Glycobiology, 2000, Vol. 10, No. 5 521-529
© 2000 Oxford University Press
Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors
Departments of Medicine and Biological Chemistry, Harvard Medical School, and the Joslin Diabetes Center, Boston, MA 02215, USA
Received on November 4, 1999; revised on December 15, 1999; accepted on December 15, 1999.
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Abstract |
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Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-
-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc1-3Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of monoglucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS–PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-ß-N-acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence. Key words: endomannosidase/glycoprotein processing/quality control of proteins/glucosylated N-linked oligosaccharides/oligosaccharide-lipids
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Glycosidases with either exo- or endo-cleavage action have been used extensively to follow the progress of newly synthesized glycoproteins from the ER to the various Golgi compartments. Among the enzymes which have been widely employed are neuraminidase, peptide:N-glycosidase and endo H; indeed the latter enzyme because of its distinct specificity for N-linked oligosaccharides in their early processing stages has proven to be a particularly useful tool in following by SDS–PAGE the migration of radiolabeled glycoproteins through the Golgi cisternae (Fries and Rothman, 1980
; Olden et al., 1980; Lodish et al., 1987; Hammond and Helenius, 1994).
The initial steps in the processing of N-linked oligosaccharides involve the removal of the triglucosyl sequence which is a determinant for cotranslational N-glycosylation in most eukaryotic cells (Spiro et al., 1979; Turco and Robbins, 1979; Murphy and Spiro, 1981). This glucose excision takes place primarily in the ER through the sequential action of glucosidases I and II (Moremen et al., 1994), as well as to a lesser extent in the intermediate and cis/medial Golgi compartments by endomannosidase action (Lubas and Spiro, 1987; C.Zuber, M.J.Spiro, B.Guhl, R.G.Spiro, and J.Roth, unpublished observations). While it has been appreciated for some time that deglucosylation is necessary for the formation of mature complex carbohydrate units, it has become evident more recently that in their monoglucosylated form N-linked oligosaccharides play a major role in protein quality control (for review, see Helenius et al., 1997). It has been shown that glycoproteins are retained in the ER by the molecular chaperones calnexin and calreticulin until proper folding and/or oligomerization takes place. This retention process appears to be determined to a large extent by the lectin-like interaction of these chaperones with N-linked monoglucosylated oligosaccharides on newly synthesized glycoproteins (Ware et al., 1995; Spiro et al., 1996). Those which do not achieve proper conformation or chain assembly because of genetic factors (for review, see Hammond and Helenius, 1995) or impairment of binding to the chaperones due to a block in the formation of monoglucosylated oligosaccharides (Moore and Spiro, 1993; Kearse et al., 1994; Liu et al., 1999) undergo degradation with the attendant release of free oligosaccharides (Moore and Spiro, 1994).
In view of the increasing biological importance which is being assigned to the glucose residues on N-linked polymannose oligosaccharides, an enzymatic probe which can be used for determining the glucosylation state of radiolabeled glycoproteins in normal or altered cells would be a valuable asset. For this purpose endomannosidase would appear to be ideally suited as it has the unique property of cleaving the 1
2 linkage between glucose substituted mannose and the remainder of the 3'-trimannosyl branch of N-linked oligosaccharides with the release of a di-, tri-, or tetrasaccharide (Glc1-3Man) from mono-, di- and triglucosylated polymannose oligosaccharides respectively (Lubas and Spiro, 1988). Indeed its capacity to act effectively on glucosylated N-linked oligosaccharides in which the non-glucose-substituted mannose chains are trimmed has been shown to be similar to the specificity observed in the lectin-like chaperones (Spiro et al., 1996).
In the present study we demonstrate that endomannosidase in its highly purified recombinant form (Spiro et al., 1997) can be used to determine the state of glucosylation of radiolabeled glycoproteins produced in cultured cells during a CST-imposed glucosidase blockade or in the glucosidase I–deficient CHO mutant, Lec 23, through an examination of their change in mobility during SDS–PAGE. Employing cells infected with the temperature sensitive VSV mutant (ts045) we also show that endomannosidase is useful in demonstrating the presence of glucosylated-N-linked oligosaccharides in the ER-retained G protein. Furthermore, the endomannosidase could be effectively employed for characterizing the glucosylation state of free and lipid-linked oligosaccharides.
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Results |
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Effect of endomannosidase treatment on VSV-G protein obtained from CHO cells under glucosidase blockade or with a glucosidase deficiency
In order to prepare a glucosylated glycoprotein to be used as substrate for in vitro endomannosidase assays, CST-treated CHO cells were chosen since this cell line has no detectable endomannosidase activity which could circumvent a glucosidase blockade (Hiraizumi et al., 1993
). Moreover, since we found (data not shown) that Lec23, the glucosidase I-deficient CHO mutant (Ray et al., 1991), has no detectable endomannosidase activity when assayed over a wide range of protein concentrations, these cells could be used in addition to the parent cell line to explore the effect of in vitro deglucosylation on electrophoretic mobility in SDS–PAGE.
When VSV-infected CHO cells were radiolabeled in the absence of glucosidase inhibitors, the G protein was found to be resistant to the action of endomannosidase as well as endo H (Figure 1), as would be anticipated from the known presence of two complex N-linked oligosaccharides on this viral glycoprotein (Hunt et al., 1978). However, the G protein obtained from CST-treated cells was found to be sensitive to the action of both endomannosidase and endo H, as evident from its increased electrophoretic mobility (Figure 1). While the size reduction of ~1.5 kDa brought about by endomannosidase was consistent with the excision of two Glc3Man tetrasaccharides, the ~4.3 kDa size alteration produced by endo H indicated that as expected two triglucosylated polymannose oligosaccharides had been released (Figure 1). The increases in electrophoretic mobility of the G protein from Lec23 cells resulting from endomannosidase and endo H treatment were similar to that observed in the CST-treated parent cells, as would be anticipated in cells which have a glucosidase I deficiency (Figure 1). Moreover, when CST was added to the Lec23 cells, the same electrophoretic pattern subsequent to the enzyme treatments was observed, which confirmed that the genetically imposed glucosidase blockade was comparable to that brought about by CST (Figure 1).
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Although 300 ng of purified endomannosidase was employed in the standard incubations with radiolabeled glycoproteins, treatment of Lec23-produced G protein at various enzyme concentrations indicated that as little as 34 ng of enzyme can result in complete trimming during the 16 h assay period (Figure 2). Similar results were obtained with the G protein from CST-treated CHO cells (data not shown). Denaturation of the protein substrates by boiling or treatment with NaSCN (0.5 M) did not enhance endomannosidase action (data not shown).
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Examination of the effect of endomannosidase on lysosomal glycoproteins
When the cellular proteins of the CST-treated CHO cells were examined by SDS–PAGE after a 16 h incubation with [35S]methionine, a prominent endomannosidase-susceptible protein was observed with an Mr of ~120 kDa (Figure 3), while in the absence of CST, no effect of treatment with this enzyme was observed (data not shown). The substantial decrease in molecular mass brought about by endomannosidase (~13 kDa) suggested the presence of a large number of N-linked oligosaccharides in this protein, since the excision of each tetrasaccharide would represent a loss of 0.65 kDa. The multiplicity of carbohydrate units and the molecular mass of the glycoprotein suggested that it might be the lysosomal glycoproteins Lamp-1 and/or Lamp-2, which are known to have an Mr values in the 120 kDa range and to contain 16 to 20 N-linked oligosaccharides (Chen et al., 1985
, Fukuda, 1991). Indeed, examination of antilysosomal immunoprecipitates of cellular lysates from CST-treated CHO cells as well as the uninhibited Lec23 cell line yielded in each case a strong radiolabeled band of Mr ~120 kDa which upon endomannosidase treatment manifested a reduction in molecular mass of ~13 kDa (data not shown), and this is furthermore consistent with our previous demonstration that this antiserum reacts with a [3H]mannose-labeled component which migrates to the position of Lamp-1/Lamp-2 (Chandra et al., 1998).
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Evaluation of the effect of endomannosidase on monoglucosylated VSV G-protein produced at nonpermissive temperature by HepG2 cells infected with ts045
Cells infected with the temperature-sensitive mutant of VSV (ts045) are known upon incubation at 39.5°C to form an abnormally folded G-protein which remains in the ER due to a quality control interaction with the molecular chaperone calnexin in which its N-linked oligosaccharides partially trimmed to their monoglucosylated state are involved (Hammond and Helenius, 1994
). This situation provided us with a model in which we could examine whether endomannosidase action on an in vivo produced glycoprotein with monoglucosylated carbohydrate units would be apparent on SDS–PAGE. Indeed, upon endomannosidase digestion a clearly perceptible increase in the mobility of the G protein from the ts045-infected cells was noted which was consistent with a decrease in Mr due to the release of two Glc1Man disaccharides (Figure 4); although this change was small it was reproducible in multiple trials. The larger increment in mobility noted with endo H (Figure 4) indicated, as anticipated, that the two N-linked oligosaccharides are still present in their immature polymannose state.
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Action of endomannosidase on oligosaccharide-lipids
Incubation of 14C-labeled oligosaccharide-lipids with endomannosidase was performed to determine whether this enzyme can be used as a probe to determine the glucosylation state of these biosynthetic intermediates which are involved in the N-glycosylation of proteins. We explored the possibility that the endomannosidase susceptibility of the oligosaccharide-lipids could be assessed by SDS–PAGE as it had previously been noted that they migrate as distinct components close to the dye front (Spiro and Spiro, 1985
). Indeed, when tightly cross-linked gels were used for the electrophoresis, it became apparent that the migration of these oligosaccharide-lipids is related to the number of sugar residues, as exemplified by the faster movement of the unglucosylated lipid-linked Man9GlcNAc2 compared to its triglucosylated form (Figure 5, Std OL). Therefore, the observation that upon incubation with endomannosidase the mobility of the Glc3Man9GlcNAc2-lipid was distinctly enhanced provided evidence that the enzyme acts upon these molecules (Figure 5). When a mixture of oligosaccharide-lipids containing the tri-, di-, mono-, and unglucosylated forms of Man9GlcNAc2 was submitted to endomannosidase treatment, the broad band which it presented on SDS–PAGE, consistent with its heterogeneity, was converted to a distinctly faster and sharper component due to the release of mono to triglucosylated mannose saccharides (Glc1-3Man).
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A less direct but more detailed evaluation of the glucosylation state of oligosaccharide-lipids before and after endomannosidase treatment could be performed by thin layer chromatography of the oligosaccharides after release by endo H. Since endo H has been shown to act on lipid-linked oligosaccharides (Chalifour and Spiro, 1984
), the two enzymes can be readily used in sequence with the formation of carbohydrate units terminating in a single GlcNAc residue. By this approach it became apparent that the lipid-linked oligosaccharide mixture evaluated directly by SDS–PAGE (Figure 5) was indeed converted from a Glc3-1Man9GlcNAc2 mixture to the Man8 state (Figure 6) by treatment with endomannosidase due to the release of tetra-, tri-, and disaccharides. The Man9GlcNAc2 component present in this mixture remained unchanged (Figure 6). When pure lipid-linked Glc3Man9GlcNAc2 was incubated with endomannosidase, it was totally converted to the Man8 species which appeared as Man8GlcNAc upon endo H treatment (Figure 6).
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Endomannosidase treatment of free oligosaccharides
The recombinant endomannosidase also proved to be useful for determining the glucosylation state of free oligosaccharides, whether present as a single component or in a mixture (Figure 7). Glucosylated Man9GlcNAc2 oligosaccharides, for example, irrespective of the number of glucose residues, are exclusively trimmed to Man8GlcNAc2 (Figure 7). If the oligosaccharide mixture had contained glucosylated Man8GlcNAc2 or Man7GlcNAc2 species, endomannosidase treatment would have yielded Man7 or Man6 components, respectively. When used in conjunction with endo H, the nature of the reducing end of the oligosaccharides (i.e., terminating in a single GlcNAc or a di-N-acetylchitobiose moiety) can be determined in addition to the glucosylation state (Figure 7).
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Evaluation of the amount of endomannosidase required to cleave Glc1Man9GlcNAc2 under the standard 60 min incubation indicated that nearly total cleavage had been achieved with 5 ng of the enzyme (data not shown). Since it has previously been reported that the endomannosidase greatly favors monoglucosylated over di- and triglucosylated oligosaccharides (Lubas and Spiro, 1988
), the time course of Glc3Man9GlcNAc2 cleavage was examined with 540 ng of the enzyme. At this enzyme level, essentially complete release of the tetrasaccharide, with concomitant formation of the Man8 component was accomplished (Figure 8).
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Discussion |
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It is evident from the present study that endomannosidase can be effectively used for assessing the glucosylation state of glycoproteins and can therefore take its place among the glycosidases which have been used to follow the intracellular processing and migration of proteins with N-linked oligosaccharides. Moreover, the capacity of the endomannosidase to act on oligosaccharide-lipids and free oligosaccharides can be used as a tool to determine the presence of glucose residues in these biosynthetic intermediates and intracellular degradative products, respectively. The importance of glucose constituents for the biosynthesis of glycoproteins has been appreciated since the time it became apparent that this sugar, present on oligosaccharide lipids in the form of a triglucosyl sequence linked to the
1,3'-branch of polymannose-di-N-acetylchitobiose, is the optimal substrate for the ER oligosaccharyl transferase (Spiro et al., 1979; Turco and Robbins, 1979; Murphy and Spiro, 1981). More recently it has been demonstrated that monoglucosylated-N-linked oligosaccharides which are produced in the ER by the excision of the outer two glucose residues by glucosidases I and II or by the glucosidase II-UDP-Glc:glycoprotein glucosyltransferase cycle (Sousa et al., 1992), bind to the molecular chaperones calnexin and calreticulin until correct folding and oligomerization of peptide chains has taken place (Helenius et al., 1997). Indeed, it has been shown that when there is an impairment in the formation of monoglucosylated N-linked oligosaccharides due to a CST-imposed glucosidase blockade or to a genetic deficiency in glucosidase II (Reitman et al., 1982), this lectin-based control mechanism does not function adequately and a pronounced accelerated degradation occurs in some glycoproteins (Moore and Spiro, 1993; Kearse et al., 1994; Liu et al., 1999). Free glucosylated oligosaccharides are released into the cytosol during ER-associated protein degradation (Moore and Spiro, 1994) through the action of N-glycanase (Weng and Spiro, 1997), and moreover such oligosaccharides can also be the product of the hydrolytic action of the oligosaccharyltransferase (Spiro and Spiro, 1991).
In evaluating the action of the recombinant endomannosidase on glucosylated glycoproteins we employed as our primary model CST-treated CHO cells, as well as its Lec23 glucosidase I-deficient mutant, since the absence of endogenous endomannosidase activity in these cells precluded their use of the alternate deglucosylation route afforded by this enzyme (Moore and Spiro, 1990) and consequently tightened the glucosidase blockade. Furthermore, we chose to study these cells after infection with VSV so that the G envelope protein with its two well defined N-linked oligosaccharides would be a major product of their biosynthetic activities. While this system provided us with the G protein containing its oligosaccharides in the Glc3Man9GlcNAc2 form, infection with temperature sensitive VSV mutant (ts045) which is retained in the ER at 39.5°C (Hammond and Helenius, 1994) gave us the opportunity to test endomannosidase with monoglucosylated N-linked oligosaccharides.
The employment of SDS–PAGE for assessing the susceptibility of [35S]methionine-labeled proteins to the endomannosidase proved to be a sensitive tool for demonstrating the presence of tri- and monoglucosylated-linked oligosaccharides (Glc3Man9GlcNAc2 or Glc1Man9GlcNAc2) in the VSV G protein with its two carbohydrate units as well as the more extensively N-glycosylated lysosome associated membrane proteins (Lamp-1 and/or Lamp-2) with their 16 to 20 oligosaccharides. Assessment of endoglycosidase sensitivity of glycoproteins on the basis of enhanced electrophoretic mobility has been extensively used after endo H (Fries and Rothman, 1980; Olden et al., 1980) or peptide:N-glycanase treatments (Hirani et al., 1987). The finding that the endomannosidase acts effectively on intact oligosaccharide lipids extends its usefulness to determining the state of glucosylation of these biosynthetic intermediates in mutants (Kornfeld et al., 1979; Quellhorst et al., 1999) or in altered metabolic states such as glucose starvation (Rearick et al., 1981; Turco and Pickard, 1982) or energy deprivation (Spiro et al., 1983). Furthermore, the release of the lipid-linked oligosaccharide by endo H following the endomannosidase treatment provided a specific determination of the mannosylation state as well as the glucosylation of the carbohydrate moiety. The usefulness of the endomannosidase is broad since in contrast to the ER processing glucosidases (Grinna and Robbins, 1980) it acts well on glucosylated oligosaccharide substrates with truncated mannose branches, such as occur in mutants in which the synthesis of dolichol-P-mannose is impaired (Kornfeld et al., 1979). When used in conjunction with endo H on free oligosaccharides we show that the endomannosidase can be used to distinguish between free glucosylated oligosaccharides terminating on the their reducing end in a GlcNAc or di-N-acetylchitobiose moiety which appear in the cytosol and ER vesicles, respectively, during the early stages of glycoprotein biosynthesis (Moore and Spiro, 1994).
The utility of the recombinant endomannosidase in probing glycoproteins with N-linked oligosaccharides is enhanced by its ability to act on undenatured protein substrates and on immunoprecipitates and this would be consistent with the exposed location of the hydrophilic oligosaccharide units.
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Materials and methods |
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Cell culture and virus infection
CHO and HepG2 cells were obtained from ATCC (Rockville, MD) while the CHO mutant, Lec23 (Ray et al., 1991
), was obtained from Dr. Pamela Stanley (Albert Einstein College of Medicine, Bronx, NY). The growth media used were MEM (Sigma) containing 1 g of glucose/liter for CHO and Lec23 cells while RPMI (Gibco) was employed for HepG2. All media contained 10% FBS, streptomycin (100 µg/ml), and penicillin (100 U/ml), and the cells were kept at 37°C in a humidified atmosphere with 5% CO2. Infection of CHO and Lec23 cells with VSV (Indiana strain, ATCC) was performed after growth to ~80% confluence on 100 mm plates. Virus (1.2 x 109 p.f.u) was added in 1 ml of growth medium containing 20 mM HEPES, pH 7.4, and the plates were rocked gently at room temperature for 90 min; the virus was then removed and replaced with growth medium for a 4 h incubation at 37°C. HepG2 cells were infected with VSV strain ts045 (a gift from Dr. William Balch, Scripps Research Institute, La Jolla, CA) in a similar manner for 90 min at room temperature followed by 16 h at 32°C.
Radiolabeling of cells
VSV-infected and uninfected cells on 100 mm plates were washed twice with methionine-free medium (Sigma) containing 20 mM HEPES, pH 7.4, and 2% dialyzed FBS and incubated in 2 ml of this medium for 15 min at 37°C. Fresh methionine-free medium with or without the 6-O-butanoyl derivative of CST (a gift of Dr. M.Kang, Merrell Dow Research Institute, Cincinnati, OH) was added for a 30 min preincubation prior to addition of 300 µCi/plate of [35S]methionine (1198 Ci/mmol, DuPont-New England Nuclear). Following a further incubation for 16 h at 37°C, unlabeled methionine was added (2 mM final concentration) and after low-speed centrifugation (500 x g for 15 min) to remove free cells, the viruses were harvested from the medium by ultracentrifugation (105,000 x g for 120 min). Solubilization of viral proteins was accomplished at 4°C by addition to the pellet a lysis buffer consisting of 100 mM Na MES, pH 6.5, buffer containing 400 mM NaCl, 2% (v/v) Triton X-100, and a mixture of protease inhibitors (5 mM EDTA, 10 µg/ml leupeptin, 10 U/ml aprotinin, 10 mM iodoacetamide, and 2 mM PMSF). Following centrifugation (14,000 x g for 20 min) in an Eppendorf model 5415C microcentrifuge, the clear supernatant was used for further study. For the examination of intracellular proteins, the plates were washed with PBS containing 2 mM methionine and protease inhibitors and the cells were scraped from the plates, pelleted by a brief centrifugation (500 x g for 15 min) and treated at 4°C with the lysis buffer to yield a clear supernatant following centrifugation in the Eppendorf centrifuge.
For study of the G-protein produced by the temperature-sensitive strain of VSV, methionine-labeling experiments (360 µCi/plate) were performed at 39.5°C employing a 10 min pulse followed by a 90 min chase. In these experiments, only the cell lysate was studied.
Immunoprecipitation
Preparation of rabbit antiserum directed against lysosomal proteins from rat liver has been previously described (Chandra et al., 1998). Radiolabeled lysates from both CHO and Lec23 cells were reacted with Protein A-Sepharose CL-4B beads (Sigma) coated with these antibodies subsequent to pretreatment with beads coated with preimmune serum. After extensive washing with buffer containing 50 mM NaMES, 300 mM NaCl, 0.1% Triton X-100, 0.02% SDS in the presence of the protease inhibitors, aliquots were dried by lyophilization for reaction with the endomannosidase.
Preparation of radiolabeled oligosaccharide-lipids and oligosaccharides
Thyroid slices were incubated with [14C]glucose for the preparation of radiolabeled oligosaccharide-lipids and oligosaccharides (Spiro et al., 1976). Free monoglucosylated oligosaccharide (Glc1Man9GlcNAc) was obtained by endo H digestion of glycopeptides (Lubas and Spiro, 1988) while Glc3Man9GlcNAc2 was isolated after mild acid hydrolysis of the triglucosylated oligosaccharide-lipid (Spiro et al., 1976). To obtain mixtures of underglucosylated oligosaccharide-lipids, thyroid slices were incubated in the presence of CCCP (Spiro et al., 1983).
Endomannosidase digestions
The recombinant enzyme employed in these studies was produced in Escherichia coli and isolated by affinity chromatography as described previously (Spiro et al., 1997). The specific activity of the purified endomannosidase was ~70 U/µg protein, where one unit of activity is defined as the amount of enzyme that catalyzes the release of 1000 d.p.m. of Glc13Man per h (Hiraizumi et al., 1993).
For the digestion of [35S]methionine-labeled glycoproteins, aliquots of the viral and cell lysates, as well as of the immunoprecipitated proteins bound to Protein A-Sepharose, were dried on a Speed-Vac (Savant Instruments, Holbrook, NY) and subsequently incubated with the endomannosidase at 37°C in a total volume of 40 µl of 0.1 M NaMES, pH 6.5, containing 0.1% Triton X-100, 10 U/ml aprotinin, 10 µg/ml of leupeptin, 0.5 mM PMSF, as well as 1.0 mM each of CST and DMJ and 50 µg/ml of BSA in the presence of toluene. The protease and exoglycosidase inhibitors were included in the incubations to block possible activities associated with the substrates. Because of its strong inhibitory activity to endomannosidase, Tris had to be excluded from the digestions (Lubas and Spiro, 1988). In the standard assay, 300 ng of endomannosidase were employed and the incubations were carried out for 16 h. Examination of the digests were performed by SDS–PAGE.
Endomannosidase digestions of 14C-labeled oligosaccharide-lipids for analysis by SDS–PAGE were carried as described above except that the Triton concentration was 0.2% and that 600 ng of purified enzyme was used. When the oligosaccharide-lipids were incubated with endomannosidase with the object of examining the carbohydrate moiety by thin layer chromatography, the incubation volume was increased to 60 µl. Furthermore at the termination of the endomannosidase treatment, the digests were treated with endo H to release the oligosaccharide from the lipid (Chalifour and Spiro, 1984).
Treatment of 14C-labeled free oligosaccharides with the endomannosidase was carried out in a manner similar to that previously described (Lubas and Spiro, 1987). The substrates were incubated for varying periods of time at 37°C with the indicated amount of endomannosidase in 60 µl of the 0.1 M NaMES buffer containing 0.2% Triton X-100. Although no glycosidase inhibitors were required in these incubations, EDTA (80 mM) and BSA (100 µg/ml) were included in the assays to stabilize the enzyme when the highly diluted enzyme was used (<10 ng/tube). After deproteinization and desalting of the samples (Lubas and Spiro, 1987) the oligosaccharide products were resolved by thin layer chromatography. The saccharide components were visualized by fluorography, and then quantitated by densitometry (model 300A Molecular Dynamics Densitometer, Sunnyvale, CA) or by scintillation counting subsequent to elution from the chromatograms.
Endo H digestions
Cellular or viral glycoproteins, after boiling for 3 min in 0.5% SDS containing 0.1 M 2-mercaptoethanol (10 µl) were incubated with 4 mU of endo H (Genzyme, Cambridge, MA) in 50 µl final volume of 0.2 M sodium citrate buffer, containing aprotinin (10 U/ml), PMSF (1 mM) for 24 h at 37°C. Subsequent to these digestions the samples were examined by SDS–PAGE.
Free oligosaccharides were treated with endo H (4 mU) for 24 h at 37°C as described previously (Anumula and Spiro, 1983) followed by thin layer chromatographic examination of the desalted digests. Endo H digestion of oligosaccharide-lipids were carried out in a manner similar to that previously reported (Chalifour and Spiro, 1984) for 48 h at 37°C with 6 mu of the enzyme in 200 µl of the pH 5.2 citrate buffer containing 0.2% (v/v) Triton followed by chromatography of the desalted samples.
Assessment of endomannosidase activity in Lec23 cells.
Enzyme measurements were carried out on various aliquots (125–400 µg of protein) on the postnuclear membranes of disrupted cells in a manner described previously (Dairaku and Spiro, 1997) employing 14C-labeled Glc1Man9GlcNAc as substrate.
SDS–PAGE
Electrophoresis was carried out by the procedure of Laemmli (1970) on 10 to 15% polyacrylamide gels which were 1.5 mm thick and overlaid by 3.5% stacking gels; the radioactive components were detected by fluorography. Since the oligosaccharide-lipids migrate close to the dye front, care was taken during their electrophoresis that this marker did not run off the gel.
Thin layer chromatography
Chromatography of large oligosaccharides was carried out on plastic sheets precoated with Silica Gel 60 (0.2 mm thickness, Merck) for 20–24 h in 1-propanol/acetic acid/water, 3:3:2 (Solvent System A) while the small saccharide products of endomannosidase action (Glc3Man and Glc1Man) were separated by chromatography in pyridine/ethyl acetate/water/acetic acid, 5:5:3:1 (Solvent System B) for 24 h on plastic sheets precoated with cellulose (0.1 mm thickness, Merck). A wick of Whatman No. 3MM paper was clamped to the thin layer plates during chromatography.
Radioactivity measurements
Liquid scintillation counting was carried out with Ultrafluor (National Diagnostics) with a Beckman LS7500 instrument. Detection of radioactive components on thin layer plates was accomplished by fluorography at –80°C on X-Omatic AR film (Eastman Kodak) after spraying with a mixture containing 2-methylnaphthalene (Spiro and Spiro, 1985). The components on electrophoretic gels were visualized at –80°C after treatment with ENHANCE (DuPont-New England Nuclear) using the X-Omatic film.
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Acknowledgments |
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This work was supported by Grant DK17477 from the National Institutes of Health. We thank Vishnu Bhoyroo for help in some aspects of this investigation.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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VSV, vesicular stomatitis virus; G-protein, VSV glycoprotein; CST, castanospermine; DMJ, 1-deoxymannojirimycin; endo H, endo-ß-N-acetylglucosaminidase; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; FBS; fetal bovine serum; MEM, minimum essential medium; pfu, plaque forming units; BSA, bovine serum albumin; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MES, 2-(N-morpholino) ethanesulfonic acid; CCCP, carbonyl cyanide chlorophenylhydrazone; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride. All sugars are in the D-configuration.
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Footnotes |
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1 To whom correspondence should be addressed at: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215
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References |
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Chalifour,R.J. and Spiro,R.G. (1984) Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-ß-N-acetylglucosaminidase H. Comparison of enzymatic and acid hydrolysis techniques for saccharide release. Arch. Biochem. Biophys., 229, 386–394.[ISI][Medline]
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