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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Key words: endomannosidase/glycoprotein processing/quality control of proteins/glucosylated N-linked oligosaccharides/oligosaccharide-lipids
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Introduction |
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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; C.Zuber, M.J.Spiro, B.Guhl, R.G.Spiro, and J.Roth, unpublished observations).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 Ideficient CHO mutant, Lec 23, through an examination of their change in mobility during SDSPAGE. 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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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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By this approach it became apparent that the lipid-linked oligosaccharide mixture evaluated directly by SDSPAGE (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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Discussion |
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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 SDSPAGE 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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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 Glc
1
3Man 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 SDSPAGE.
Endomannosidase digestions of 14C-labeled oligosaccharide-lipids for analysis by SDSPAGE 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 SDSPAGE.
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 (125400 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.
SDSPAGE
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 2024 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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Abbreviations |
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Footnotes |
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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, 386394.[ISI][Medline]
Chandra,N.C., Spiro,M.J. and Spiro,R.G. (1998) Identification of a glycoprotein from rat liver mitochondrial inner membrane and demonstration of its origin in the endoplasmic reticulum. J. Biol. Chem., 273, 1971519721.
Chen,J.W., Murphy,T.L., Willingham,M.C., Pastan,I. and August,J.T. (1985) Identification of two lysosomal membrane glycoproteins. J. Cell Biol., 101, 8595.[Abstract]
Dairaku,K. and Spiro,R.G. (1997) Phylogenetic survey of endomannosidase indicates late evolutionary appearance of this N-linked oligosaccharide processing enzyme. Glycobiology, 7, 579586.[Abstract]
Fries,E. and Rothman,J.E. (1980) Transport of vesicular stomatitis virus glycoprotein in a cell-free extract. Proc. Natl Acad. Sci. USA, 77, 38703874.[Abstract]
Fukuda,M. (1991) Lysosomal membrane glycoproteins. Structure, biosynthesis and intracellular trafficking. J. Biol. Chem., 266, 2132721330.
Grinna,L.S. and Robbins,P.W. (1980) Substrate specificities of rat liver microsomal glucosidases which process glycoproteins. J. Biol. Chem., 255, 22552258.
Hammond,C. and Helenius,A. (1994) Quality control in the secretory pathway: retention of a misfolded viral membrane glycoprotein involves cycling between the ER, intermediate compartment and Golgi apparagus. J. Cell Biol., 126, 4152.[Abstract]
Hammond,C. and Helenius,A. (1995) Quality control of the secretory pathway. Curr. Opin. Cell Biol., 7, 523529.[ISI][Medline]
Helenius,A., Trombetta,E.S., Hebert,D.N. and Simons,J.F. (1997) Calnexin, calreticulin and the folding of glycoproteins. Trends Cell Biol., 7, 193200.[ISI]
Hiraizumi,S., Spohr,U. and Spiro,R.G. (1993) Characterization of endomannosidase inhibitors and evaluation of their effect on N-linked oligosaccharide processing during glycoprotein biosynthesis. J. Biol. Chem., 268, 99279935.
Hirani,S., Bernasconi,R.J. and Rasmussen,J.R. (1987) Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis. Anal. Biochem., 162, 485492.[ISI][Medline]
Hunt,L.A., Etchison,J.R. and Summers,D.F. (1978) Oligosaccharide chains are trimmed during synthesis of the envelope glycoprotein of vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA, 75, 754758.[Abstract]
Kearse,K.P., Williams,D.B. and Singer,A. (1994) Persistence of glucose residues on core oligosaccharides prevents association of TCR and TCRß proteins with calnexin and results specifically in accelerated degradation of nascent TCR
proteins within the endoplasmic reticulum. EMBO J., 13, 36783686.[Abstract]
Kornfeld,S., Gregory,W. and Chapman,A. (1979) Class E Thy-1 negative mouse lymphoma cells utilize an alternate pathway of oligosaccharide processing to synthesize complex type oligosaccharides. J. Biol. Chem., 254, 1164911654.[Medline]
Laemmli,U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Liu,Y., Choudhury,P., Cabral,C.M. and Sifers,R.N. (1999) Oligosaccharide modification in the early secretory pathway directs the selection of misfolded glycoproteins. J. Biol. Chem., 274, 58615867.
Lodish,H.F., Kong,N., Hirani,S. and Rasmussen,J. (1987) A vesicular intermediate in the transport of hepatoma secretory proteins from the rough endoplasmic reticulum to the Golgi complex. J. Cell Biol., 104, 221230.[Abstract]
Lubas,W.A. and Spiro,R.G. (1987) Golgi endo--D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme. J. Biol. Chem., 262, 37753781.
Lubas,W.A. and Spiro,R.G. (1988) Evaluation of the role of rat liver endo--D-mannosidase in processing N-linked oligosaccharides. J. Biol. Chem., 263, 39903998.
Moore,S.E.H. and Spiro,R.G. (1990) Demonstration that Golgi endo--D-mannosidase provides a glucosidase independent pathway for the formation of complex-N-linked oligosaccharides of glycoproteins. J. Biol. Chem., 265, 1310413112.
Moore,S.E.H. and Spiro,R.G. (1993) Inhibition of glucose trimming by castanospermine results in rapid degradation of unassembled major histocompatibility complex class I molecules. J. Biol. Chem., 268, 38093812.
Moore,S.E.H. and Spiro,R.G. (1994) Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis. J. Biol. Chem., 269, 1271512721.
Moremen,K.W., Trimble,R.B. and Herscovics,A. (1994) Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology, 4, 113125.[ISI][Medline]
Murphy,L.A. and Spiro,R.G. (1981) Transfer of glucose to oligosaccharide-lipid intermediates by thyroid microsomal enzymes and its relationship to the N-glycosylation of proteins. J. Biol. Chem., 256, 74877494.
Olden,K., Hunter,V.A. and Yamada,K.M. (1980) Biosynthetic processing of the oligosaccharide chains of cellular fibronectin. Biochim. Biophys. Acta, 632, 408416.[ISI][Medline]
Quellhorst,G.J., Jr., ORear,J.L, Cacan,R., Verbert,A. and Krag,S.S. (1999) Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells. Glycobiology, 9, 6572.
Ray,M.K., Yang,J., Sundaram,S. and Stanley,P. (1991) A novel glycosylation phenotpe expressed by Lec23, a Chinese hamster ovary mutant deficient in -glucosidase I. J. Biol. Chem., 266, 2281822825.
Rearick,J.I., Chapman,A. and Kornfeld,S. (1981) Glucose starvation alters lipid-linked oligosaccharide biosynthesis in Chinese hamster ovary cells. J. Biol. Chem., 256, 62556261.
Reitman,M.L., Trowbridge,I.S. and Kornfeld,S. (1982) A lectin-resistant mouse lymphoma cell line is deficient in glucosidase II, a glycoprotein-processing enzyme. J. Biol. Chem., 257, 1035710363.
Sousa,M.C., Ferrero-Garcia,M.A. and Parodi. A.J. (1992) Recognition of the oligosaccharide and protein moieties of glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. Biochemistry, 31, 97105.[ISI][Medline]
Spiro,M.J. and Spiro,R.G. (1985) Effect of anion-specific inhibitors on the utilization of sugar nucleotides for N-linked carbohydrate unit assembly by thyroid endoplasmic reticulum vesicles. J. Biol. Chem., 260, 58085815[Abstract]
Spiro,M.J. and Spiro,R.G. (1991) Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle. J. Biol. Chem., 266, 53115317.
Spiro,M.J., Spiro,R.G. and Bhoyroo,V.D. (1976) Lipid-saccharide intermediates in glycoprotein biosynthesis. I. Formation of an oligosaccharide-lipid by thyroid slices and evaluation of its role in protein glycosylation. J. Biol. Chem., 251, 64006408.[Abstract]
Spiro,M.J., Spiro,R.G. and Bhoyroo,V.D. (1979) Glycosylation of proteins by oligosaccharide-lipids. Studies on a thyroid enzyme involved in olligosaccharide transfer and the role of glucose in this reaction. J. Biol. Chem., 254, 76687674.[ISI][Medline]
Spiro,R.G., Spiro,M.J. and Bhoyroo,V.D. (1983) Studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids. Effect of energy deprivation. J. Biol. Chem., 258, 94699476.
Spiro,R.G., Zhu,Q., Bhoyroo,V. and Söling,H.-D. (1996) Definition of the lectin-like properties of the molecular chaperone, calreticulin and demonstration of its copurification with endomannosidase from rat liver Golgi. J. Biol. Chem., 271, 1158811594.
Spiro,M.J., Bhoyroo,V.D. and Spiro,R.G. (1997) Molecular cloning and expression of rat liver endo--mannosidase, an N-linked oligosaccharide processing enzyme. J. Biol. Chem., 272, 2935629363.
Turco,S.J. and Pickard,J. (1982) Altered G-protein glycosylation in vesicular stomatitis virus-infected glucose-deprived baby hamster kidney cells. J. Biol. Chem., 257, 86748679.
Turco,S.J. and Robbins,P.W. (1979) The initial stages of processing of protein-bound oligosaccharides in vitro. J. Biol. Chem., 254, 45604567.[Abstract]
Ware,F.E., Vassilakos,A., Peterson,P.A., Jackson,M.R., Lehrman,M.A. and Williams,D.B. (1995) The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins. J. Biol. Chem., 270, 46974704.
Weng,S. and Spiro,R.G. (1997) Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver. Biochem. J., 322, 655661.[ISI][Medline]