| Glycobiology | Pages |
Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity
Endo-[alpha]-d-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-[beta]-N-acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glc[alpha]1->3(1-deoxy)mannojirimycin, the conversion of the G protein into an endo H-resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.
Key words: endomannosidase/glycoprotein processing/vesicular stomatitis virus G protein/influenza virus hemagglutinin/ glucosidase inhibition/N-linked oligosaccharides
Introduction
Since the discovery of endomannosidase in rat liver Golgi membranes (Lubas and Spiro, 1987, 1988) it has become evident that this unique enzyme, which takes part in the processing of N-linked carbohydrate units by cleavage of the internal [alpha]1->2 linkage between the glucose- substituted mannose and the remainder of the polymannose oligosaccharide, can provide a pathway for circumventing glucosidase blockade imposed by inhibitors (Moore and Spiro, 1990) or genetic factors (Fujimoto and Kornfeld, 1991; Moore and Spiro, 1992). Although it has recently been shown that endomannosidase normally participates in the early trimming of N-linked oligosaccharides (Weng and Spiro, 1996), its distinct capacity for achieving deglucosylation by means other than ER glucosidase-mediated scissions has provided the most convincing demonstration of its in vivo action. As removal of the three glucose residues on the nascent N-linked oligosaccharide is a prerequisite for the formation of complex N-linked carbohydrate units (for review, see Moremen et al., 1994), the finding that continued synthesis of glycoproteins bearing these mature units takes place during a glucosidase blockade provides a sensitive demonstration that the endomannosidase route is being employed. Indeed, in studies with glucosidase inhibited HepG2 cells, it could be shown that several secretory glycoproteins could be processed to the endo H-resistant form characteristic of complex oligosaccharides (Rabouille and Spiro, 1992).
The wide distribution of endomannosidase in mammalian cells and indeed in all classes of vertebrates (Dairaku and Spiro, 1997) suggests that this well characterized enzyme, which has recently been cloned (Spiro et al., 1997), has to be taken into account in studies in which glucosidase inhibitors are employed to explore the role of this sugar in regulating intracellular movement of glycoproteins as well as the quality control machinery (for reviews, see Elbein, 1991; Helenius, 1994). The latter event in particular has gained increased attention in recent years since it has become evident that chaperones like calnexin and calreticulin interact in a lectin-like manner with monoglucosylated N-linked carbohydrate units (Hebert et al., 1995; Ware et al., 1995, Spiro et al., 1996). Attempts to control human immunodeficiency virus replication with the use of glucosidase inhibitors have further focused attention on the deglucosylation process (for review, see Feizi and Larkin, 1990).
The present study was undertaken to explore whether endomannosidase can facilitate the processing of N-linked oligosaccharides on membrane glycoproteins during a glucosidase blockade and to determine to what extent the use of this alternate deglucosylation route is cell specific. For this purpose we have chosen as a model the G protein of VSV since it bears two well characterized complex triantennary N-linked oligosaccharides (Reading et al., 1978) and moreover this virus has the capacity to infect a large variety of cells. Indeed, this property of the virus enabled us to demonstrate that the utilization of the endomannosidase pathway for production of the G protein with mature carbohydrate units was clearly host cell dependent but did not necessarily correlate with the in vitro determined cellular level of this enzyme. These observations may prove useful in guiding the choice of cell lines for future studies in which glucosidase blockades are employed.
Results
Assessment of the capacity of various cell types to process the VSV G protein by the endomannosidase route
In order to evaluate the ability of VSV-infected cells to process the G protein of the virus by the endomannosidase pathway, we determined the endo H susceptibility of this envelope glycoprotein during a bCST-imposed glucosidase blockade. Since N-linked oligosaccharides cannot be converted into the complex carbohydrate units, which are known to be present on the native G protein (Reading et al., 1978), without prior removal of the glucose residues, the appearance in the presence of a glucosidase inhibitor of endo H-resistant form of this glycoprotein provides a sensitive index of in vivo endomannosidase action.
When this procedure was applied to HepG2 cells (Figure
Figure 1. Evaluation of endomannosidase processing of VSV G protein by HepG2 and MDCK cells in the presence of glycosidase inhibitors on the basis of its susceptibility to endo H treatment. Virus infected cells were radiolabeled with 60 µCi [35S]methionine in the absence (None) or presence of either 0.2 mM bCST (CST) or 0.18 mM kifunensine (KIF) for 15 h as described in Materials and methods. Subsequently, free virus was harvested from the medium and aliquots of the virus lysate were submitted to SDS-PAGE with (+) or without (-) prior endo H digestion. The components were visualized by fluorography and the position of migration of the G protein as well as the other VSV constituents are indicated to the left of the gels. The migration of the G protein (Mr = 70 kDa) was confirmed by immunoprecipitation as described in Materials and methods. Electrophoretic examination of the VSV produced by MDCK cells revealed a distinctly different response of G protein processing to a glucosidase blockade (Figure In order to evaluate to what extent the alternate processing route provided by endomannosidase is specified by the host cell, we examined the endo H responsiveness of the G protein in a number of additional VSV-infected cells. The electrophoretograms of the viruses harvested from these bCST-treated cells demonstrated a widely diverse utilization of the endomannosidase pathway, as exemplified in Figure Figure 2. Variation in the susceptibility to endo H digestion of the VSV G protein produced by various infected cell lines during glucosidase inhibition. The radiolabeling was carried out under the same conditions as in Figure 1 in the absence (Control) and presence of bCST (CST) or KIF. SDS-PAGE of the virus lysates was performed with (+) or without (-) prior endo H digestion. The components were visualized by fluorography. Figure 3. Assessment of the extent of endomannosidase processing of the VSV G protein by various cell lines as determined by the formation of endo H resistant N-linked oligosaccharides in the presence of glucosidase blockade. The designated VSV-infected host cells were radiolabeled with 60 µCi [35S]methionine for 15 h in the presence of 0.2 mM bCST. After harvesting of the virus from the medium, the susceptibility of its G protein to endo H digestion was examined by SDS-PAGE (see Figure 1) and quantitated after fluorography by densitometry of G protein bands. The values are plotted as the percent of the G protein which was resistant to the enzyme treatment and accordingly indicates processing of N-linked oligosaccharides beyond the glucosidase blockade. Determination of the radiolabel incorporated from the [35S]methionine into the VSV recovered from the medium after a 15 h incubation indicated that neither bCST nor KIF had any perceptible effect on virus production in any of the cell lines examined. In the presence of the inhibitors, an average of 96% and 103%, respectively, of the control value was observed.
Effect of endomannosidase inhibition on the processing of VSV G protein
In order to seek confirmation that the development of endo H resistance during glucosidase blockade is an index of G protein processing by endomannosidase, we preincubated VSV-infected BHK cells with GDMJ prior to radiolabeling. It was apparent that in the presence of this specific endomannosidase inhibitor (Hiraizumi et al., 1993), the G protein could not be processed to an endo H-resistant form as it was in the control and bCST-treated cells (Figure
Figure 4. Effect of endomannosidase inhibition on the processing of VSV G protein by BHK cells as assessed by endo H treatment. Virus infected BHK cells were radiolabeled with [35S] methionine in the absence (None) or presence of bCST (CST), KIF, or GDMJ (4 mM); in the last mentioned incubation, bCST was also included to prevent possible cleavage of the endomannosidase inhibitor. SDS-PAGE of the virus lysates was carried out with (+) or without (-) prior endo H digestion. The components were visualized by fluorography.
In vitro measurements of endomannosidase activity in various cell lines
In order to evaluate whether the capacity of the various host cells to process the VSV G protein by the endomannosidase route reflects the cellular level of this enzyme, in vitro assays were undertaken (Table I). While the complete lack of G protein processing in CHO cells was consistent with the absence of in vitro measured endomannosidase activity (cf. Figure
Table I.
| Cell type | Enzyme activitya d.p.m./mg × 10-2 |
| BRL | 57 |
| HepG2 | 16 |
| CHO | 0b |
| BW5147.3 | 29 |
| MDBK | 20 |
| MDCK | 13 |
| NRK | 21 |
| LLC-PK1 | 2 |
| PtK1 | 14c |
| BHK | 79 |
Evaluation of endomannosidase processing of influenza virus HA
Although influenza virus has a much narrower range of host cell infectivity than VSV, its capacity to infect MDCK cells gave us the opportunity to compare endomannosidase processing of envelope glycoproteins from two viruses in the same cell line. Such an examination indicated that processing of the HA glycoprotein by influenza virus infected MDCK cells was prevented by bCST treatment as observed for the VSV G protein; indeed, HA like the G protein remained susceptible to endo H digestion during the glucosidase blockade (Figure
Figure 5. valuation of influenza virus HA process by MDCK cells during glucosidase blockade. Virus infected cells were radiolabeled with 75 µCi [35S]methionine in the absence (Control) or presence of either 0.2 mM bCST (CST) for 15 h as described in Materials and methods. Aliquots of the lysed virus isolated from the medium were submitted to SDS-PAGE with (+) or without (-) prior endo H or PNGase digestion. The components were visualized by fluorography and the position of the HA (Mr = 85 kDa) as well as the NP and NA components are indicated to the left of the gels. The migration of HA was confirmed by immunoprecipitation as described in Materials and methods.
Discussion
It is apparent from the present study that endomannosidase can participate in the processing of the VSV G protein so as to relieve a glucosidase blockade and make possible the continued formation of complex N-linked oligosaccharides on this envelope glycoprotein. Our investigations, however, indicate that the endomannosidase route for the processing of the G protein is host cell dependent. Indeed when we evaluated the in vivo action of endomannosidase by its unique capacity of circumventing the arrest imposed on the early processing sequence by a glucosidase inhibitor, through an assessment of the extent to which the G protein was converted to an endo H-resistant form, we observed a cell specific variation which ranged from greater than 90% in PtK1 and HepG2 cells to undetectable in the CHO as well as the MDBK and MDCK cell lines. Only partial conversion of the G protein to an endo H-resistant state was evident in several cell lines and the two-banded electrophoretic pattern seen after treatment with endo H suggests that only one of the two N-linked oligosaccharides had undergone processing. As anticipated, incubation of cells in the presence of the specific endomannosidase inhibitor, GDMJ, resulted in a complete blockage of the conversion of the G protein into an endo H-resistant form.
In vivo evidence of G protein processing by endomannosidase was evident in seven of the ten cell lines examined. As would be expected CHO cells, which stand alone in having no detectable endomannosidase activity, kept the G protein exclusively in the immature endo H-sensitive state during glucosidase blockade. However, surprisingly, MDBK and MDCK cells, although endowed with a substantial complement of the enzyme, were also unable to process the VSV envelope glycoprotein by the endomannosidase route. This disparity prompted us to examine influenza virus HA processing in the MDCK cell line, and this again indicated that in vivo endomannosidase action on this viral glycoprotein did not occur to any extent. Since MDCK have previously been shown to utilize the endomannosidase pathway when total glycoprotein processing was evaluated in vivo (Hiraizumi et al., 1993), it would appear that the enzyme exhibits some molecular selectivity in its action or possibly that the virus envelope glycoproteins do not pass through the appropriate cellular compartment.
Our observation that the VSV G protein can be extensively processed (>90%) by the HepG2 cells indicates that this cell line has a high capacity for utilizing the endomannosidase route; previous studies from our laboratory had shown that at least five distinct secretory glycoproteins can employ this alternate deglucosylation pathway (Rabouille and Spiro, 1992).
The host cell dependence of endomannosidase utilization demonstrated in the present study which focused on a single viral envelope glycoprotein has relevance to the interpretation of studies which aim to explore the role which the polymannose-linked glucose residues may exert on intracellular movement or quality control of various glycoproteins. Indeed in the latter instance, the lectin-like interaction of the molecular chaperones, calnexin and calreticulin, have been implicated. Although in our present study we found no evidence that retention of glucose inhibited viral replication, which is consistent with previous studies on influenza virus (Pan et al., 1983) as well as the fowl plague virus (Romero et al., 1983), a number of reports have indicated that glucosidase inhibitors can function as inhibitors of human immunodeficiency virus proliferation and syncytium formation (Feizi and Larkin, 1990). While the mechanism of this antiviral action is currently unknown, any approaches which rely on the retention of glucose residues should be conducted in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.
Materials and methods
Culture of cells
All cell lines were obtained from ATCC (Rockville, MD). The cells were grown in the following media: RPMI-1640 (GIBCO) supplemented with 10% FBS for HepG2 cells; Coon's modified F-12 medium (Sigma) supplemented with 5% FBS for BRL 3A buffalo rat liver cells; DMEM containing 1.0 g/l glucose and supplemented with 10% FBS for MDBK cells; DMEM containing 4.5 g/l glucose and supplemented with 5% FBS for MDCK cells and NRK-45F normal rat kidney fibroblast cells; Medium 199 (GIBCO) supplemented with 3% FBS for LLC-PK1 pig kidney cells; DMEM containing 4.5 g/l glucose and supplemented with 10% tryptose phosphate broth (DIFCO) and 10% FBS for BHK-21 baby hamster kidney cells; DMEM containing Earle's balanced salt solution and supplemented with 10% FBS for PtK1 kangaroo rat kidney cells; Ham's F-12 (GIBCO) supplemented with 10% FBS for CHO cells; and DMEM containing 4.5 g/l glucose and supplemented with 10% FBS for BW5147.3 mouse lymphoma cells. Penicillin (100 U/ml) and streptomycin (100 µg/ml) were included in all cell cultures which were carried out at 37°C in an atmosphere of 95% air and 5% CO2 on 60 mm dishes (Falcon) with the exception of the BW5147.3 cells which were grown in suspension.
Virus infection and radiolabeling of cells
VSV (Indiana strain) was obtained from ATCC and propagated in BHK-21 cells; the stock virus (3 × 109 pfu/ml) was stored at -80/C. Influenza virus, PR8/MS (2.4 × 1011 pfu/ml) was a gift from Dr. Karl S. Matlin (Massachusetts General Hospital, Boston, MA). The cultured cells were infected with either virus at a multiplicity of infection of 20-50 pfu/cell.
Cell monolayers (90% confluent) on 60 mm dishes or in suspension (1 × 107 BW5147.3 cells per dish) were infected with VSV or influenza virus and at 3 h or 5 h postinfection, respectively, washed with methionine-free DMEM containing 3% dialyzed FBS. Following a 30 min equilibration with this medium at 37°C, 1 ml of fresh methionine-free DMEM with or without specified inhibitors, which included 0.18 mM KIF (Toronto Research Chemicals), 0.2 mM bCST (a gift from Dr. M. Kang, Merrell Dow Research Institute, Cincinnati, OH) or 4 mM GDMJ (Hiraizumi et al., 1993), was added for a further 40 min incubation. Subsequent to these preincubations, the infected cells were radiolabeled for 15 h with 60-75 µCi [35S]methionine (1110 Ci/mmol, DuPont-New England Nuclear) in 1 ml of methionine-free DMEM; the indicated inhibitors continued to be present during this period.
Viruses were harvested from the medium at the end of the incubations by ultracentrifugation (105,000 × g for 120 min) subsequent to a low-speed centrifugation (500 × g for 15 min) to remove free cells. Solubilization of protein was accomplished at 4°C by adding to the viral pellet 0.2 ml of lysis buffer consisting of 100 mM Tris/HCl, pH 7.6, buffer containing 400 mM NaCl, 2% (v/v) Triton X-100, and a mixture of protease inhibitors (Moore and Spiro, 1993).
Endo H and PNGase digestions
The solubilized virus was digested with endo H (4 mU, Genzyme) or PNGase F (1 U, Oxford GlycoSystems) as previously described (Nayak and Spiro, 1991) for 48 h and 24 h, respectively, at 37°C prior to SDS-PAGE.
SDS-PAGE
The electrophoresis of VSV and influenza virus was carried out in SDS on 10% and 8% polyacrylamide gels (1.5 mm thick), respectively, which were overlaid by 2.5% stacking gels, according to the procedure of Laemmli (1970). The radioactive components were visualized by fluorography at -80°C after treatment with ENHANCE (Du Pont-New England Nuclear) using X-Omatic AR film and quantitated by scanning with a laser densitometer (model 300A, Molecular Dynamics).
Endomannosidase assay
Measurements of this enzyme were carried out on postnuclear membranes of the various cell lines. For this purpose the harvested, washed cells were suspended in 4 volumes of 0.1 M NaMES, pH 6.5, containing 0.5 mM dithiothreitol and disrupted with a Branson sonifier as previously described (Weng and Spiro, 1993). Postnuclear supernatants (600 × g, 10 min) of the homogenates were centrifuged for 60 min at 160,000 × g to obtain the membrane pellets which after a wash with the homogenizing buffer were suspended in the same at a protein concentration of about 15 mg/ml. Endomannosidase activity was then determined in a manner similar to that previously described (Lubas and Spiro, 1987) by incubating aliquots of the postnuclear membranes with 14C-labeled Glc1Man9GlcNAc (10,000 d.p.m.) in 50 µl of 0.1 M NaMES, pH 6.5, containing 0.2% (v/v) Triton X-100, 10 mM EDTA, 2 mM CST, and 2 mM DMJ. The desalted products of the enzyme action were separated by thin layer chromatography and after visualization by fluorography the Glc[alpha]1->3Man component was quantitated by scintillation counting subsequent to elution from the plates. The substrate included in the incubations can yield a maximum of 2610 d.p.m. as the disaccharide.
Immunoprecipitation procedure
To confirm the identity of the radiolabeled VSV G protein and the HA component of the influenza virus, immunoprecipitation of the lysed viruses was carried out with antisera and protein A-Sepharose as previously described (Rabouille and Spiro, 1992) prior to SDS-PAGE. For the VSV G protein, rabbit polyclonal serum (5 µl) generously provided by Dr. John K. Rose (Yale University, New Haven, CT) was used, while HA was immunoprecipitated with a monoclonal antibody (5 µl) which was a gift of Dr. Karl S. Matlin (Massachusetts General Hospital, Boston, MA); in the latter case, a 2 h incubation with rabbit anti-mouse IgG (Sigma) preceded the addition of the protein A-Sepharose.
Protein determination
The quantitation of protein was achieved with the Bio-Rad dye binding assay (Bradford, 1976) and bovine serum albumin as a standard.
Acknowledgments
This work was supported by Grants DK17325 and DK17477 from the National Institutes of Health; V.K.K. was supported by an American Diabetes Association Mentor-based postdoctoral fellowship.
Abbreviations
VSV, vesicular stomatis virus; G protein, VSV glycoprotein; HA, hemagglutinin; CST, castanospermine; bCST, 6-O-butanoyl-CST; KIF, kifunensine; DMJ, 1-deoxymannojirimycin; GDMJ, Glc[alpha]1->3DMJ; endo H, endo-[beta]-N-acetylglucosaminidase; PNGase, peptide:N-glycosidase; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney; MDBK, Madin-Darby bovine kidney; FBS; fetal bovine serum; DMEM, Dulbecco's modified Eagle medium; pfu, plaque forming units; MES, 2-(N-morpholino)ethanesulfonic acid. All sugars are in the d-configuration.
References
1To whom correspondence should be addressed at: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215
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