from uninfected cells and infected cells (Figure 6). Extracts from uninfected cells produce two peaks (1 and 2) representing the two early GPI intermediates, GlcNAc-PI and GlcNH2-PI, based on their sensitivity to PI-PLC (not shown) with a ratio of 4:1, respectively (Figure 6A). This ratio drops to 14:1 following infection (Figure 6B) indicating that the synthesis of the glycolipid 2 is affected. To discriminate between GlcNAc-PI and GlcNH2-PI, HNO2 treatment, which cleaves only GlcNH2-PI, was performed followed by butanol phase partition. The butanol phase was then analyzed by TLC chromatography. Following this treatment the ratio of glycolipid 1 to glycolipid 2 shifted to 8:1 indicating that glycolipid 2 contains a non N-acetylated glucosamine (Figure 6C). Bio-Gel P4 analysis of the water soluble fraction showed, that the labeled carbohydrate released from glycolipid 2 by HNO2 treatment, coeluted at 1.7 Gu (Figure 6D), corresponding to the elution position of anhydromannitol (Ferguson, 1993
). These data clearly show that in infected cells the deacetylation of GlcNAc-PI leading to the formation of GlcNH2-PI is affected, thus depriving the GPI biosynthesis machinery of the substrate for the subsequent elongation by the three mannoses necessary for the completion of the evolutionary conserved GPI-core-glycan.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentAbbreviationsReferences |
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The data presented here demonstrate, that expression of GPI-proteins in their anchored form cannot be achieved in the baculovirus system. By using the Toxoplasma gondii SAG1 surface antigen as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein, it was demonstrated, that radiolabeling of the recombinant SAG1 protein was only achieved in the absence of tunicamycin indicating that no GPI-anchors are present on the recombinant protein. Since it was shown that GPI-anchoring can be reduced or even abolished in heterologous systems, especially in the case of protozoan proteins, by poor recognition of the GPI-cleavage/attachment site (Moran and Caras, 1994
), we determined the capability of the baculovirus system to synthesize GPI anchors. Biochemical analysis, by using a cell-free system, showed that GPI biosynthesis is repressed upon baculovirus infection (either with wild type or recombinant virus). This repression occurs at a time point during infection that coincides with the switch from the early- to late-phase of the baculovirus infection cycle (Knudson and Harrap, 1976), thus explaining why the bulk of recombinant GPI-proteins expressed under the control of the standard late polyhedrin (polh) promoter cannot be isolated in a GPI-anchored form. TLC analysis showed that synthesis of virtually all GPI glycolipids is reduced to almost background levels while Dol-P-Man is still being produced in normal amounts. To elucidate if this reduction in GPI biosynthesis is due to a block in mannose utilization or lack of the mannose acceptor GlcNH2-PI, we labeled in vitro the early GPI-glycolipids (GlcNAc-PI and GlcNH2-PI) followed by HNO2 treatment and TLC-analysis of these two glycolipids. These experiments clearly showed, that following baculovirus infection the deacetylation of GlcNAc-PI, leading to the formation of GlcNH2-PI is reduced. Since the early intermediate GlcNAc-PI is still being made, it is obvious that GlcNAc-PI deacetylation is blocked which may cause a lack of substrate (GlcNH2-PI) for the subsequent mannosylation steps. Future analyses will have to determine the nature of this phenomenon, which may be due to enzyme inhibition or degradation as it has been shown that baculovius infection leads to a specific decrease in the activity of ß1
4-N-acetylgalactosaminyltransferase of lepidopteran cells (van Die et al., 1996) or even inhibition of gene expression for enzymes involved in the early steps of GPI-anchor biosynthesis. Earlier reports of GPI-anchored recombinant proteins in this expression system (Murphy et al., 1990; Davies and Morgan, 1993; Richardson et al., 1993; Chaudhri et al., 1994; Longacre et al., 1994), may be due to the residual GPI-biosynthetic activity observed during the early phase of the infection, sufficient for detection of GPI by using, e.g., the highly specific and sensitive CRD-antibody as an analytical tool (Chaudhri et al., 1994). Thus the baculovirus system appears to be unsuitable for the expression of proteins under the control of the polh promoter in these cell lines, in cases, where their GPI-anchor is required for downstream applications and analyses. Therefore, data obtained with GPI proteins expressed in the baculovirus system may have to be evaluated. |
Materials and methods |
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Insect cell culture
All techniques concerning insect cell culture and baculovirus propagation were performed as described (Summers and Smith, 1987
). Trichoplusia ni High Five (H5) insect cells (Invitrogen) were grown in monolayer cultures at 27°C in TC 100 medium (Gibco) supplemented with 10% fetal calf serum (Gibco).
Construction of a SAG 1 baculovirus transfer vector
The complete coding region for the T.gondii SAG1 gene was PCR-amplified from a T. gondii cDNA library and cloned into pVL1392 (Invitrogen) via BglII and EcoRI, omitting the first in-frame ATG codon, which was shown to decrease translation efficiency protein (Burg et al., 1988; Soldati and Boothroyd, 1993; Kim et al., 1994). The constructs were analyzed and confirmed by fine restriction enzyme-mapping and sequencing using an automated sequencer (ABI PRISM 377 DNA sequencer, Perkin Elmer). The recombinant transfer vector pVL1392SAG1 was cotransfected with a linear baculovirus DNA (Baculogold, Pharmingen) in the presence of lipofectin (Gibco) as a transfection reagent.
Analysis of recombinant protein expression
For protein production H5 cells were routinely infected with recombinant virus (moi = 10) and incubated at 27°C for 3–5 days in complete medium TC 100. After removing the culture supernatant by centrifugation, the cell pellet was washed twice with PBSdef and resuspended in sample buffer (1% glycerol, 1.5% SDS, 125 mM Tris/HCL (pH 6.8), 0.05% bromophenol blue) and resolved by 12% SDS–PAGE under nonreducing conditions, transferred to nitrocellulose, and probed with specific monoclonal antibodies raised against T.gondii SAG1 surface antigen. The secondary antibody (alkaline phosphatase-conjugated goat anti-mouse IgG (Promega)) was used according to the manufacture’s instructions and antibody-positive bands were visualized by the NBT/BCIP color development System (Promega).
Metabolic labeling of insect cells with [3H]-mannose and [3H]-glucosamine
About 5 x 106 H5 cells were infected with recombinant virus (moi = 10) and incubated at 27°C for 2 days in complete medium. For labeling, the medium was replaced with minimal medium (TC 100 without tryptose and glucose) in the presence or absence of 2 µg/ml tunicamycin (Calbiochem). The cells were incubated at 27°C for 2 h, and then the minimal medium was replaced with labeling mix (2 ml TC 100 without tryptose and glucose containing 500 µCi [2-3H] D-mannose or 500 µCi [6-3H] D-glucosamine (Amersham)) in the presence or absence of tunicamycin. The incubation was continued overnight. The cells were pelleted (1500 r.p.m./10 min/4°C, Heraeus Minifuge), washed twice with PBSdef., lysed for 1 h at 4°C in 1 ml 0.5% NP-40 lysis buffer containing fresh protease inhibitors. The lysate was analyzed by SDS–PAGE followed by autoradiography. Aliquots were used to generate core-glycan fragment as described below.
Biosynthesis and extraction of GPI–glycolipids in vitro
Cells were prepared as described above, except that the medium was supplemented with 10 µg/ml tunicamycin. After 1 h at 27°C, the cells were hypotonically lysed as described previously (Masterson et al., 1989). These cell lysates were centrifuged (13,000 r.p.m./10 min/4°C). The pellet was washed (three times) with 100 mM Na-HEPES (pH 7.4), 100 mM KCl, 10 mM MgCl2, 0.1 mM TLCK, 1 µg/ml leupeptin. After centrifugation, the pellet was resuspended in washing buffer supplemented with 5 mM MnCl2, 0.2 µg/ml tunicamycin, 1 mM ATP, 1 mM CoA, and 2 µCi of [3H]-labeled nucleotide sugars. Assays were supplemented with 1 mM GDP-mannose for experiments involving UDP-[3H]-N-acetyl-D-6-[3H]glucosamine or 1 mM UDP-N-acetylglucosamine for experiments involving GDP-D-6-[3H]mannose and incubated at 27°C for 1 h. For mannose labeling, glycolipids were extracted in one step by adding chloroform/methanol (C/M: 1:1, by volume) to the reaction mixtures to yield the final ratio of chloroform/methanol/water as C/M/W (10:10:3, by volume). For glucosamine labeling, glycolipids were extracted first with chloroform/methanol (C/M: 2:1, by volume) followed by extraction with C/M/W (10:10:3, by volume). Extracted glycolipids were analyzed by TLC on silica Gel 60 plates (Merck) using solvent system A: chloroform/methanol/0.25% KCl (10:10:3, by volume) or solvent system B: chloroform/methanol/acetic acid/water (25:15:4:2). After chromatography, the plates were dried and scanned for radioactivity with a Berthold LB 2842 automatic scanner. Bacillus cereus PI-PLC (Boehringer-Mannheim) and nitrous acid (HNO2) treatments were performed as described previously (Azzouz et al., 1995).
Bio-Gel P4 analysis
Glycolipids were deaminated (HNO2 treatment) as described previously (Mayor and Menon, 1990) and sized on Bio-Gel P4 column (1 130 cm, –400 mesh) equilibrated and eluted with 0.2 M ammonium acetate containing 0.02% sodium azide. Glucose oligomers from partially hydrolyzed dextran were included as internal standards and detected by oxidation after adding an aliquot of 25 µl from each fraction to 100 µl orcinol (2 mg/ml in concentrated sulfuric acid). Radioactivity was monitored by liquid scintillation counting.
Generation and analysis of the neutral core–glycans
Bulk glycolipids were dephosphorylated, deaminated and reduced as described (Mayor and Menon, 1990). The resulting material was desalted on AG3WX4 (OH–) and AG50WX12 (H+) tandem ion-exchange columns and filtered through a 0.2 µm filter. Neutral glycans were analyzed by high pH anion exchange chromatography (HPAEC) on a Dionex Basic Chromatography System. (Dionex Corp). The analysis was accomplished using a gradient elution program as described (Mayor and Menon, 1990). -Mannosidase digestions were performed as described (Azzouz et al., 1995). For generation of the neutral core glycan from labeled GPI-proteins, the lysates were first delipidated (Schmidt et al., 1998) and acetone-precipitated.
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Acknowledgment |
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We thank R.Dennis and M.Westermann for their critical reading of the manuscript. We thank A.Lander for providing the H5 cells. This work was supported by the Deutsche Forschungsgemeinschaft, PROCOPE/DAAD, Fonds der Chemischen Industrie, Hessisches Ministerium für Wissenschaft und Kunst and P.E.Kempkes Foundation Marburg, Germany. M.H.K. thanks the DAAD and the Hessische Graduierten Förderung for doctoral fellowships.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentAbbreviationsReferences |
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Dol-P-Man, dolichol phosphate mannose; GlcNAc, N-acetylglucosamine; GlcNH2, glucosamine; GPI, glycosylphosphatidyl-inositol; Gu, glucose units; HPAEC, high pH anion exchange chromatography; PI-PLC, phosphatidyl-inositol phospholipase C; SAG1, Toxoplasma gondii surface antigen; TLC, thin layer chromatography.
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Footnotes |
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1 These authors contributed equally to this work.
2 To whom correspondence should be addressed
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