Lepidopteran insect cells are used routinely as hosts for recombinant glycoprotein expression by using baculovirus vectors. The major properties of the baculovirus system include high expression levels and the ability of insect cell lines to synthesize N-glycans. But the major glycans bound to recombinant N-glycoproteins produced in such a system are unsuitable for use in human therapy.
The N-glycosylation pathway in higher eukaryotes starts by the transfer en bloc in the rough endoplasmic reticulum (ER) of a tetradecasaccharide (Glc3Man9GlcNAc2) from a lipid intermediate (oligosaccharide-PP-Dol) to an Asn residue in the Asn-X-Ser/Thr consensus sequence of a nascent protein. This process is immediately followed by sequential deglycosylation steps. The Man8GlcNAc2-protein is the key structure which leaves the ER to the Golgi apparatus. This oligomannoside-type glycoprotein is further processed in the different Golgi stacks, being trimmed by mannosidase I producing a Man5GlcNAc2 structure. Following the addition of a single GlcNAc residue by GlcNAc transferase I (GnTI), two mannose residues are removed by mannosidase II. This constitutes a prerequisite step for elongation by GlcNAc transferase II (GnTII), galactosyl- and sialyltransferases to give a variety of complex-type glycans (for a review, see Kornfeld and Kornfeld, 1985).
By contrast, although the occurrence of Glc3Man9GlcNAc2-PP-Dol has been demonstrated (Quesada Allue and Belocopitow, 1978; Butters et al., 1981; Sagami and Lennarz, 1987; Parker et al., 1991), the processing of N-glycans in insect cells remains poorly defined. Taking the processing occurring in mammalian cells as a model, some enzymatic activities have been detected and in some cases, cDNAs have even been isolated. Several lines of evidence have shown that these cells possess mannosidase I (Davidson et al., 1991; Kerscher et al., 1995; Ren et al., 1995; Kawar et al., 1997), mannosidase II (Altmann and März, 1995; Jarvis et al., 1997; Ren et al., 1997), GnTI and GnTII (Altmann et al., 1995; Velardo et al., 1993), and fucosyltransferase (Staudacher et al., 1992). Furthermore, some information on insect cell processing has been provided by structural studies on foreign recombinant proteins, suggesting that they can produce glycoproteins with galactose and sialic acid (Davidson et al., 1990; Davidson and Castellino, 1991a,b; Ogonah et al., 1996; Hsu et al., 1997).
The only evidence for the processing pathway have been obtained by the effects of inhibitors on electrophoretic mobility and/or endoglucosaminidase H sensitivity of glycoproteins (Jarvis and Summers, 1989; Jarvis et al., 1990; Jarvis and Garcia, 1994). In this report we provide a more direct analysis by looking at the effect of glycosidase inhibitors (castanospermine, swainsonine) and an intracellular trafficking inhibitor (monensin) on the glycans themselves. This allowed us to demonstrate in vivo the trimming pathway leading from the glycan precursor Glc3Man9GlcNAc2, to the final product, Man3(Fuc)GlcNAc2.
Figure 1. Metabolic labeling of Sf9 cells with 2-[3H]mannose. Sf9 cells were labeled for 30 or 120min with 2-[3H]mannose under the conditions described under Materials and methods. (A) shows the HPLC analysis of glycan moieties linked to oligosaccharides-PP-Dol after 120 min labeling. (B) and (C) show the HPLC analysis of glycan moieties linked to glycoproteins after 30 and 120 min labeling, respectively. G3M9Gn2, G2M9Gn2, and G1M9Gn2 indicate oligomannoside species possessing two GlcNAc and nine mannose residues with three, two, or one glucose residues, respectively. M9Gn2 and M8Gn2, indicate oligomannoside species possessing two GlcNAc residues and nine or eight mannose residues, respectively. M3 indicates the elution time of an oligomannoside possessing three mannose residues and two GlcNAc residues. Labeling of Sf9 cells with 2-[3H]Man
Sf9 cells were incubated under standard conditions in the presence of 2-[3H]Man. Figure
Figure 2. Effect of tunicamycin on the incorporation of 2-[3H]mannose on oligosaccharide-PP-Dol and on glycoproteins. Sf9 cells were labeled with 2-[3H]mannose under the conditions described in Materials and methods for 60 min in the absence (0) or in the presence of various concentrations(1, 2.5, and 5 µg/ml) of tunicamycin.
Since most of the oligosaccharide structures synthesized by Sf9 cells have been shown to be fucosylated, we investigated the fucosylation status of the product migrating as Man3GlcNAc2 species. In an attempt to specifically label fucosylated compounds, Sf9 cells were incubated with 6-[3H]Fuc. Whatever the incubation time from 30 min to 2 h, no significant radioactivity was detected either in the lipid or in the glycoprotein fraction. However, when the product migrating as Man3GlcNAc2 prepared from mannose-labeled glycoproteins was acid-treated under conditions designed to release fucosyl residues or treated with [alpha]-fucosidase from bovine epididymis, radioactive fucose could be detected (Figure
Figure 3. Fucosylation status of metabolically labeled Man3GlcNAc2 species. Sf9 cells were labeled for 120 min with 2-[3H] mannose under the conditions described under Materials and methods. The "M3" (see Figure 1) species was isolated by preparative HPLC from the labeled glycans of the glycoprotein fraction. The isolated material was analyzed by HPLC using isocratic conditions and two successive columns. Control (A) indicates the migration of "M3" species isolated from glycoproteins. This material was treated by [alpha]-fucosidase or submitted to mild acid hydrolysis as described under Materials and methods (B and C, respectively). A cochromatography of acid treated material with the control was also performed (D). M3Gn2 indicates the elution time of an oligomannoside possessing three mannose residues and two GlcNAc residues. M3(F)Gn2 indicates the elution time of an oligomannoside possessing three mannose residues and two GlcNAc residues, the terminal reducing one being fucosylated. F, Free fucose; arrow indicates the elution position for the free mannose.
To study the sequential events leading from Glc3Man9GlcNAc2 to Man3(Fuc)GlcNAc2, we used processing and intracellular trafficking inhibitors which have been shown to impair some key reactions in the mammalian N-glycosylation pathway. Effect of castanospermine
Figure Characterization of an hexosaminidase-sensitive intermediate accumulated in the presence of swainsonine
Swainsonine is known to impair the biosynthesis of complex glycoproteins by inhibition of mammalian Golgi mannosidase II (Tulsiani et al., 1982). It is also known that Sf9 mannosidase II is sensitive to swainsonine (Jarvis et al.,1997; Ren et al., 1997). Labeling of Sf9 cells under standard conditions in the presence of 5 µM swainsonine inhibits the formation of Man3(Fuc)GlcNAc2 as shown in figure
Figure 4. Effect of castanospermine on the pattern of oligomannosides bound to glycoproteins. Sf9 cells were labeled with 2-[3H]mannose under the conditions described in Materials and methods for 60 min in the presence of 150 µg/ml castanospermine. (A) represents the HPLC analysis of glycan moieties bound to the glycoprotein fraction. (B) shows the same material after digestion with [alpha]-mannosidase and purification on Biogel P2. G3M9Gn2 indicates oligomannoside species possessing two GlcNAc, nine mannose, and three glucose residues. G3M5Gn2, G2M5Gn2, and G1M5Gn2 indicate oligomannoside species possessing two GlcNAc and five mannose residues with three, two, or one glucose residues, respectively. M3(F)Gn2 indicates the elution time of an oligomannoside possessing three mannose residues and two GlcNAc residues, the terminal reducing one being fucosylated.
Figure 5. Effect of swainsonine on the processing of glycans bound to glycoproteins. Sf9 cells were labeled with 2-[3H]mannose under the conditions described in Materials and methods for 60 min in the presence of 5 µM swainsonine. The oligosaccharides obtained after PNGase digestion of glycoproteins were isolated by preparative HPLC (hatched peaks). The isolated peak A and Man9GlcNAc2 were analyzed before (Control) and after hexosaminidase or [alpha]-mannosidase treatment in the conditions described in materials and methods. Peak A was also successively submitted to hexosaminidase and to [alpha]-mannosidase. It was also treated with [alpha]-fucosidase. G1M9Gn2 indicates oligomannoside species possessing two GlcNAc, nine mannose, and one glucose residues. M9Gn2, M8Gn2, and M5Gn2 indicate oligomannoside species possessing two GlcNAc residues and nine, eight, or five mannose residues, respectively. M3(F)Gn2 indicates the elution time of an oligomannoside possessing three mannose residues and two GlcNAc residues, the terminal reducing one being fucosylated. F, Free fucose; M, free mannose; peak B has been identified as GlcNAcMan3GlcNAc2 species.
As this structure has been demonstrated in mammalian cells to be an acceptor substrate for the core [alpha]1,6-fucosyltransferase (Wilson et al., 1976), the presence of fucosyl residue was checked. Both trifluoroacetic acid hydrolysis (not shown) and bovine epididymis [alpha]-fucosidase digestion (Figure Effect of monensin
Monensin has been used to obtain information about the terminal steps of the glycosylation process. This carboxylic ionophore has been shown to impede the exit of secretory and membrane glycoproteins (Tartakoff and Vassalli, 1978), to lead to the dilation of Golgi vesicles, and to enhance the accumulation of sugar nucleotides (Cecchelli et al., 1986), and also to be effective on insect cells as shown by the blockage of secretion of an immune protein from insect fat body cells (Gunne and Steiner, 1993). When Sf9 cells were incubated under standard conditions in the presence of 10 µM monensin, the level of incorporation of 2-[3H] mannose on glycoproteins was decreased to 20% of the control, indicating that the N-glycosylation process was disturbed. Figure
Figure 6. Effect of monensin on protein synthesis, on the pattern of labeled glycoproteins and on radioactive incorporation onto glycoprotein after labeling with 2-[3H] mannose. Sf9 cells were labeled with [35S]Met and [35S]Cys mixture under the conditions described in Materials and methods for 15 min in the absence (open circles) or in the presence (solid circles) of 10 µM monensin. After precipitation and filtration, the radioactivity of the acido-precipitable material was determined by liquid scintillation (A). For electrophoresis, cells were labeled for 60 min with 2-[3H]mannose under the conditions described in Materials and methods in the absence (control) or in the presence (+ monensin) of 10 µM monensin. After lysis in the presence of 1% Triton X-100, the samples were run on an SDS-PAGE gel. Autoradiography was performed for the control. For quantification of the radioactivity, the proteins were transferred from the gel onto nitrocellulose membranes. The membranes were cut into 2 mm fractions along the electrophoretic path. The radioactivity of each fraction was determined by liquid scintillation counting (B). For the kinetic analysis after labeling with 2-[3H] mannose (C), SF9 were labeled under the conditions described in Materials and methods for 2, 4, or 6 h in the absence (open circles) or in the presence (solid circles) of 10 µM monensin. After sequential extraction the radioactivity of the protein pellet was measured by liquid scintillation.
Figure 7. Effect of monensin on the pattern of oligomannosides bound to glycoproteins. Sf9 cells were labeled with 2-[3H]mannose under the conditions described in Materials and methods for 30 min in the presence of 10 µM monensin. The oligosaccharides obtained after PNGase digestion of glycoproteins were isolated by preparative HPLC (hatched peaks). The isolated Man3(F)Gn2 and peak B were analyzed before (Control) and after mild acid hydrolysis, hexosaminidase, and jack bean [alpha]-mannosidase treatments under the conditions described in Materials and methods. G1M9Gn2 indicates oligomannoside species possessing two GlcNAc, nine mannose, and one glucose residues. M9Gn2 and M8Gn2 indicate oligomannoside species possessing two GlcNAc residues and nine or eight mannose residues, respectively. M3(F)Gn2 and M1(F)Gn2 indicate oligomannosides possessing two GlcNAc residues, the terminal reducing one being fucosylated and three or one mannose residues, respectively. F, Free fucose; M, free mannose. Conclusions
So far, the different steps of the N-glycan processing pathway in insect cells have been deduced by analogy to mammalian systems either from structural studies of baculovirus expressed recombinant glycoproteins (Hsu et al., 1997) or from the detection of glycosyltransferases and glycosidases. In this work, using metabolic labeling with 2-[3H]Man of non infected Sf9 cells we were able to demonstrate in vivo the sequential events of N-glycoprotein processing, leading from a Glc3Man9GlcNAc2 precursor to the Man3(Fuc)GlcNAc2 final product. The newly synthesized, labeled glycans were isolated from the bulk of the cell glycoproteins, making the pattern more relevant of major events than when glycans were studied from a single exogenous glycoprotein. More important is to note that in our case, the insect cell glycosylation process was not disturbed, in contrast to what was observed in the baculovirus infection. Indeed, highly modified glycosylation of recombinant human plasminogen expressed in baculovirus system during infection has been reported by Davidson and Castellino (1991a,b).
Use of castanospermine, swainsonine, and monensin allowed us to identify metabolic intermediates similar to the ones observed in mammalian cells: glucosylated species in the case of inhibition of glucosidases, GlcNAcMan5(Fuc)GlcNAc2 as a consequence of mannosidase II inhibition by swainsonine, and GlcNAcMan3(Fuc)GlcNAc2 which accumulated in the presence of monensin. The jack bean [alpha]-mannosidase specificity strongly suggests the following structure for this latter compound: GlcNAc[beta]1,2Man[alpha]1,3(Man[alpha]1,6)Man[beta]1,4GlcNAc[beta]1,4(Fuc[alpha]1,6)GlcNAc. These observations are in good agreement with the occurrence of mannosidase I (Davidson et al., 1991; Kerscher et al., 1995; Ren et al., 1995; Kawar et al., 1997), GnTI (Altmann et al., 1993; Velardo et al., 1993), mannosidase II (Altmann and März, 1995; Jarvis et al., 1997; Ren et al., 1997) and fucosyltransferase activities (Staudacher et al., 1992). A low GnTII activity has been measured by Altmann et al. (1993), but we could not detect significant amounts of products bearing a GlcNAc residue bound to the [alpha]1,6 core mannose, although their presence in low quantities could not be excluded.
Compared to mammalian cells, the difference of N-glycan processing in Sf9 cells is the removal of the GlcNAc residue previously transferred by GnTI. In fact, an unusual, membrane-bound [beta]-N-acetylglucosaminidase activity has been reported by Altmann et al. (1995) and proposed to be involved in the last step of N-glycans processing. Our results support the idea that this enzymatic activity is of physiological relevance to the glycosylation process and is Golgi-located. In fact in the presence of monensin which is known to block vesicular trafficking between Golgi stacks, we observed accumulation of both GlcNAcMan3(Fuc)GlcNAc2 species and Man3(Fuc)GlcNAc2 species. This indicates that the [beta]-N-acetylglucosaminidase activity must be located in Golgi vesicles that are different from the ones where are located the previous steps of the processing. Figure
Figure 8. Proposed pathway for the processing of endogenous glycoproteins of Sf9 cells. The sequence of events leading from Glc3Man9GlcNAc2 bound to asparaginyl residue of glycoproteins to the final Man3(F)GlcNAc2 has been deduced from intermediates characterized after the action of castano-spermine, swainsonine, and monensin. Only the intermediates which have been detected in this work have been represented. Cells and cell cultures
Sf9 cells were cultured in TC 100 medium (Gibco-BRL Life Sciences, Grand Island, NY, USA) supplemented with 5% fetal calf serum. The pH was adjusted to 6.2. Cells were cultured as monolayer at 28°C in 25cm2 flasks (Falcon, Becton Dickinson). Cell labeling, sequential extraction, and SDS-PAGE
Labelings were performed when cells had reached confluency. After removal of the culture medium, 2 ml of labeling medium was added (the labeling medium consisted of Grace medium (Gibco-BRL Life Sciences, Grand Island, NY) without glucose, sucrose, and fructose). It has been established that removal of glucose, sucrose, and fructose from Grace medium did not modify the glycosylation pattern of lipid intermediates and glycoproteins but led to an increased incorporation of label. Under standard conditions, incubations were performed at 28°C for 2 h in the presence of 50 µCi/ml 2-[3H]Man (specific radioactivity: 429GBq/mmol, Amersham, Bucks, UK). When inhibitors were added, the following concentrations were used: tunicamycin (from 1 to 5 µg/ml), castanospermine (150 µg/ml), swainsonine (5 µM), and monensin (10 µM). A 30 min preincubation was performed in the presence of the inhibitor before adding the radioactive precursor. In the case of fucose labeling, 50 µCi/ml of 6-[3H]Fuc (specific radioactivity: 1.1 TBq/mmol, Amersham, Bucks, UK) were used in the same standard conditions.
After incubation cells were washed with cold PBS pH 6.5, scraped from the flask and resuspended in 350 µl of a mixture containing : sodium cacodylate 0.1 M pH 7.4, immunoglobulin G 0.65%, and 5 mM MgCl2, then 800 µl of methanol and 1200 µl of chloroform were added. After mixing, a sequential extraction was performed as described by Cacan and Verbert (1995). Briefly, the interphase obtained after centrifugation of the chloroform/methanol/water mixture was extracted with chloroform/methanol/water 10:10:3 (by vol) to obtain the oligosaccharide-PP-Dol fraction and the residual glycoproteins.
For SDS-PAGE electrophoresis, cells were lysed after the incubation period in a Tris/HCl 50 mM buffer pH 7.4 containing 5 mM EDTA, 150 mM NaCl, and 1% Triton X100. After boiling in reducing sample buffer containing SDS, the samples were run on an SDS-PAGE gel. Hyperfilm from Amersham (Bucks, UK) was used for autoradiography of intensified gels. For quantification of the radioactivity, the proteins were transferred from the gel onto nitrocellulose membranes. The membranes were cut into 2 mm fractions along the electrophoretic path. The radioactivity of each fraction was determined by liquid scintillation counting. Release of glycan moieties and HPLC analysis
The glycan moieties were released from oligosaccharide-PP-Dol fraction by a mild acid hydrolysis treatment (HCl 0.1M in tetrahydrofuran, 80°C, 2 h).
The glycoprotein pellet was first trypsinized (300 µg TPCK-treated trypsin (Sigma, Saint-Louis, MO) in 300µl NaHCO3 0.1 M pH7.9) overnight at room temperature. After 10 min boiling, the trypsinate was dried and treated with 500 mU PNGase F (Boehringer Mannheim, Mannheim, Germany) in 100 µl phosphate buffer (20 mM pH 7.5, with 50 mM EDTA) overnight at 37°C.
Glycan moieties from oligosaccharide-PP-Dol or glycoprotein hydrolysis were purified on a Biogel P2 column (Bio-Rad, USA) using a 0.1 M acetic acid as solvent.
Radioactive oligosaccharides were recovered and analyzed by HPLC using an amino-derivatized column ASAHIPAK NH2P-50 (250 × 4.6 mm; Asahi, Kawasaki-ku, Japan). Oligosaccharide species were separated using a gradient of acetonitrile and water from 70/30 by vol to 50/50 by vol at a flow rate of 1 ml/min for 90 min. Peaks are detected using a continuous flow liquid scintillation (Flo-one [beta] detector, Packard, France). Identification of peaks was made by comparing their retention time with standard oligomannosides prepared from lipid intermediates obtained from labeled wild type CHO cells or the Man-P-Dol deficient CHO cells B3F7 (Kmiécik et al., 1995). The separation was achieved according to the number of Glc, Man, and GlcNAc residues from Man1GlcNAc2 to Glc3Man9GlcNAc2. In the case of separation of fucosylated from nonfucosylated species (Man3(Fuc)GlcNAc2 from Man3GlcNAc2) and of free mannose from fucose, isocratic conditions (acetonitrile/water, 70/30 by vol) and two successive columns were used.
When preparative chromatography was performed, 1 ml fractions were collected from the column and 50 µl aliquots were counted by liquid scintillation. Cleavage of fucose by acid hydrolysis
Fucose was released from fucosylated oligosaccharides by mild acid treatment (trifluoroacetic acid 0.05 M at 100°C during 3 h) according to Michalski (1995). After drying under nitrogen, defucosylated samples were analyzed by HPLC. Exoglycosidase digestions
Oligosaccharide samples (from 50,000 to 200,000 d.p.m.) were incubated with one of the following mixtures at 37°C for 24 h. (1) N-Acetyl-[beta]-hexosaminidase digestion: enzyme from jack bean (200 mU, Oxford Glycosciences, UK) in 100 mM sodium citrate phosphate pH 5 (15 µl total volume). (2) [alpha]-Mannosidase digestion: enzyme from jack bean (500 mU, Oxford Glycosciences, UK) in 50 mM sodium acetate pH 5, 1 mM ZnCl2 (20 µl total volume). (3) [alpha]-Fucosidase digestion: enzyme from bovine epididymis (40 mU, Oxford Glycosciences, UK) in 100 mM sodium citrate phosphate pH 6 (15 µl total volume), or enzyme from almond meal (20 µU, Oxford Glycosciences, UK) in 50 mM sodium acetate pH 5 (15 µl total volume). After incubation, enzyme was precipitated by cold ethanol and removed by filtration through 0.45 µm Millipore filter, and then the samples were analyzed by HPLC. Labeling of proteins
After being preincubated during 30 min with Met/Cys free medium, cell cultures were incubated with 100 µCi of radioactive [35S]Met-[35S]Cys mixture: (Amersham, Bucks, UK). After 30 or 60 min of incubation, the cells were lysed by 1% SDS and precipitated by a mixture of 0.6% PTA and 12% TCA. After filtration on glass fiber filters (Whatman GF/A) and washings by 10% TCA, the precipitates were counted by liquid scintillation.
We are very thankful to Drs. Martine Cérutti and Gérard Devauchelle (Station de Pathologie Comparée, INRA CNRS URA 2209, 30380 Saint Christol Lèz Alès, France) for their generous gift of the Sf9 cell line. We are thankful to the Professor Donald Jarvis for his critical reading of the manuscript. This work was supported in part by EEC contract number ERB FMRX CT96 0025 (Carenet 2), by CNRS (Programme Physique Chimie du Vivant, Réseau GT-rec) and by USTL.
ER, endoplasmic reticulum; GnTI, N-acetylglucosaminyltransferase I; GnTII, N-acetylglucosaminyltransferase II; HPLC, high performance liquid chromatography; oligosaccharide-PP-Dol, oligosaccharide pyrophosphodolichol; PBS, phosphate-buffered saline; PNGase, peptide N-glycanase; TPCK, N-tosyl-l-phenyl-alanine chloromethyl ketone.
Results
Materials and methods
Acknowledgments
Abbreviations
References
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