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• W2075491039 abstract "Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc1Man7–9GlcNAc2) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates. Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc1Man7–9GlcNAc2) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptide-based interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates. endoplasmic reticulum castanospermine calnexin calreticulin influenza hemagglutinin heavy chain of class I histocompatibility molecule polyacrylamide gel electrophoresis Chinese hamster ovary monoclonal antibody 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Glycoprotein folding within the endoplasmic reticulum (ER)1 is facilitated in part by the membrane-bound chaperone calnexin (CNX) and its soluble homolog calreticulin (CRT) (1Parodi A.J. Biochem. J. 2000; 348: 1-12Crossref PubMed Scopus (283) Google Scholar). These proteins are unique among molecular chaperones in that they utilize a lectin site as a means to associate with unfolded glycoproteins (2Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (721) Google Scholar, 3Ware 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, 4Spiro R.G. Zhu Q. Bhoyroo V. Soling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The lectin site is specific for Glc1Man7–9GlcNAc2oligosaccharides, which exist transiently as intermediates during the processing of Asn-linked glycoproteins. It is a widely held view that the removal and re-addition of the terminal glucose residue of these oligosaccharides, catalyzed, respectively, by the ER enzymes glucosidase II and UDP-glucose:glycoprotein glucosyltransferase, regulate cycles of CNX and CRT binding to glycoproteins (5Cannon K.S. Helenius A. J. Biol. Chem. 1999; 274: 7537-7544Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). In this model, CNX and CRT do not function as classical molecular chaperones that prevent aggregation by binding to hydrophobic polypeptide segments. Rather, they are thought to promote folding by recruiting other chaperones and folding enzymes, such as the thiol oxidoreductase ERp57 (7High S. Lecomte F.J. Russell S.J. Abell B.M. Oliver J.D. FEBS Lett. 2000; 476: 38-41Crossref PubMed Scopus (136) Google Scholar, 8Van der Wal F.J. Oliver J.D. High S. Eur. J. Biochem. 1998; 256: 51-59Crossref PubMed Scopus (35) Google Scholar), to the glycoprotein substrate. The concept that CNX and CRT associate with glycoproteins solely through lectin-oligosaccharide interactions is based primarily on experiments wherein cultured cells were treated with tunicamycin to block Asn-linked oligosaccharide addition or with the glucosidase I and II inhibitors castanospermine or deoxynojirimycin to prevent the conversion of the Glc3Man9GlcNAc2precursor to the monoglucosylated Glc1Man9GlcNAc2 species. Subsequent immunoprecipitation with anti-CNX or anti-CRT antibodies frequently revealed a dramatic reduction in the amounts of various glycoproteins co-isolating as complexes with these chaperones (2Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (721) Google Scholar, 9Nauseef W.M. McCormick S.J. Clark R.A. J. Biol. Chem. 1995; 270: 4741-4747Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 10Otteken A. Moss B. J. Biol. Chem. 1996; 271: 97-103Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 11Ou W.J. Cameron P.H. Thomas D.Y. Bergeron J.J. Nature. 1993; 364: 771-776Crossref PubMed Scopus (488) Google Scholar, 12Peterson J.R. Ora A. Van P.N. Helenius A. Mol. Biol. Cell. 1995; 6: 1173-1184Crossref PubMed Scopus (266) Google Scholar, 13Toyofuku K. Wada I. Hirosaki K. Park J.S. Hori Y. Jimbow K. J. Biochem. (Tokyo). 1999; 125: 82-89Crossref PubMed Scopus (63) Google Scholar, 14Vassilakos A. Cohen-Doyle M.F. Peterson P.A. Jackson M.R. Williams D.B. EMBO J. 1996; 15: 1495-1506Crossref PubMed Scopus (170) Google Scholar). Similar results were obtained with mutant cell lines that lack the glucosidases involved in producing the monoglucosylated oligosaccharide (15Balow J.P. Weissman J.D. Kearse K.P. J. Biol. Chem. 1995; 270: 29025-29029Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In addition, glucosidase inhibitors added after complexes with CNX or CRT were formed prevented or slowed glycoprotein dissociation, thus implicating glucosidase activity in the dissociation process (5Cannon K.S. Helenius A. J. Biol. Chem. 1999; 274: 7537-7544Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar,17Wada I. Kai M. Imai S. Sakane F. Kanoh H. EMBO J. 1997; 16: 5420-5432Crossref PubMed Scopus (79) Google Scholar). In contrast with the preceding results, many lines of evidence have suggested that CNX and CRT can also associate with non-native proteins via protein-protein interactions. First, several studies show that complexes between CNX and either membrane-bound or soluble glycoproteins cannot be dissociated by enzymatic removal of oligosaccharides (3Ware 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, 18Zhang Q. Tector M. Salter R.D. J. Biol. Chem. 1995; 270: 3944-3948Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Arunachalam B. Cresswell P. J. Biol. Chem. 1995; 270: 2784-2790Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Second, there are several examples of CNX interacting with proteins that either lack Asn-linked oligosaccharides naturally (20Rajagopalan S. Xu Y. Brenner M.B. Science. 1994; 263: 387-390Crossref PubMed Scopus (210) Google Scholar) or have lost them through mutagenesis or under-glycosylation (21Carreno B.M. Schreiber K.L. McKean D.J. Stroynowski I. Hansen T.H. J. Immunol. 1995; 154: 5173-5180PubMed Google Scholar, 22Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Crossref PubMed Scopus (168) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Third, after treatment of cells with castanospermine to block the formation of monoglucosylated oligosaccharides, CNX or CRT have been detected in association with specific glycoproteins such as invariant chain (24Zhang Q. Salter R.D. J. Immunol. 1998; 160: 831-837PubMed Google Scholar), CD3δ subunit (25van Leeuwen J.E. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), coagulation factors V and VIII (26Pipe S.W. Morris J.A. Shah J. Kaufman R.J. J. Biol. Chem. 1998; 273: 8537-8544Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), acid phosphatase (27Jannatipour M. Callejo M. Parodi A.J. Armstrong J. Rokeach L.A. Biochemistry. 1998; 37: 17253-17261Crossref PubMed Scopus (29) Google Scholar), and the α subunit of the nicotinic acetylcholine receptor (28Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1998; 273: 17064-17072Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Fourth, both CNX and CRT have been shown to bind specifically to nonglycosylated peptides both in vitro and in vivo (29Spee P. Subjeck J. Neefjes J. Biochemistry. 1999; 38: 10559-10566Crossref PubMed Scopus (57) Google Scholar, 30Nair S. Wearsch P.A. Mitchell D.A. Wassenberg J.J. Gilboa E. Nicchitta C.V. J. Immunol. 1999; 162: 6426-6432PubMed Google Scholar, 31Basu S. Srivastava P.K. J. Exp. Med. 1999; 189: 797-802Crossref PubMed Scopus (210) Google Scholar, 32Jorgensen C.S. Heegaard N.H. Holm A. Hojrup P. Houen G. Eur. J. Biochem. 2000; 267: 2945-2954Crossref PubMed Scopus (32) Google Scholar). Fifth, using purified components in vitro, it has been demonstrated that CNX and CRT are capable of functioning as molecular chaperones to suppress the aggregation and enhance the folding not only of glycoproteins bearing monoglucosylated oligosaccharides but of nonglycosylated proteins as well (33Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 34Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (218) Google Scholar). Despite this accumulated information, the concept that CNX and CRT are capable of associating in vivo with unfolded proteins via polypeptide-based interactions in addition to lectin-oligosaccharide binding has been largely discounted. It has been speculated that the lack of dissociation of CNX-substrate complexes after complete deglycosylation may be due to the trapping of the two species within the same detergent micelle (1Parodi A.J. Biochem. J. 2000; 348: 1-12Crossref PubMed Scopus (283) Google Scholar, 35Rodan A.R. Simons J.F. Trombetta E.S. Helenius A. EMBO J. 1996; 15: 6921-6930Crossref PubMed Scopus (139) Google Scholar) or that the substrate, being nonnative, might become insoluble upon dissociation (1Parodi A.J. Biochem. J. 2000; 348: 1-12Crossref PubMed Scopus (283) Google Scholar, 35Rodan A.R. Simons J.F. Trombetta E.S. Helenius A. EMBO J. 1996; 15: 6921-6930Crossref PubMed Scopus (139) Google Scholar, 36Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Similarly, it has been suggested that the association of CNX with nonglycosylated proteins in vivo may arise through nonspecific inclusion of CNX within misfolded protein aggregates (1Parodi A.J. Biochem. J. 2000; 348: 1-12Crossref PubMed Scopus (283) Google Scholar,36Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 37Cannon K.S. Hebert D.N. Helenius A. J. Biol. Chem. 1996; 271: 14280-14284Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, apart from a single instance in which CNX was detected in association with aggregates of nonglycosylated vesicular stomatitis virus G protein (37Cannon K.S. Hebert D.N. Helenius A. J. Biol. Chem. 1996; 271: 14280-14284Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), there has been no direct evidence to support such speculations. Finally, the in vitro studies demonstrating direct binding of nonglycosylated peptides to CRT or the molecular chaperone functions of CNX and CRT with non-glycoproteins have been questioned in terms of their relevance to the in vivo situation (1Parodi A.J. Biochem. J. 2000; 348: 1-12Crossref PubMed Scopus (283) Google Scholar). In an effort to address the question of the existence of polypeptide-based interactions between CNX and its diverse substratesin vivo, we chose to utilize the same methodology used most commonly in previous studies to demonstrate the apparent exclusivity of lectin-oligosaccharide interactions, i.e. block the formation of monoglucosylated oligosaccharides and assess by co-immuno-isolation if complexes between diverse substrates and CNX can be detected. We reasoned that upon loss of the lectin-oligosaccharide interaction, any remaining polypeptide-based association might be too weak to survive rigorous immuno-isolation conditions. Consequently, care was taken to employ mild, yet highly specific isolation procedures. Using either pharmacologic or genetic methods to block the formation of the Glc1Man9GlcNAc2oligosaccharide in diverse cell types, we show that although many complexes were lost, a large number of CNX-substrate complexes remained readily detectable. Complementary results were also obtained using a substrate that lacked oligosaccharides through mutation of its Asn-X-(Ser/Thr) sequence. Interactions with CNX (and CRT) were maintained in the absence of any detectable aggregation. We conclude that in addition to the well established lectin-oligosaccharide interaction, polypeptide-based association does indeed exist in vivo between CNX or CRT and a diverse array of protein substrates. Murine BW5147 thymoma cells, their glucosidase II-deficient variant PhaR2.7 (38Reitman M.L. Trowbridge I.S. Kornfeld S. J. Biol. Chem. 1982; 257: 10357-10363Abstract Full Text PDF PubMed Google Scholar) (both provided by Dr. R. Kornfeld, Washington University), and L cells were grown in Dulbecco's modified Eagle's minimum essential medium. Murine EL-4 thymoma cells and the human C1R cell line, that stably expresses the HLA-B27 molecule (39Anderson K.S. Cresswell P. EMBO J. 1994; 13: 675-682Crossref PubMed Scopus (112) Google Scholar) (provided by Dr. P. Cresswell, Yale University), were cultured in RPMI 1640. CHO-K1 and its glucosidase I-deficient variant CHO-Lec23 (40Ray M.K. Yang J. Sundaram S. Stanley P. J. Biol. Chem. 1991; 266: 22818-22825Abstract Full Text PDF PubMed Google Scholar) were obtained from Dr. A. Helenius, Swiss Federal Institute of Technology, and were grown in α-minimum essential medium. Stably transfectedDrosophila melanogaster Schneider cells that express CNX along with H-2Kb heavy (H) chains in the presence of mouse β2-microglobulin (41Danilczyk U.G. Cohen-Doyle M.F. Williams D.B. J. Biol. Chem. 2000; 275: 13089-13097Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) were maintained in Schneider's insect medium (Sigma). All media were supplemented with 10% fetal bovine serum and antibiotics. A rabbit antiserum (anti-8) directed against the C terminus of the H-2Kb H chain, which reacts with all conformational states of Kb, was provided by Dr. Brian Barber, University of Toronto. Antiserum UCSF#2 reacts with the cytoplasmic tail of class I HLA H chains and was provided by Dr. Frances Brodsky, Stanford University. mAb PIN1.1, which reacts with invariant chain, was obtained from Dr. Tania Watts, University of Toronto. Two rabbit antisera were used to isolate CNX. One was directed against the C-terminal 14 amino acids (anti-C-CNX), and the second was raised against the entire 462-residue ER luminal domain (anti-N-CNX). mAb 12CA5, which reacts with the influenza hemagglutinin (HA) epitope tag on CNX(HA) and the CNX 1–387(HA), mutant was provided by Dr. Paul Hamel, University of Toronto. The N-glycosylation mutants of the H-2Kb H chain were generated by mutating the consensus glycosylation sequence, Asn-X-(Ser/Thr), using the QuikChangeTM site-directed mutagenesis kit (Stratagene) and full-length H-2Kb cDNA in pcDNA3 (Invitrogen) as template. To remove the glycosylation site at residue 176, Asn-176 was changed to Lys using the mutagenic oligonucleotide (mutated base in lowercase) 5′-GC AGA TAT CTG AAG AAC GGG AAg GCG ACG CTG CTG CGC-3′. The glycosylation site at residue 86 was removed by substituting Asn-86 with Lys using 5′-C CTG CTC GGC TAC TAC AAg CAG AGC AAG GGC GGC-3′ as the mutagenic oligonucleotide. To add a glycosylation site at position 256, Tyr-256 was changed to Asn with the mutagenic oligonucleotide 5′-GGG AAG GAG CAG aAT TAC ACA TGC CAT GTG TAC C-3′. Recombinant plasmids were introduced into L cells using the SuperFectTM transfection reagent (Qiagen), and stably transfected cells were established by G418 selection. Transient expression of HA-tagged calnexin (CNX(HA) and a truncated ER luminal segment of calnexin (CNX-(1–387(HA))) in L cells was conducted as described previously (41Danilczyk U.G. Cohen-Doyle M.F. Williams D.B. J. Biol. Chem. 2000; 275: 13089-13097Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). BW5147, PhaR2.7, CHO-K1, CHO-Lec23, or L cells at a density of 1 × 107 cells/100-mm dish were incubated in Met-free medium for 60 min at 23 °C to deplete intracellular Met pools. They were then radiolabeled at 23 °C for the times indicated in the various figure legends by the addition of 400 μCi/ml [35S]Met. Castanospermine (CAS), when added, was present throughout the prelabeling and labeling periods. Cells were lysed for 30 min at 4 °C in 1 ml of lysis buffer containing either 1% digitonin or 1% CHAPS in PBS, pH 7.4, 10 mm iodoacetamide, 60 μg/ml PefablocR (Roche Molecular Biochemicals), and 10 μg/ml each of leupeptin, antipain, and pepstatin. For isolation of CNX and associated molecules, lysates were incubated with preimmune or anti-CNX antibodies for 2 h. Immune complexes were collected for 1 h using protein A-agarose beads and analyzed by SDS-PAGE as described previously (41Danilczyk U.G. Cohen-Doyle M.F. Williams D.B. J. Biol. Chem. 2000; 275: 13089-13097Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). For detection of calnexin-associated Kb, Db, HLA-B27, and invariant chain, digitonin lysates were subjected to sequential immunoprecipitation (42Suh W.K. Derby M.A. Cohen-Doyle M.F. Schoenhals G.J. Fruh K. Berzofsky J.A. Williams D.B. J. Immunol. 1999; 162: 1530-1540PubMed Google Scholar). Briefly, CNX-substrate complexes were recovered with anti-CNX antiserum, dissociated by heating at 42 °C for 1 h in 0.2% SDS, adjusted to 2% Nonidet P-40, 5% skim milk, and incubated with anti-class I H chain or anti-invariant chain antibodies. Immune complexes were collected and analyzed as above. Radiolabeling of transfected Drosophila cells with [35S]Met, lysis, and immuno-isolation was carried out as described previously (14Vassilakos A. Cohen-Doyle M.F. Peterson P.A. Jackson M.R. Williams D.B. EMBO J. 1996; 15: 1495-1506Crossref PubMed Scopus (170) Google Scholar). Briefly, after induction of the metallothionein promoter with 1 mm CuSO4 for 16 h, Drosophila cells were incubated for 1 h in Met-free Schneider's medium in the presence or absence of 1 mm CAS. Cells were then radiolabeled with 0.5 mCi/ml [35S]Met for 5 min in the presence or absence of CAS and lysed in digitonin lysis buffer. Lysates were incubated with anti-class I H chain or anti-C-CNX antibodies, and immune complexes were collected on protein A-agarose followed by SDS-PAGE analysis using 10% gels (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Radioactive proteins were visualized by fluorography. L cells (1 × 107) expressing wild type or nonglycosylated H-2Kb were radiolabeled with [35S]Met for 30 min, lysed in 1 ml of 1% digitonin buffer, and centrifuged briefly at top speed in an Eppendorf microcentrifuge to remove insoluble material. A 0.5-ml aliquot of lysate was loaded onto a 12-ml, 10–40% (w/v) linear glycerol gradient prepared in digitonin buffer. The gradients were centrifuged at 4 °C for 15 h at 35,000 rpm using a Beckman SW 41 rotor. Fractions (0.75 ml) were collected from the top of the gradients, and Kb H chains were immuno-isolated from each fraction using anti-8 antiserum. As a control for the total amount of Kb molecules loaded onto the gradient, an additional 0.5-ml sample of lysate was also immuno-isolated with anti-8 antiserum. For detection of H-2Kb-CNX complexes by immunoblotting, 1 × 107 L cells expressing wild type Kb or various Kbglycosylation mutants were lysed in digitonin lysis buffer and immuno-isolated with anti-8 antiserum and protein A-agarose. After SDS-PAGE analysis, proteins were transferred to nitrocellulose membrane (44Suh W.K. Cohen-Doyle M.F. Fruh K. Wang K. Peterson P.A. Williams D.B. Science. 1994; 264: 1322-1326Crossref PubMed Scopus (274) Google Scholar), and the membrane was incubated with rabbit anti-N-CNX antiserum at 1:5,000 dilution followed by donkey anti-rabbit IgG horseradish peroxidase conjugate at 1:10,000 dilution (Jackson Laboratories). Immune complexes were visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). To establish whether CNX associates with its substrates only via its lectin site or whether protein-protein interactions also contribute to this association, we examined the formation of CNX-substrate complexes in glucosidase I- or glucosidase II-deficient cell lines and in the presence of the glucosidase inhibitor, CAS. In an effort to minimize protein aggregation and to preserve potentially weak protein-protein interactions, metabolic radiolabeling of cells was conducted at 25 °C, and lysis was performed using the mild detergents digitonin and CHAPS. Furthermore, immune complexes were washed for the minimum number of times (typically three) required to preserve CNX-substrate interactions while minimizing recovery of nonspecifically associated proteins. Initially, the BW5147 mouse lymphoma cell line and its glucosidase II-deficient mutant, PhaR2.7, were radiolabeled with [35S]Met, and digitonin lysates were subjected to immuno-isolation with two separate anti-CNX antisera. The anti-C-CNX antibody recognizes the last 14 residues of the cytoplasmic tail of CNX, and the anti-N-CNX antibody is directed against the entire ER luminal domain (residues 1–462). As reported previously (15Balow J.P. Weissman J.D. Kearse K.P. J. Biol. Chem. 1995; 270: 29025-29029Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), in addition to CNX, which appeared as a major band of 90 kDa, a large number of newly synthesized proteins co-isolated as complexes with CNX from the parental BW5147 cells (Fig.1A). A similar pattern of proteins was observed with the two independent anti-CNX antisera (Fig.1A, lanes 2 and 3). A substantial number of these proteins were lost or reduced in intensity in the glucosidase II-deficient PhaR2.7 cells, reflecting their apparent requirement for monoglucosylated oligosaccharides for stable association with CNX. However, it is noteworthy that many other proteins remained firmly associated with CNX (Fig. 1A,lanes 5 and 6). A similar result was obtained when parental BW5147 cells were treated with CAS to block glucosidase activity (Fig. 1A, compare lanes 2 and3 with lane 7). Indeed, the patterns of CNX-associated proteins were remarkably similar in the PhaR2.7 and CAS-treated BW5147 cells. It is conceivable that these proteins arise as a result of nonspecific interactions either with the precipitating immunoglobulins or with protein A-agarose beads. However, we consider this possibility unlikely since a similar spectrum of proteins was obtained with the two independently generated anti-CNX antibodies (containing different arrays of immune globulins), and they were absent from control isolations performed with preimmune serum and protein A-agarose beads (Fig. 1A, lanes 1 and 4). To confirm these findings, we also compared glucosidase I-deficient Lec23 cells to their parental CHO cell line (Fig. 1B). Lec23 cells have been shown to possess little or no glucosidase I activity, and no monoglucosylated oligosaccharides could be detected on glycoproteins (40Ray M.K. Yang J. Sundaram S. Stanley P. J. Biol. Chem. 1991; 266: 22818-22825Abstract Full Text PDF PubMed Google Scholar). Remarkably, despite the block in formation of monoglucosylated oligosaccharides, there was no obvious reduction in the number of CNX-associated proteins recovered with each antiserum, although some differences in the patterns of recovered proteins were apparent (Fig. 1B, compare lane 2with lane 5 and lane 3 with lane 6). Again, these proteins were absent in control isolations performed with preimmune antiserum. To further exclude the possibility that the proteins remaining associated with CNX in glucosidase-deficient cells or after CAS treatment were due to nonspecific associations with immune complexes, HA-epitope tagged CNX (CNX(HA)) and a soluble variant (CNX-(1–387(HA))) were prepared and transfected into mouse L cells. We showed previously that the CNX-(1–387(HA)) variant fails to form complexes with newly synthesized proteins (41Danilczyk U.G. Cohen-Doyle M.F. Williams D.B. J. Biol. Chem. 2000; 275: 13089-13097Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The transfectants were radiolabeled with [35S]Met, lysed in 1% CHAPS, and subjected to immuno-isolation with anti-HA mAb. As shown in Fig.1C, lane 1, a large number of newly synthesized proteins were recovered with the anti-HA mAb from cells expressing full-length CNX(HA). Consistent with the experiments presented in Fig.1, panels A and B, many proteins were also recovered in association with CNX(HA) after treatment of cells with CAS (Fig. 1C, lane 3). In contrast, only trace levels of proteins were recovered in association with the binding-impaired CNX-(1–387(HA)) variant (Fig. 1C, lane 2). This was also the case when the CNX-(1–387(HA)) variant was isolated from CAS-treated cells (data not shown). That the CNX-(1–387(HA)) variant was recovered under identical conditions of immune isolation as the CNX(HA) construct establishes that the CNX-associated proteins remaining after CAS treatment are indeed bona fide complexes and not merely proteins nonspecifically adsorbed to anti-HA immune precipitates. Our finding that CNX remains capable of specific association with many proteins under conditions where the formation of monoglucosylated oligosaccharides is blocked contrasts with a number of previous studies. For example, Ora and Helenius (16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) compared CNX-associated proteins in CHO versus glucosidase I-deficient Lec23 cells and showed that many fewer proteins associated with CNX in parental CHO cells than we observed and that none of these could be detected in the Lec23 mutant cells. To determine if the disparity in results arises from differences in the radiolabeling and immune isolation methods, we directly compared CNX-associated proteins in CHO and Lec23 cells using our mild conditions and those of Ora and Helenius (16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The major differences in methodology included our radiolabeling of cells at 23 °C versus 37 °C, our use of digitoninversus CHAPS during cell lysis and washing of immune complexes, our elimination of a pre-clearance step with fixedStaphylococcus aureus cells, and our recovery of immune complexes over a 3-h period versus the overnight isolation employed by Ora and Helenius (16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The results of this comparison are depicted in Fig. 1D. Consistent with the results presented in Fig. 1B, we observed few differences in the patterns of CNX-associated proteins recovered from CHO cells relative to glucosidase I-deficient Lec23 cells (compare lanes 2 and 4). In contrast, the procedure of Ora and Helenius (16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed" @default.
• W2075491039 created "2016-06-24" @default.
• W2075491039 creator A5035802962 @default.
• W2075491039 creator A5058688987 @default.
• W2075491039 date "2001-01-01" @default.
• W2075491039 modified "2023-10-11" @default.
• W2075491039 title "The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo" @default.
• W2075491039 cites W1518996810 @default.
• W2075491039 cites W1607176255 @default.
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