TY - JOUR
AU1 - Marchal,, Ingrid
AU2 - Mir,, Anne-Marie
AU3 - Kmiécik,, Daniel
AU4 - Verbert,, André
AU5 - Cacan,, René
AB - ABSTRACT The most frequent type of N-glycan synthesized by lepidopteran Sf9 cells appears to be fucosylated Man3GlcNAc2, and this has been a limitation for a large scale production and utilization of therapeutic glycoproteins in cultured insect cells. The current knowledge of the protein glycosylation pathway derived from structural studies on recombinant glycoproteins expressed by using baculovirus vectors. In this work we provide more direct evidence for the sequential events occurring in the processing of endogenous N-glycoproteins of noninfected Sf9 cells. By metabolic labeling with radioactive mannose, we characterized the glycan structures which accumulated in the presence of processing inhibitors (castanospermine and swainsonine) and in the presence of an intracellular trafficking inhibitor (monensin). We thus demonstrated that from the glycan precursor Glc3Man9GlcNAc2 to β intermediate, the processing pathway in Sf9 cells paralleled the one demonstrated in mammalian cells. By using monensin, we demonstrated the formation of Man3(Fuc)GlcNAc2 from GlcNAcMan3(Fuc)GlcNAc2, a reaction which has not been described in mammalian cells. Our results support the idea that the hexosaminidase activity is of physiological relevance to the glycosylation pathway and is Golgi located. glycoproteins, insect cells, N-linked oligosaccharides, processing inhibitors Introduction 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. Results Labeling of Sf9 cells with 2-[3H]Man Sf9 cells were incubated under standard conditions in the presence of 2-[3H]Man. Figure 1 represents HPLC analyses of the oligosaccharide-PP-Dol and glycoprotein fractions. As has already been demonstrated, the major species occurring in lipid intermediates was Glc3Man9GlcNAc2 (Figure 1A). The pattern obtained with glycoproteins (Figure 1B,C) revealed Glc1Man9GlcNAc2, Man9GlcNAc2 and Man8GlcNAc2, which have been described in many biological models to originate from Glc3Man9GlcNAc2 by a trimming located in the rough ER. Besides these oligosaccharides, a species migrating like Man3GlcNAc2 could be detected. The substrate product relationships between the radioactivity bound to oligosaccharide-PP-Dol and glycoproteins is shown Figure 2. When the labeling was performed in the presence of tunicamycin (an inhibitor of the first step in the synthesis of oligosaccharide-PP-Dol), a 97% inhibition was observed onto lipid intermediates and a 90% inhibition onto glycoproteins. This indicated that Glc3Man9GlcNAc2 from lipid intermediates was the precursor of glycan species linked to proteins. It could be assumed that the product migrating as Man3GlcNAc2 was the end-product of the trimming of glycoproteins in Sf9 cells, since its quantity increased with the incubation time (Figure 1B,C) and chase experiments (not shown) indicated that it was formed at the expense of larger species. Fig. 1. Open in new tabDownload slide 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. Fig. 1. Open in new tabDownload slide 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. Fig. 2. Open in new tabDownload slide 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. Fig. 2. Open in new tabDownload slide 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 mannoselabeled glycoproteins was acid-treated under conditions designed to release fucosyl residues or treated with α-fucosidase from bovine epididymis, radioactive fucose could be detected (Figure 3). Indeed, as for many cells, the major origin of GDP-L-fucose is GDP-D-mannose (Ginsburg, 1961) rather than exogenous fucose which accounts for less than 10% of the GDP-L-fucose intracellular pool (Yurchenko and Atkinson, 1977). In fact, using isocratic conditions, Man3GlcNAc2 could be separated from Man3(Fuc)GlcNAc2, as demonstrated by cochromatography analysis of the Man3 species obtained before and after acid treatment (Figure 3D). The facts that the enzyme from bovine epididymis cleaves α1±6 fucose residues more efficiently than other α-fucose linkages and that the α1±3,4 fucosidase from almond meal was inefficient in releasing fucose (data not shown), demonstrated that fucose residues were α1±6 linked. 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 4 represents the pattern obtained on glycoproteins when the labeling was performed under standard conditions in the presence of 150 µg/ml castanospermine (Figure 4A), an inhibitor of rough ER glucosidases I and II (Pan et al., 1983). Compared to the control without castanospermine (Figure 1B,C), the Glc3Man9GlcNAc2 species was observed as expected, but also the final Man3(Fuc)GlcNAc2 was lacking. The glycan moieties observed on proteins were mainly glucosylated, as demonstrated by the action of α-mannosidase which led to the formation of a major Glc3Man5GlcNAc2 peak (Figure 4B), since the glucosylated α1,3 branch of the oligosaccharides was protected from the digestion by the exoglycosidase. It has been well described (Kobata, 1976) that the Manα1,6 in the trimannosyl core of the N-linked sugar chain was not cleaved by jack bean α-mannosidase digestion, unless the Manα1,3 of the core was removed. Thus, the α-mannosidase treatment of Glc3Man9GlcNAc2 produced Glc3Man5GlcNAc2. In fact the pattern obtained after α-mannosidase treatment (Figure 4B) was a mixture of different Man5 oligomannosides with additional 1, 2, or 3 glucose residues as they comigrated with authentic Glc3Man5GlcNAc2, Glc2Man5GlcNAc2, and Glc1Man5GlcNAc2 obtained from Man-P-Dol deficient cell line B3F7 (see Material and methods). The presence of species having a size higher than Glc3Man5GlcNAc2 (20% of the radioactivity) after α-mannosidase digestion was presumably due to incomplete hydrolysis. Thus, as in mammalian cells, the processing of N-glycans starts by the removal of glucose residues by the action of castanospermine-sensitive glucosidases. Similar conclusions have been drawn from SDS-PAGE mobility and endoglucosaminidase H sensitivity changes in glycoproteins after castanospermine treatment of the cells (Jarvis et al., 1990; Jarvis and Garcia, 1994). Fig. 3. Open in new tabDownload slide 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 α-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. Fig. 3. Open in new tabDownload slide 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 α-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. 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 5 (preparative HPLC). Under these conditions, intermediates are observed between the expected Man5GlcNAc2 species and Glc1Man9-, Man9-, and Man8GlcNAc2 species. Among these peaks, the peak A, migrating as Man6GlcNAc2 was the only one sensitive to N-acetyl-β-hexosaminidase, leading to Man5GlcNAc2 (see as control, the behavior of Man9GlcNAc2 species) indicating the presence of GlcNAcMan5GlcNAc2 species. Whatever the incubation time, a part of the material contained in peak A was not affected by the action of hexosaminidase, suggesting also the presence of Man6GlcNAc2 (25% of the radioactivity). Peak A was incompletely hydrolyzed by α-mannosidase, leading to a product (peak B) which has been identified as GlcNAcMan3GlcNAc2 by its susceptibility to hexosaminidase and resistance to jack bean α-mannosidase (see also Figure 7, peak B). Indeed, jack bean α-mannosidase was not able to cleave a single α1,6-linked mannose from the core β-mannose as it has been clearly demonstrated for GlcNAcMan3GlcNAc2 isolated from bovine rhodopsin (Liang et al., 1979). In contrast peak A was totally hydrolyzed by sequential treatment with hexosaminidase and jack bean α-mannosidase (Figure 5). This demonstrated that peak A contained also GlcNAcMan5GlcNAc2 species. Fig. 4. Open in new tabDownload slide 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 α-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. Fig. 4. Open in new tabDownload slide 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 α-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. As this structure has been demonstrated in mammalian cells to be an acceptor substrate for the core α1,6-fucosyltransferase (Wilson et al., 1976), the presence of fucosyl residue was checked. Both trifluoroacetic acid hydrolysis (not shown) and bovine epididymis α-fucosidase digestion (Figure 5) led to the release of radioactive fucose. This confirmed that, as observed above, radioactive fucose was synthesized by Sf9 cells from radioactive GDP-Man. Thus, three intermediates accumulated in the presence of swainsonine were present in peak A: Man6GlcNAc2, GlcNAcMan5GlcNAc2, and β. 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 6A shows that in the presence of monensin, the synthesis of proteins was greatly affected as demonstrated by a 97% decrease of labeled methionine incorporation. When the mannose labeled glycoproteins were analyzed by SDS-PAGE, it clearly appeared that the mobility of glycoproteins labeled in the presence of monensin was different from those labeled in the control without monensin (Figure 6B). These results indicated that newly synthesized glycoproteins are only weakly labeled when cells were incubated in the presence of the inhibitor. In Figure 7 is reported the HPLC analysis of the glycan moieties bound to proteins synthesized under these conditions. Relative to the final product Man3(Fuc)GlcNAc2, the radioactivity incorporated in rough ER specific species (Glc1Man9-, Man9-, and Man8GlcNAc2) was low. More interesting was the presence of a new intermediate, referred to as peak B, migrating as Man4GlcNAc2. Fucose was released by trifluoroacetic acid hydrolysis of the two isolated peaks, demonstrating that these two oligosaccharides are fucosylated. It is worth mentioning that defucosylation led to a loss of radioactivity of the defucosylated material, indicating a poor incorporation of radioactive mannose residues due to low protein synthesis. This result is in agreement with our previous observations (Figure 6 and D.Jarvis, personal communication) that monensin dramatically inhibited overall protein synthesis. Thus, the poor mannose labeling reflected inhibition of protein synthesis and the observed labeling resulted mainly from fucosylation of nonlabeled, previously synthesized, glycoprotein acceptors, retained in subterminal Golgi compartments as an effect of traffic inhibition by monensin. This is demonstrated by kinetic studies of incorporation of the radioactivity from 2-[3H] mannose labeling onto glycoprotein acceptor in the presence or in the absence of monensin (Figure 6C). In the absence of monensin the incorporation of 2-[3H] mannose was linear up to 6 h, but in the presence of monensin the incorporation reached a plateau at 2 h, demonstrating the glycosylation of preexisting structures which are blocked in the secretion pathway. As fucose was the major radioactive sugar incorporated in the presence of monensin (Figure 7) it can be assumed that the material labeled in the presence of monensin was trapped in fucosyltransferase containing vesicles, i.e., Golgi complex. Compared to Man3(Fuc)GlcNAc2, peak B was susceptible to N-acetyl-β-hexosaminidase digestion leading to a product migrating as Man3(Fuc)GlcNAc2, indicating that the substrate was GlcNAcMan3(Fuc)GlcNAc2. As expected Man3(Fuc)GlcNAc2 was totally digested by α-mannosidase, releasing both low labeled free mannose and fucose labeled Man1(Fuc)GlcNAc2. As shown in Figure 7, the migration of peak B was not affected by jack bean α-mannosidase treatment. This is in agreement with the structure we proposed: GlcNAcMan3(Fuc)GlcNAc2, which has been demonstrated, as mentioned above, not to be susceptible to jack bean α-mannosidase digestion (Kobata, 1976; Liang et al., 1979). Fig. 5. Open in new tabDownload slide 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 α-mannosidase treatment in the conditions described in materials and methods. Peak A was also successively submitted to hexosaminidase and to α-mannosidase. It was also treated with α-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. Fig. 5. Open in new tabDownload slide 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 α-mannosidase treatment in the conditions described in materials and methods. Peak A was also successively submitted to hexosaminidase and to α-mannosidase. It was also treated with α-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. 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 Fig. 6. Open in new tabDownload slide 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. modified glycosylation of recombinant human plasminogen expressed in baculovirus system during infection has been reported by Davidson and Castellino (1991a,b). Fig. 6. Open in new tabDownload slide 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. modified glycosylation of recombinant human plasminogen expressed in baculovirus system during infection has been reported by Davidson and Castellino (1991a,b). Fig. 7. Open in new tabDownload slide 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 α-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. Fig. 7. Open in new tabDownload slide 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 α-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. 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, β as a consequence of mannosidase II inhibition by swainsonine, and GlcNAcMan3(Fuc)GlcNAc2 which accumulated in the presence of monensin. The jack bean α-mannosidase specificity strongly suggests the following structure for this latter compound: GlcNAcβ1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4(Fucα1,6)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 α1,6 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, membranebound β-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 β-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 8 proposes a scheme of the processing of N-glycoproteins in Sf9 cells based on our observations. It is interesting to note that when looking at the glycosylation pattern of recombinant glycoprotein produced in the baculovirus expression system, a larger variety of structures can be detected (oligo-and paucimannosidic, hybrid, and complex types). This reinforces the idea that baculovirus infection greatly affects the glycosylation pathway of the recipient insect cells. Materials and methods 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. Fig. 8. Open in new tabDownload slide 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 castanospermine, swainsonine, and monensin. Only the intermediates which have been detected in this work have been represented. Fig. 8. Open in new tabDownload slide 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 castanospermine, swainsonine, and monensin. Only the intermediates which have been detected in this work have been represented. 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 TPCKtreated 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 β 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-β-hexosaminidase digestion: enzyme from jack bean (200 mU, Oxford Glycosciences, UK) in 100 mM sodium citrate phosphate pH 5 (15 µl total volume). (2) α-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) α-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. Acknowledgments 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. 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TI - Use of inhibitors to characterize intermediates in the processing of N-glycans synthesized by insect cells: a metabolic study with Sf9 cell line
JF - Glycobiology
DO - 10.1093/glycob/9.7.645
DA - 1999-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/use-of-inhibitors-to-characterize-intermediates-in-the-processing-of-n-eI0W20Tm4s
SP - 645
EP - 654
VL - 9
IS - 7
DP - DeepDyve
ER -