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• W2056404010 abstract "Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc1Man5–9GlcNAc2, interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains ∼50% of binding compared with full-length controls. This site is in addition to a Zn2+-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin. Calnexin and calreticulin are membrane-bound and soluble chaperones, respectively, of the endoplasmic reticulum (ER) which interact transiently with a broad spectrum of newly synthesized glycoproteins. In addition to sharing substantial sequence identity, both calnexin and calreticulin bind to monoglucosylated oligosaccharides of the form Glc1Man5–9GlcNAc2, interact with the thiol oxidoreductase, ERp57, and are capable of acting as chaperones in vitro to suppress the aggregation of non-native proteins. To understand how these diverse functions are coordinated, we have localized the lectin, ERp57 binding, and polypeptide binding sites of calnexin and calreticulin. Recent structural studies suggest that both proteins consist of a globular domain and an extended arm domain comprised of two sequence motifs repeated in tandem. Our results indicate that the primary lectin site of calnexin and calreticulin resides within the globular domain, but the results also point to a much weaker secondary site within the arm domain which lacks specificity for monoglucosylated oligosaccharides. For both proteins, a site of interaction with ERp57 is centered on the arm domain, which retains ∼50% of binding compared with full-length controls. This site is in addition to a Zn2+-dependent site located within the globular domain of both proteins. Finally, calnexin and calreticulin suppress the aggregation of unfolded proteins via a polypeptide binding site located within their globular domains but require the arm domain for full chaperone function. These findings are integrated into a model that describes the interaction of glycoprotein folding intermediates with calnexin and calreticulin. endoplasmic reticulum calnexin calreticulin citrate synthase glutathione S-transferase malate dehydrogenase N-hydroxysulfosuccinimidyl As the site of synthesis of proteins destined for secretion, cell surface expression, and residency in the secretory pathway, the endoplasmic reticulum (ER)1contains an array of folding enzymes and molecular chaperones that facilitate the folding of newly synthesized proteins. Peptidylprolylcis-trans-isomerase and members of the protein disulfide isomerase family enzymatically catalyze rate-limiting steps in the folding pathway of polypeptides, whereas molecular chaperones such as Grp94 and BiP function by preventing aggregation through cycles of binding and release of unfolded polypeptides. Another set of chaperones present in the ER, calnexin (CNX) and calreticulin (CRT), interact preferentially with glycoproteins that bear Asn-linked oligosaccharides, enhancing their folding and subunit assembly (1Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 2Leach M.R. Williams D.B. Michalak M. Eggleton P. Calreticulin in Health and Disease. 2nd Ed. Landes Bioscience, Georgetown, TX2002Google Scholar, 3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Trombetta E.S. Helenius A. Curr. Opin. Struct. Biol. 1998; 8: 587-592Crossref PubMed Scopus (210) Google Scholar). This preferential binding is caused by the presence within CNX and CRT of a lectin site with specificity for the oligosaccharide-processing intermediate, Glc1Man9GlcNAc2 (5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar, 6Spiro R.G. Zhu Q. Bhoyroo V. Soling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 7Vassilakos A. Michalak M. Lehrman M.A. Williams D.B. Biochemistry. 1998; 37: 3480-3490Crossref PubMed Scopus (227) Google Scholar, 8Ware 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 (381) Google Scholar). However, oligosaccharide binding is not an absolute requirement for their association with diverse glycoproteins that transit the ER. Both molecules have been shown to bind in vitro and in vivo to nonglycosylated proteins and peptides as well as to glycoproteins lacking the Glc1Man9GlcNAc2 oligosaccharide (9Basu S. Srivastava P.K. J. Exp. Med. 1999; 189: 797-802Crossref PubMed Scopus (208) Google Scholar, 10Carreno B.M. Schreiber K.L. McKean D.J. Stroynowski I. Hansen T.H. J. Immunol. 1995; 154: 5173-5180PubMed Google Scholar, 11Jannatipour M. Callejo M. Parodi A.J. Armstrong J. Rokeach L.A. Biochemistry. 1998; 37: 17253-17261Crossref PubMed Scopus (29) Google Scholar, 12Jorgensen C.S. Heegaard N.H. Holm A. Hojrup P. Houen G. Eur. J. Biochem. 2000; 267: 2945-2954Crossref PubMed Scopus (32) Google Scholar, 13Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1998; 273: 17064-17072Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 14Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Crossref PubMed Scopus (168) Google Scholar, 15Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 16Nair S. Wearsch P.A. Mitchell D.A. Wassenberg J.J. Gilboa E. Nicchitta C.V. J. Immunol. 1999; 162: 6426-6432PubMed Google Scholar, 17Pipe S.W. Morris J.A. Shah J. Kaufman R.J. J. Biol. Chem. 1998; 273: 8537-8544Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 18Rajagopalan S., Xu, Y. Brenner M.B. Science. 1994; 263: 387-390Crossref PubMed Scopus (207) Google Scholar, 19van Leeuwen J.E. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Zhang Q. Salter R.D. J. Immunol. 1998; 160: 831-837PubMed Google Scholar, 21Spee P. Subjeck J. Neefjes J. Biochemistry. 1999; 38: 10559-10566Crossref PubMed Scopus (56) Google Scholar, 22Danilczyk U.G. Williams D.B. J. Biol. Chem. 2001; 276: 25532-25540Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). CNX, a type I transmembrane protein, and its soluble paralog CRT are Ca2+-binding proteins that share sequence similarity that is most pronounced in a central segment containing two proline-rich sequence motifs repeated in tandem (23David V. Hochstenbach F. Rajagopalan S. Brenner M.B. J. Biol. Chem. 1993; 268: 9585-9592Abstract Full Text PDF PubMed Google Scholar, 24Fliegel L. Burns K. MacLennan D.H. Reithmeier R.A. Michalak M. J. Biol. Chem. 1989; 264: 21522-21528Abstract Full Text PDF PubMed Google Scholar, 25Smith M.J. Koch G.L. EMBO J. 1989; 8: 3581-3586Crossref PubMed Scopus (215) Google Scholar, 26Wada I. Rindress D. Cameron P.H., Ou, W.J. Doherty J.J., Jr. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar). Motifs 1 and 2, which are repeated three times each in CRT and four times each in CNX, have consensus sequences of I-DP(D/E)A-KPEDWD(D/E) and G-W-P-IN-P-Y, respectively. In addition, there are three segments of high sequence similarity, A, B, and C, with the last two flanking the repeat motifs (Fig. 1).In cultured cell systems, the association of CNX and CRT with newly synthesized glycoproteins is transient, with dissociation occurring at or near acquisition of a native structure. CNX and CRT also associate in prolonged fashion to misfolded or incompletely assembled glycoproteins (for review, see Refs. 1Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 2Leach M.R. Williams D.B. Michalak M. Eggleton P. Calreticulin in Health and Disease. 2nd Ed. Landes Bioscience, Georgetown, TX2002Google Scholar, 3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Trombetta E.S. Helenius A. Curr. Opin. Struct. Biol. 1998; 8: 587-592Crossref PubMed Scopus (210) Google Scholar). These interactions have been shown to promote proper glycoprotein folding and subunit assembly, either to enhance or inhibit the degradation of non-native glycoprotein conformers, and to retain misfolded or incompletely folded glycoproteins in the ER (1Bergeron J.J. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 2Leach M.R. Williams D.B. Michalak M. Eggleton P. Calreticulin in Health and Disease. 2nd Ed. Landes Bioscience, Georgetown, TX2002Google Scholar, 3Parodi A.J. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar, 4Trombetta E.S. Helenius A. Curr. Opin. Struct. Biol. 1998; 8: 587-592Crossref PubMed Scopus (210) Google Scholar). How these diverse functions are accomplished is controversial. In one model, the interactions of glycoproteins with CNX and CRT are controlled entirely by the presence of the single terminal glucose residue on the Glc1Man9GlcNAc2 oligosaccharide (5Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (708) Google Scholar,27Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (486) Google Scholar). Cycles of release and rebinding are regulated by glucose removal and readdition catalyzed by the action of the ER enzymes glucosidase II and UDP-glucose:glycoprotein glucosyltransferase, respectively. In this model, CNX and CRT do not function as classical molecular chaperones but are proposed to retain unfolded glycoproteins and coordinate the activities of other ER chaperones and folding enzymes. Such a view is supported by the finding that CNX and CRT interact with the ER resident thiol oxidoreductase, ERp57 (28Oliver J.D. Roderick H.L. Llewellyn D.H. High S. Mol. Biol. Cell. 1999; 10: 2573-2582Crossref PubMed Scopus (273) Google Scholar). Alternatively, a second “dual binding” model has been proposed in which unfolded glycoproteins interact with both the lectin site and a polypeptide binding site in CNX and CRT (8Ware 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 (381) Google Scholar, 29Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this model, the polypeptide component of the interaction serves a molecular chaperone function to suppress aggregation of the unfolded substrate. Support for this model comes from in vitro studies demonstrating that CNX and CRT can discriminate between folded and unfolded conformers of nonglycosylated proteins and that they can suppress the aggregation of unfolded proteins as potently as members of the HSP60, HSP70, HSP90, and small heat shock families of chaperones (29Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar, 31Stronge V.S. Saito Y. Ihara Y. Williams D.B. J. Biol. Chem. 2001; 276: 39779-39787Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).The multiple binding functions attributed to CNX and CRT prompted us to investigate how these binding sites are organized. Previous studies have suggested that their lectin sites might be centered on the repeat motifs, although oligosaccharide binding by this segment represents only a small fraction of that observed with the full-length proteins (7Vassilakos A. Michalak M. Lehrman M.A. Williams D.B. Biochemistry. 1998; 37: 3480-3490Crossref PubMed Scopus (227) Google Scholar, 32Peterson J.R. Helenius A. J. Cell Sci. 1999; 112: 2775-2784Crossref PubMed Google Scholar). Furthermore, CRT has been shown to interact with ERp57 in a Zn2+-dependent manner via a segment located near its N terminus (33Corbett E.F. Oikawa K. Francois P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and, in the absence of Zn2+, via a segment within its repeat motifs (34Frickel E.M. Riek R. Jelesarov I. Helenius A. Wuthrich K. Ellgaard L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1954-1959Crossref PubMed Scopus (238) Google Scholar). Comparable studies on ERp57 binding by CNX have not been undertaken. Finally, there have been no reports on the location of a polypeptide binding site for either protein. Recently, the crystal structure of CNX (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) has shown the protein to consist of a globular β-sandwich domain as well as an elongated hairpin “arm” domain formed by the repeat motifs (Fig. 1) An NMR structure of the CRT repeat motifs (36Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar) has shown that this segment also forms a hairpin arm domain (Fig. 1). With this structural information as a guide, we employed extensive deletion mutagenesis to localize the lectin sites of CNX and CRT, their ERp57 binding sites, and the sites whereby they suppress the aggregation of nonglycosylated proteins. The results necessitate a re-evaluation of previous localization studies and offer a more coherent view of the functional organization of these complex proteins.DISCUSSIONA recently reported x-ray crystal structure shows that the ER luminal portion of canine CNX consists of two domains, a β-sandwich globular domain that contains boxes A, B, and C and a 140 Å hairpin extension (arm domain) formed by the repeat motifs (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). A structurally similar arm domain is seen in the NMR structure (36Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar) of the CRT repeat motifs (Fig. 7). In both proteins, the motif 1 repeats form one strand of the hairpin, and the motif 2 repeats form the other strand. Each motif 1 repeat interacts with a corresponding motif 2 repeat in a head-to-tail fashion. Upon soaking the CNX crystal in glucose, the monosaccharide was detected within a concave depression in the globular domain (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Six glucose contact residues were identified, four in box A, one between boxes A and B, and one in box C, all highly conserved among CNXs and CRTs (Fig. 7). The presence of a lectin site within the concave surface of the globular domain is consistent with its structural similarity to the galectin and legume lectin families. A Ca2+ binding site was also identified within the globular domain (Fig. 7A), which is different from a site mapped previously to the repeat motifs using45Ca2+ overlay and ruthenium red binding (42Tjoelker L.W. Seyfried C.E. Eddy R.L., Jr. Byers M.G. Shows T.B. Calderon J. Schreiber R.B. Gray P.W. Biochemistry. 1994; 33: 3229-3236Crossref PubMed Scopus (92) Google Scholar). Also, there are two disulfide bonds in the CNX structure, one in the globular domain, shown to be labile, and the second near the tip of the arm domain (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Given this structural information, it remained to be established how CNX and CRT utilize their two domains to function as molecular chaperones for glycoproteins. Consequently, we set out to map the lectin, ERp57 binding, and polypeptide binding sites of CNX and CRT.FIG. 7Functional sites on CNX and CRT.A, locations of the lectin, ERp57 binding, and polypeptide binding sites on CNX and CRT as determined in this study. Shown are the x-ray crystal structure of the ER luminal domain of CNX (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and one of the NMR structures determined for the arm domain of CRT (36Ellgaard L. Riek R. Herrmann T. Guntert P. Braun D. Helenius A. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3133-3138Crossref PubMed Scopus (155) Google Scholar). A presumed CNX-like globular domain has been added to the CRT structure to facilitate depiction of functional sites. CNX residues identified as contacting glucose in the crystal structure are depicted instick representation. B, model of the interaction of monoglucosylated folding intermediates with CNX or CRT. The monoglucosylated substrate binds to the chaperone through both lectin-oligosaccharide interactions as well as polypeptide-based associations (29Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar). This enables CNX and CRT to function more effectively with glycoprotein substrates than traditional polypeptide-based chaperones (31Stronge V.S. Saito Y. Ihara Y. Williams D.B. J. Biol. Chem. 2001; 276: 39779-39787Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The terminal glucose residue of the substrate engages the lectin sites of CNX and CRT within their globular domains. Polypeptide-based contacts occur primarily between the substrate and the globular domain, but additional contacts may occur through the arm domain as well. The flexible arm domain also binds ERp57 and brings it into proximity with the substrate, where it catalyzes disulfide bond formation and isomerization.View Large Image Figure ViewerDownload (PPT)When CNX deletion mutants were assayed for their binding to the natural oligosaccharide ligand [3H]Glc1Man9GlcNAc2, we found that 75% of the CNX oligosaccharide binding capability could be localized to its globular domain (CNX 1–255/390–461). This is consistent with the location of bound glucose in the x-ray crystal structure of CNX (35Schrag J.D. Bergeron J.J., Li, Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Although a corresponding globular lectin domain has not yet been demonstrated for CRT, we showed for the first time that a CRT deletion construct lacking the repeat motifs and corresponding to the globular domain of CNX (CRT 1–189/284–401) also retained 75% of the oligosaccharide binding observed with full-length CRT. This finding, coupled with the sequence similarity between CNX and CRT, suggests that CRT possesses a globular lectin domain akin to that of CNX (Fig. 7A). In agreement with previous results (7Vassilakos A. Michalak M. Lehrman M.A. Williams D.B. Biochemistry. 1998; 37: 3480-3490Crossref PubMed Scopus (227) Google Scholar,32Peterson J.R. Helenius A. J. Cell Sci. 1999; 112: 2775-2784Crossref PubMed Google Scholar), we detected a weak lectin site within the repeat motifs (arm domains) of both CNX and CRT. Deletion constructs that correspond to the repeat motifs alone (CNX 257–387 and CRT 191–282) were able to bind oligosaccharide, albeit much more weakly than the full-length controls. However, through the use of competitor oligosaccharides that either possess or lack a terminal α1–3-linked glucose we could demonstrate that oligosaccharide binding by the repeats differed from that observed by the globular domain in that it lacked specificity for monoglucosylated oligosaccharides. Collectively, these findings establish that the globular domain contains the lectin site for the physiologically relevant oligosaccharide and help to clarify previous studies that reported oligosaccharide binding by the repeat motifs.Using a similar approach, we localized the Zn2+-independent ERp57 binding site to the arm domains of both CNX and CRT. We found that although the globular domains exhibited minimal binding, the arm domains retained about 50% of the ERp57 binding exhibited by the full-length controls. Additional deletion mutants revealed that the first repeat 1 motif and the last motif 2 repeat, which exhibit paired interactions at the base of the arm domains of both CNX and CRT, are not required for ERp57 binding (Fig. 7A). This suggests that ERp57 binds to segments of the arm domain which are located distal to the globular domain. During the preparation of this manuscript, Ellgaard and co-workers (34Frickel E.M. Riek R. Jelesarov I. Helenius A. Wuthrich K. Ellgaard L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1954-1959Crossref PubMed Scopus (238) Google Scholar) described the use of NMR chemical shift mapping to localize ERp57 interaction with CRT residues 225–251. This segment corresponds to the last copy of motif 1 and the first copy of motif 2, representing the tip of the arm domain. In conjunction with the present findings, it is likely that ERp57 binds primarily to the tips of the arm domains of both CRT and CNX. However, because the arm domains retain only 50% of the binding observed with full-length controls, there appears to be a role for the globular domains as well, presumably in constraining the orientation or conformation of the arm domains. Taken together, the oligosaccharide and ERp57 mapping studies show that the globular domain is the main site of interaction with monoglucosylated Asn-linked oligosaccharide, whereas the arm domain acts to tether the ERp57 oxidoreductase.In addition to the Zn2+-independent ERp57 binding site described above, we observed that in the presence of 100 μm Zn2+ ERp57 bound to the N-terminal portion of the CRT globular domain (CRT 1–182). This corresponds to a site of interaction previously described by Corbett et al. (33Corbett E.F. Oikawa K. Francois P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In their study, Zn2+-independent ERp57 was not observed, which may reflect differences in the binding assays employed. Using 1 mm Zn2+, they measured changes in fluorescence of Cascade Blue-labeled ERp57 in response to CRT addition, which requires significant changes in the environment of the fluorophore. We also show that CNX has a Zn2+-dependent site of interaction with ERp57 localized to the N-terminal portion of its globular domain (CNX 1–256). It should be noted that the addition of 10–100 μm Zn2+ causes a conformational change in CRT resulting in increased hydrophobicity (43Khanna N.C. Tokuda M. Waisman D.M. J. Biol. Chem. 1986; 261: 8883-8887Abstract Full Text PDF PubMed Google Scholar), increased chaperone activity (30Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar), and, above 2 mm, quantitative precipitation (44Baksh S. Spamer C. Heilmann C. Michalak M. FEBS Lett. 1995; 376: 53-57Crossref PubMed Scopus (51) Google Scholar). In the presence of 100 μmZn2+, CNX aggregates rapidly at elevated temperatures and exhibits reduced solubility at room temperature, suggesting that like CRT, it exposes hydrophobic stretches upon Zn2+binding. 2M. Leach and D. Williams, unpublished observations. Given these solubility changes, we considered the possibility that Zn2+-dependent ERp57 binding to CRT and CNX is nonspecific. However, Zn2+-dependent ERp57 binding was preserved despite the inclusion in our assays of 0.1 mg/ml bovine serum albumin and 0.1% Nonidet P-40 to minimize nonspecific absorption. The physiological significance of this second mode of ERp57 interaction is unclear. It is known that Zn2+ is present within the ER (45Reddy A.G. Devi B.G. Rao S.B. Gupta P.D. Cytobios. 1989; 60: 21-26PubMed Google Scholar), but whether its concentration is sufficient to induce conformational changes in CRT and CNX and to influence ERp57 binding remains an open question.Efforts to localize a polypeptide binding site within CRT and CNX identified their globular lectin domains as the region primarily responsible for suppressing the aggregation of nonglycosylated proteins. This was supported by the finding that N- and C-terminal deletion mutants lacking either of the conserved boxes B or C exhibited impaired aggregation suppression and by the fact that the globular domain constructs (CRT 1–189/284–401 and CNX 1–255/390–461) retained the ability to suppress aggregation, whereas the arm domains (CRT 191–282 and CNX 257–387) did not. However, although the arm domains themselves were incapable of suppressing aggregation, they clearly enhanced aggregation suppression by the globular domains. This suggests that polypeptide segments of non-native folding intermediates may interact with both domains of CRT and CNX (Fig. 7A). Because molecular chaperones interact with folding intermediates through binding sites that possess some hydrophobic character (46Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar), we examined the surface of CNX for hydrophobic patches. Candidate hydrophobic interaction sites do exist on the same face of the globular domain as the lectin site, and additional sites are present along the length of the arm domain. Further site-directed mutagenesis within these regions will be required to determine more precisely how CRT and CNX bind to nonglycosylated folding intermediates.Taken together, our findings suggest a model whereby CRT and CNX interact with non-native conformers of glycoproteins during folding within the ER. As shown in Fig. 7B, we envision that a monoglucosylated glycoprotein substrate could occupy the large cavity formed by the globular domain and the arm domain. This would permit contacts through the lectin site as well as through polypeptide binding sites residing within both domains. The arm domain, which is presumably quite flexible, would perform the additional function of bringing ERp57 in proximity to free thiol groups to form mixed disulfide intermediates in both oxidation and isomerization reactions. The arm may also physically constrain the glycoprotein, impeding rapid diffusion away from the chaperone. In this context, the previously described enhancement of aggregation suppression by CRT and CNX in the presence of ATP (29Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Saito Y. Ihara Y. Leach M.R. Cohen-Doyle M.F. Williams D.B. EMBO J. 1999; 18: 6718-6729Crossref PubMed Scopus (216) Google Scholar) might be caused by the closing of the arm domain over substrate in a manner analogous to the α-helical “lid” segment of HSP70 (47Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1049) Google Scholar). Given progress in delineating the lectin, ERp57 binding, and polypeptide binding sites of CRT and CNX, we are in a position to test how these functions are integrated into a functional chaperone and in turn regulated by cofactors such as Ca2+, Zn2+, and ATP. Selective ablation of each of these functions by mutagenesis and assessing the consequences using in vitro and in vivo assays of glycoprotein folding and quality control should be most informative. As the site of synthesis of proteins destined for secretion, cell surface expression, and residency in the secretory pathway, the endoplasmic reticulum (ER)1contains an array of folding enzymes and molecular chaperones that facilitate the folding of newly synthesized proteins. Peptidylprolylcis-trans-isomerase and members of the prote" @default.
• W2056404010 created "2016-06-24" @default.
• W2056404010 creator A5030685650 @default.
• W2056404010 creator A5035802962 @default.
• W2056404010 creator A5045887929 @default.
• W2056404010 creator A5066252683 @default.
• W2056404010 date "2002-08-01" @default.
• W2056404010 modified "2023-10-18" @default.
• W2056404010 title "Localization of the Lectin, ERp57 Binding, and Polypeptide Binding Sites of Calnexin and Calreticulin" @default.
• W2056404010 cites W109442298 @default.
• W2056404010 cites W1498310106 @default.
• W2056404010 cites W1503394109 @default.
• W2056404010 cites W1517838566 @default.
• W2056404010 cites W1546074605 @default.
• W2056404010 cites W1586011497 @default.
• W2056404010 cites W1593830682 @default.
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• W2056404010 cites W1970007744 @default.
• W2056404010 cites W1982972673 @default.
• W2056404010 cites W1986129246 @default.
• W2056404010 cites W1992864681 @default.
• W2056404010 cites W1994947913 @default.
• W2056404010 cites W1999456217 @default.
• W2056404010 cites W1999466952 @default.
• W2056404010 cites W2017035228 @default.
• W2056404010 cites W2025610536 @default.
• W2056404010 cites W2028674462 @default.
• W2056404010 cites W2031192254 @default.
• W2056404010 cites W2038303530 @default.
• W2056404010 cites W2038857470 @default.
• W2056404010 cites W2048384162 @default.
• W2056404010 cites W2062613923 @default.
• W2056404010 cites W2075018537 @default.
• W2056404010 cites W2075491039 @default.
• W2056404010 cites W2075808686 @default.
• W2056404010 cites W2076945013 @default.
• W2056404010 cites W2079118456 @default.
• W2056404010 cites W2081327685 @default.
• W2056404010 cites W2102041044 @default.
• W2056404010 cites W2107671070 @default.
• W2056404010 cites W2121217914 @default.
• W2056404010 cites W2123311867 @default.
• W2056404010 cites W2128788229 @default.
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• W2056404010 cites W2163309884 @default.
• W2056404010 cites W2169145132 @default.
• W2056404010 cites W2172066208 @default.
• W2056404010 cites W2188206162 @default.
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