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• W1991396089 abstract "The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human α1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded α1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded α1-antitrypsin for degradation by the proteasome. The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human α1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded α1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded α1-antitrypsin for degradation by the proteasome. endoplasmic reticulum α1-antitrypsin polyacrylamide gel electrophoresis UDP-glucose:glycoprotein glucosyltransferase The endoplasmic reticulum (ER)1 functions as the intracellular site where nascent polypeptides enter the central vacuolar system (1Walter P. Gilmore R. Blobel G. Cell. 1984; 38: 5-8Abstract Full Text PDF PubMed Scopus (395) Google Scholar) and fold into their correct functional conformation (2Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar), which is dictated by the primary amino acid sequence (3Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5217) Google Scholar). Quality control machinery resident to that compartment facilitates the selective elimination of incompletely folded proteins to ensure that only functional molecules are deployed to distal sites (4Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (457) Google Scholar, 5Hammond E. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (589) Google Scholar). As such, the role of conformation-based quality control is fundamental to normal cell physiology. Although initially unexpected, it is now recognized that the 26 S proteasome, a constituent of the cytosol (6Rivett J.A. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (383) Google Scholar), is responsible for the degradation of many ER-situated proteins (7Ward C.L. Omura S. Kopito Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1133) Google Scholar, 8Hiller M.M. Finger A. Schweiger M. Wolf D. Science. 1996; 273: 1725-1728Crossref PubMed Scopus (619) Google Scholar, 9Galan J.-M. Cantegrit B. Garner C. Namy O. Haguenauer-Tsapis R. FASEB J. 1998; 12: 315-323Crossref PubMed Scopus (25) Google Scholar, 10Werner E.D. Brodsky J.L. McCracken A.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (394) Google Scholar). Indeed, the cytoplasmic delivery of proteasomal substrates has been reported (11Wiertz E.J.H.J. Jones R.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 12Wiertz E.J.H.J. Tortortella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-443Crossref PubMed Scopus (955) Google Scholar, 13Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 14de Virgilio M. Weninger H. Ivessa E. J. Biol. Chem. 1998; 273: 9734-9743Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). As yet, however, the molecular basis by which proteins of the ER are selected for proteasomal degradation remains unclear, although the molecular chaperone calnexin, which binds monoglucosylated oligosaccharides (15Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), has been implicated as a possible participant in the sorting process (16McCracken A.A. Brodsky J.L. J. Cell Biol. 1996; 132: 291-298Crossref PubMed Scopus (348) Google Scholar, 17Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The molecular pathogenesis of several human diseases, including cystic fibrosis, familial hypercholesterolemia, and a heritable form of pulmonary emphysema, are caused, in part, by the participation of conformation-based quality control factors (for reviews, see Refs. 18Thomas P.J. Qu B.-H. Pedersen P.L. Trends Biol. Sci. 1995; 20: 456-459Abstract Full Text PDF PubMed Scopus (484) Google Scholarand 19Choudhury P. Liu Y. Sifers R.N. News Physiol. Sci. 1997; 12: 162-165Google Scholar). The latter disorder is caused by the impaired secretion of misfolded genetic variants of human α1-antitrypsin (AAT) from liver hepatocytes. Human AAT is a monomeric glycoprotein that is a member of the serine proteinase inhibitor superfamily that protects lung elastin fibers from elastolytic destruction. Defective intracellular transport of the aberrantly folded glycoprotein diminishes circulating levels of the inhibitor, resulting in the elastolytic destruction of lung elastin (reviewed in Ref.20Sifers R.N. Shen R.-F. Woo S.L.C. Mol. Biol. Med. 1989; 6: 127-135PubMed Google Scholar). Defective secretion of AAT has been investigated by us (21Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar, 22Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar, 23Le A. Ferrell G.A. Dishon D.S. Le Q.-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar, 24Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar, 26Choudhury P. Liu Y. Bick R. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and others (10Werner E.D. Brodsky J.L. McCracken A.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13797-13801Crossref PubMed Scopus (394) Google Scholar, 17Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 27Teckman J.H. Perlmutter D.H. J. Biol. Chem. 1996; 271: 13215-13220Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 28Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (235) Google Scholar) as a model to dissect the involvement of quality control machinery in the molecular pathogenesis of human disease. As observed for other hepatic secretory glycoproteins (29Ou W.J. Cameron P.H. Thomas D.Y. Bergeron J.J. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar), rounds of binding to calnexin are predicted to facilitate the folding of the newly synthesized AAT into its functional conformation (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). Our analyses have shown that prolonged physical association with calnexin accompanies intracellular retention of the terminally misfolded glycoprotein (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar) and that the removal of mannose from asparagine-linked oligosaccharides is essential for subsequent degradation (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). The focus of the present study was to investigate the intracellular degradation of misfolded AAT as a model to elucidate the role of intracellular mannosidase activity in glycoprotein quality control, a phenomenon recently observed by us (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar) and others (30Su K. Stoller T. Rocco J. Zemsky J. Green R. J. Biol. Chem. 1993; 268: 14301-14309Abstract Full Text PDF PubMed Google Scholar, 31Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar, 32Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (304) Google Scholar, 33Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (563) Google Scholar). Results from the present investigation are consistent with a model in which modification by ER mannosidase I mediates the proteasomal degradation of terminally misfolded AAT by abrogating its dissociation from calnexin in response to the attenuated deglucosylation of asparagine-linked oligosaccharides. This finding provides evidence that ER-situated mannosidase activity occupies a previously unrecognized elevated hierarchical position among known members of the glycoprotein folding and quality control machinery and extends the role of calnexin beyond that of a simple molecular chaperone. All salts, buffers, and protease inhibitors were purchased from Sigma, except for lactacystin, which was synthesized by Dr. E. J. Corey (Harvard University). Endoglycosidase H, jack bean mannosidase, and all oligosaccharide processing inhibitors were purchased from Boehringer Mannheim, with the exception of castanospermine (CalBiochem) and kifunensine (Toronto Research Chemicals, Inc). All tissue culture media were purchased from ICN Biochemicals and Life Technologies, Inc. Fetal bovine serum was procured from Summit Biotechnology. Polyclonal rabbit antiserum against a synthetic peptide homologous to the cytoplasmic tail of canine calnexin was purchased from StressGen, and an IgG fraction of polyclonal goat anti-human AAT was procured from Organon Teknika-Cappel. Mouse hepatoma cells were stably transfected with DNA encoding human AAT in which 33 amino acids are absent from the carboxyl terminus of the 394-amino acid polypeptide (cell line H1A/N13) (21Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1988; 263: 7330-7335Abstract Full Text PDF PubMed Google Scholar). Unless stated otherwise, incubation of cells with inhibitors was performed for 60 min in regular growth medium (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar) prior to a 15-min pulse in methionine-free medium containing [35S]methionine (NEN Life Science Products) (23Le A. Ferrell G.A. Dishon D.S. Le Q.-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar). Cell lysis was performed with buffered Nonidet P-40 (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar), and protein immunoprecipitation was accomplished by a 2-h incubation of the soluble cell lysate with an excess of specific antiserum immobilized to protein G-agarose, as described (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). Following immunoprecipitation, radiolabeled proteins were resolved by SDS-PAGE and detected by fluorographic enhancement of vacuum-dried gels. Increased electrophoretic mobility of radiolabeled AAT, resulting from oligosaccharide modification, was resolved in 20-cm gels (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). Calnexin-associated AAT was separated from released monomers by velocity sedimentation of Nonidet P-40 cell lysates in linear 5–20% sucrose gradients as described previously (24Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). Physical interaction with calnexin was confirmed by sequential coimmunoprecipitation of radiolabeled molecules with antiserum against the molecular chaperone as described previously (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). The relative amount of AAT in each sedimenting species was assessed by scintillation spectrophotometry of immunoprecipitated protein excised from the gel. Prior to treatment, calnexin was dissociated from immunoprecipitated AAT by incubation with 5 mm EDTA at 37 °C (24Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). Digestions were performed for 16 h at 37 °C with 0.03 μg of jack bean mannosidase in 50 μl of 10 mmcitrate, pH 5.0, followed by an additional 8-h incubation upon the addition of another aliquot of enzyme. Resolution of AAT glycoforms was accomplished by SDS-PAGE and fluorography. Electrophoretic AAT standards in which all three attached oligosaccharides contain glucose was generated by preincubating cells with 0.2 μg/ml castanospermine prior to pulse-radiolabeling and immunoprecipitation. The standard in which one of the attached oligosaccharides contain glucose was generated by chasing pulse-radiolabeled cells in medium containing metabolic poisons to disrupt the physical interaction with calnexin (26Choudhury P. Liu Y. Bick R. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), resulting in the hydrolysis of glucose residues prior to immunoprecipitation. In the absence of COOH-terminal amino acids, human AAT is unable to progress to the correctly folded conformation (34Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (394) Google Scholar). In pulse-chase studies using [35S]methionine, the terminally misfolded molecules were completely eliminated from stably transfected mouse hepatoma cells within 4 h of chase (t 12 = 2 h) (Fig.1 a). Although unaffected by the lysosomotrophic amine chloroquine, intracellular disposal was inhibited >85% upon incubation of cells with the membrane-permeable peptide aldehydes N-acetyl-leu-leu-norleucinal andN-acetyl-leu-leu-methioninal (Fig. 1 a), both of which are nonspecific inhibitors of proteasomal degradation (35Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg A.L. Riordan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (775) Google Scholar). Nearly complete inhibition occurred upon incubation with lactacystin, a specific irreversible covalent inhibitor of the proteasome (36Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar), confirming the participation of the multicatalytic proteolytic complex in the disposal process. Incubation of cells with the glucosidase inhibitor castanospermine prior to pulse-radiolabeling with [35S]methionine was performed to maintain asparagine-linked oligosaccharides in the original Glc3Man9GlcNAc2 structure, preventing posttranslational assembly between the newly synthesized glycoprotein and calnexin (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar). Under these conditions, intracellular turnover was unaffected by each of the three proteasome inhibitors (Fig. 1 a). Because no insoluble radioactive AAT was detected under the latter conditions (not shown), these findings indicate that misfolded molecules are substrates for degradation by an alternative proteolytic system under conditions that prevent physical interaction with calnexin. Simultaneous detection of calnexin-associated (6.8 S) and released AAT monomers (4.5 S) by velocity sedimentation (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar) has provided evidence for the partitioning of the unfolded glycoprotein between the chaperone-associated and unbound state. A sedimentation coefficient of 6.8 S was exhibited by the entire population of misfolded AAT molecules following a 4-h chase with lactacystin (Fig.2 a, LCT), and the observation was duplicated in two additional experiments. This anomaly coincided with the quantitative coimmunoprecipitation of radiolabeled AAT with antiserum against calnexin (Fig. 2 b). An identical manipulation did not hinder the secretion of wild-type AAT (not shown), indicating that the ability of lactacystin treatment to prevent the release of the glycoprotein from calnexin was restricted to the terminally misfolded molecule. Because the detection of 4.5 S AAT monomers requires posttranslational release from calnexin (25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar), it became apparent that the abrogated dissociation of the 6.8 S complex precedes proteasomal degradation, possibly as a normal step in the disposal process. Enhanced electrophoretic mobility of radiolabeled molecules in SDS-PAGE (Fig. 1 b, CO), which results from the removal of mannose from attached oligosaccharides (24Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar, 25Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Crossref PubMed Scopus (116) Google Scholar), was arrested, as was intracellular disposal, by incubating cells with 1-deoxymannojirimycin (Fig. 1 b, DMJ), an inhibitor of ER mannosidases (37Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (350) Google Scholar). However, of the two events, only intracellular disposal was arrested with lactacystin (Fig. 1 b, LCT), confirming that the removal of mannose from asparagine-linked oligosaccharides precedes proteolysis. The importance of this finding was revealed in the next set of experiments, in which a population of released misfolded monomers, in addition to the 6.8 S complex, was detected following coincubation of cells with both lactacystin and the mannosidase inhibitor (Fig. 2 a, LCT+DMJ). Under these conditions, the relative ratio of the 6.8 and 4.5 S species was identical to that observed when disposal had been arrested with 1-deoxymannojirimycin alone (Fig. 2 a, DMJ). These findings demonstrate that intracellular mannosidase activity is responsible for abrogating the dissociation of misfolded AAT from calnexin prior to proteolysis. In the absence of sufficient material to characterize the mannose content of attached oligosaccharides, we examined the effect of processing inhibitors on AAT disposal as a means to identify the mannosidase activity essential for degradation. Because the oligosaccharide-dependent size shift has been localized to the ER (24Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar), we examined the involvement of two processing α-mannosidases, each of which can initiate the removal of mannose from asparagine-linked oligosaccharides in higher eukaryotes (38Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (325) Google Scholar) and is inhibited with 1-deoxymannojirimycin (39Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar). Of the two enzymes, ER mannosidase I catalyzes the removal of the outermost mannose unit from the middle branch of Man9GlcNAc2 (Fig.3 a), and is inhibited by the plant alkaloid kifunensine (39Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar). A separate mannose unit is excised by the kifunensine-resistant ER mannosidase II (40Weng S. Spiro R.G. J. Biol. Chem. 1993; 268: 25656-25663Abstract Full Text PDF PubMed Google Scholar), which is sensitive to both swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol (38Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (325) Google Scholar). Incubation of cells with kifunensine arrested the intracellular disposal of misfolded AAT (Fig. 3 b, KIF), whereas no demonstrable effect was exerted by either of the ER mannosidase II inhibitors (Fig. 3 b, SWN, DIM), indicating that trimming of attached glycans to Man8GlcNAc2, by ER mannosidase I, is responsible for mediating the onset of misfolded AAT degradation by the proteasome in these cells. Reversible glucosylation of asparagine-linked oligosaccharides mediates the partitioning of glycoproteins between the calnexin-associated and unbound state (41Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,42Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). Results from separate in vitro studies have demonstrated that whereas glucose transfer by UDP-glucose:glycoprotein glucosyltransferase (UGTR) to asparagine-linked Man8GlcNAc2 remains 70% as efficient as for the Man9GlcNAc2 precursor (43Parodi A.J. Mendelzon D.H. Lederkremer G.H. J. Biol. Chem. 1983; 258: 8260-8265Abstract Full Text PDF PubMed Google Scholar), subsequent deglucosylation of Glc1Man8GlcNAc2by glucosidase II is diminished to only 21% of that observed for Glc1Man9GlcNAc2 substrate (44Grinna L.S. Robbins P.W. J. Biol. Chem. 1980; 255: 2255-2258Abstract Full Text PDF PubMed Google Scholar). Because the deglucosylation of Glc1Man8GlcNAc2 is very inefficient, at least for the protein-free oligosaccharide (44Grinna L.S. Robbins P.W. J. Biol. Chem. 1980; 255: 2255-2258Abstract Full Text PDF PubMed Google Scholar), in the next set of experiments we tested the hypothesis that the removal of a single mannose unit from multiple oligosaccharides attached to the unbound monomer prevents complete dissociation of the complex following assembly with calnexin, as depicted in Fig.4. The accuracy of this prediction was first addressed by asking whether any of the oligosaccharides of calnexin-associated AAT contained glucose following inhibition of proteolysis with lactacystin. Because the removal of mannose by jack bean mannosidase is restricted by terminal glucose units (Fig.5 a), differential migration of mannosidase-digested glycoproteins in SDS-PAGE was used as a method to identify the number of oligosaccharides attached to [35S]methionine-radiolabeled AAT that are glucosylated (45Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar). For AAT, a total of four glycoforms can be generated by this methodology in which either 0, 1, 2, or all 3 attached oligosaccharides contain glucose. However, two predominant bands were resolved by SDS-PAGE following lactacystin treatment (Fig. 5 b, LCT). When compared with the migration of known electrophoretic mobility standards (Fig. 5 b, Stds), it was concluded that these species represented populations of calnexin-associated AAT in which either two or all three asparagine-linked oligosaccharides contain glucose (Fig. 5 b, LCT). A slight variation in this pattern has been observed in four additional experiments, including the infrequent detection of a faster migrating band in which only a single glycan contains glucose (not shown). Importantly, the latter species has routinely represented <10% of the total glycoform population, suggesting that it represents a minor species. Finally, radiolabel from [14C]galactose (46Godelaine D. Spiro M.J. Spiro R.G. J. Biol. Chem. 1981; 256: 10161-10168Abstract Full Text PDF PubMed Google Scholar) was incorporated as glucose into asparagine-linked oligosaccharides of the misfolded glycoprotein during cycloheximide treatment (not shown), indicating that the sugar units had been added posttranslationally, as would be expected for substrates of UGTR.Figure 5Detection and quantitation of glucosylated asparagine-linked oligosaccharides. a, strategy for detecting glycoprotein bearing glucosylated oligosaccharides. A terminal glucose unit (open square) hinders the removal of mannose residues (closed circles) by jack bean mannosidase (JBM). b, electrophoretic mobility in SDS-PAGE of radiolabeled misfolded AAT. The differential electrophoretic mobility of standards (Stds) is shown in which all three or none of the attached oligosaccharides contain glucose (see under “Materials and Methods”). Following a 4-h chase with lactacystin (LCT), radiolabeled misfolded AAT was immunoprecipitated from cells and then was either mock-digested (lane 1), digested with jack bean mannosidase (lane 2), or digested with endoglycosidase for total deglycosylation (lane 3) prior to fractionation by SDS-PAGE and detection by fluorography. For this experiment, all samples were subjected to SDS-PAGE on the same gel, and the predicted number of asparagine-linked oligosaccharides that contain glucose is shown to the right of each panel.View Large Image Figure ViewerDownload (PPT) Next, we attempted to establish a causal relationship between the attenuation of oligosaccharide deglucosylation and proteasomal degradation of misfolded AAT. Because a dynamic interaction with calnexin allows for the liberation of bound glycoprotein substrates in response to the deglucosylation of asparagine-linked oligosaccharides (41Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), we took advantage of the 2-h half-life of the misfolded protein to test the effect of a posttranslational glucosidase blockade as a means to bypass the predicted role of mannose removal to attenuate oligosaccharide deglucosylation. For this, pulse-radiolabeled cells were chased with medium containing the glucosidase inhibitor castanospermine to directly inhibit the activity of glucosidase II following cotranslational assembly of molecules with calnexin. Disposal of misfolded AAT was accelerated >2-fold under these conditions (Fig.6 a, lane 4) as compared with control cells (lane 2). Accelerated intracellular turnover was also observed when pulse-radiolabeled cells were chased with deoxynojirimycin (Fig. 6 b, DNJ), an alternative glucosidase inhibitor and glucose analog (37Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (350) Google Scholar). In contrast, incubation with the galactose analog, deoxygalactojirimycin, had no influence on the rate of degradation (Fig. 6 b, DgalJ). A ∼1.5-h lag preceded the acceleration of degradation (not shown), possibly reflecting the time required for either inhibitor to attain an appropriate concentration in the ER. It is unlikely that accelerated degradation resulted from the displacement of misfolded AAT by castanospermine because the compound is a transition state analog of the glucosidase reaction, rather than a true structural analog of glucose (37Elbein A.D. FASEB J. 1991; 5: 3055-3063Crossref PubMed Scopus (350) Google Scholar). Coincubation of cells with 1-deoxymannojirimycin during the posttranslational glucosidase blockade had no inhibitory effect on the accelerated rate of disposal (Fig.6 a, lane 5), as compared with when the mannosi" @default.
• W1991396089 created "2016-06-24" @default.
• W1991396089 creator A5011531521 @default.
• W1991396089 creator A5012582534 @default.
• W1991396089 creator A5021293751 @default.
• W1991396089 creator A5089194955 @default.
• W1991396089 date "1999-02-01" @default.
• W1991396089 modified "2023-10-18" @default.
• W1991396089 title "Oligosaccharide Modification in the Early Secretory Pathway Directs the Selection of a Misfolded Glycoprotein for Degradation by the Proteasome" @default.
• W1991396089 cites W1486018834 @default.
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