Definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver Golgi.

Calreticulin was identified by immunochemical and sequence analyses to be the higher molecular mass (60 kDa) component of the polypeptide doublet previously observed in a rat liver Golgi endomannosidase preparation obtained by chromatography on a Glc alpha 1 --> 3Man-containing matrix. The affinity for this saccharide ligand, which paralleled that of endomannosidase and was also observed with purified rat liver calreticulin, suggested that this chaperone has lectin-like binding properties. Studies carried out with immobilized calreticulin and a series of radiolabeled oligosaccharides derived from N-linked carbohydrate units revealed that interactions with this protein were limited to monoglucosylated polymannose components. Although optimal binding occurred with Glc1Man9GlcNAc, substantial interaction with calreticulin was retained after sequential trimming of the polymannose portion down to the Glc1Man5GlcNAc stage. The alpha 1 --> 6-mannose branch point of the oligosaccharide core, however, appeared to be essential for recognition as Glc1Man4GlcNAc did not interact with the calreticulin. The carbohydrate-peptide linkage region had no discernible influence on binding as monoglucosylated oligosaccharides in N-glycosidic linkage interacted with the chaperone to the same extent as in their unconjugated state. The immobilized calreticulin proved to be a highly effective tool for sorting out monoglucosylated polymannose oligosaccharides or glycopeptides from complex mixtures of processing intermediates. The copurification of calreticulin and endomannosidase from a Golgi fraction in comparable amounts and the strikingly similar saccharide specificities of the chaperone and the processing enzyme have suggested a tentative model for the dissociation through glucose removal of calreticulin-glycoprotein complexes in a post-endoplasmic reticulum locale; in this scheme, deglucosylation would be brought about by the action of endomannosidase rather than glucosidase II.

It has become apparent in recent years that N-linked oligosaccharides at an early stage of processing can play an impor-tant role in the quality control of the secretory pathway by influencing the folding and assembly of the proteins to which they are attached (1)(2)(3). More specifically, attention has been focused on the glucose residues that are initially present on the N-linked carbohydrate unit (Glc 3 Man 9 GlcNAc 2 ), since it has been noted that inhibition of glucose trimming through a blockade of the ER 1 -situated glucosidases may lead to accelerated degradation (4,5) or delayed secretion (6 -8) of various glycoproteins. The finding that retention of the triglucosyl sequence can result in such a profound effect on newly synthesized glycoproteins can be rationalized by reports that proteins with N-linked oligosaccharides bind to certain molecular chaperones only subsequent to the processing of the carbohydrate units to the monoglucosylated state (1). Most of the evidence for this lectin-like activity has been obtained through studies on calnexin (9, 10), a membrane-associated chaperone of the ER, although very recent observations, made while our investigations were in progress, have suggested on the basis of electrophoretic examinations that the lumenal chaperone calreticulin can also bind glycoproteins after partial deglucosylation (11).
Our attention was drawn to the latter chaperone by the quite unexpected finding, from sequence analyses that the larger (mass of 60 kDa) of the two previously observed polypeptide components present in the ligand affinity chromatographypurified rat liver Golgi endomannosidase preparation (12) was indistinguishable from calreticulin. The copurification of endomannosidase and calreticulin on a Glc-Man-Affi-Gel suggested that the chaperone has a saccharide affinity and provided the impetus for undertaking a detailed definition of its lectin-like properties toward N-linked oligosaccharides at various processing stages. The results obtained from studies with immobilized calreticulin provided some striking parallels between the saccharide specificity of this chaperone to those previously observed for endomannosidase (13,14) and suggested the possibility that the two proteins may work in tandem in a post-ER location.

EXPERIMENTAL PROCEDURES
Preparation of Rat Liver Membranes-Golgi and rough ER membrane fractions were prepared from the livers of fasted male rats (150 -200 g, CD strain, Taconic, Inc.) as described previously (13) by the method of Leelavathi et al. (15) modified by Tulsiani et al. (16), and the procedure of Depierre and Dallner (17), respectively.
Ligand Affinity Chromatography of Triton-solubilized Golgi Membranes-Solubilized Golgi membranes (12) and calreticulin, isolated from rat liver by a slightly modified version of the procedure described by Van et al. for a protein then termed CaBP3 (18), were chromatographed on a Glc-Man-Affi-Gel column under the conditions previously described (12). Aliquots of the eluted fractions were assayed for endomannosidase activity and were submitted to immunoblotting subsequent to polyacrylamide gel electrophoresis. Prior to carrying out the latter procedure, the protein in the sample to be examined (ϳ300 l) was precipitated by the addition of 20 volumes of acetone at Ϫ20°C and after 3 h at that temperature collected by centrifugation (2,000 ϫ g for 20 min). For subsequent study, the remainder of the neutralized glycine HCl buffer-eluted fractions that contained the endomannosidase activity were concentrated in an Ultrafree-CL filtration unit (Millipore) as described previously (12).
Peptide Sequencing-The Glc-Man-Affi-Gel purified endomannosidase preparation was submitted to 12% polyacrylamide gel electrophoresis in SDS and then electroblotted onto a polyvinylidene difluoride membrane (19) from Bio-Rad for 6 h (60 V) at 4°C in 10 mM CAPS, pH 10.6 buffer. After visualizing the two protein bands (60 and 56 kDa) by a brief (1 min) exposure to 0.1% Ponceau S in 1% acetic acid, they were separately excised, washed with water, and sent frozen to the Harvard University Microchemistry Facility. Under the direction of William S. Lane, solid phase-trypsin digestion was carried out on each protein band (3-4 g), followed by reverse-phase high performance liquid chromatography of the resulting peptides. Several of the latter were then selected for amino acid sequencing by automated Edman degradation (20).
Polyacrylamide Gel Electrophoresis and Immunoblotting-The electrophoresis was carried out on 12% polyacrylamide gels (1.5 mm thick) overlaid with a 3.5% stacking gel according to the procedure of Laemmli (21), and the protein bands were visualized by silver staining. For immunological identification, the proteins were transferred onto a nitrocellulose sheet (22), which was incubated with rabbit antiserum raised against rat liver calreticulin at 1:800 dilution, and after washing exposed to 125 I-labeled protein A as described previously (23). The radioactive bands on the nitrocellulose were detected by autoradiography, while the standards were stained by India ink.
Preparation of Radiolabeled Oligosaccharides and Glycopeptides-Metabolically radiolabeled [ 14 C]Glc 1 Man 9 GlcNAc and [ 14 C]Man 5-9GlcNAc were prepared from thyroid glycoproteins after incubation of slices with [ 14 C]glucose as described previously (14,24); in this procedure the oligosaccharides released from Pronase-generated glycopeptides by endo H are purified by preparative thin layer chromatography. To achieve complete homogeneity of the Glc 1 Man 9 GlcNAc oligosaccharide and remove any contaminating Man 9 GlcNAc, it was submitted to a second chromatography.
For the preparation of Glc 1 Man 8 GlcNAc isomers, the purified [ 14 C]Glc 1 Man 9 GlcNAc (ϳ100,000 dpm) was incubated with rat liver ER membranes (750 g of protein) for 20 h at 37°C in 100 l of 0.1 M NaMES buffer, pH 6.5, containing 0.2% (v/v) Triton X-100 in the presence of 2 mM castanospermine (a gift from Dr. M. Kang, Merrell Dow Research Institute, Cincinnati, OH) and either 2 mM 1,4-dideoxy-1,4imino-D-mannitol (Oxford Glycosystems) or 5 M kifunensine (Toronto Research Chemical). In presence of the 1,4-dideoxy-1,4-imino-D-mannitol, which inhibits ER mannosidase II (25), Glc 1 Man 8 GlcNAc, isomer B, in which the terminal mannose of the middle chain is missing, is generated while kifunensine by blocking the action of ER mannosidase I (26) results in the formation of Glc 1 Man 8 GlcNAc, isomer C, in which the residue terminating the ␣1,6-linked chain has been excised. The respective Glc 1 Man 8 GlcNAc isomers were isolated from the digests by preparative thin layer chromatography after deproteinization with 80% ethanol and desalting by passage through Dowex 50 (H ϩ ) and Dowex 1 (acetate) as described previously (26).
For the preparation of radiolabeled Glc 1 Man 7 GlcNAc, Glc 1 -Man 6 GlcNAc, and Glc 1 Man 5 GlcNAc, digestion of [ 14 C]Glc 1 Man 9 GlcNAc (85,000 dpm) was carried out with jack bean ␣-mannosidase (2 units, Sigma) in 300 l of 0.15 M sodium citrate buffer, pH 5.2, for 72 h at 37°C in the presence of toluene. The oligosaccharides were isolated from the deproteinized and desalted digests by preparative thin layer chromatography, and in each case their monoglucosylated state was verified by the release of Glc 1 Man 1 through endomannosidase digestion (14).
The heptasaccharide fraction obtained by preparative thin layer chromatography from the cytosolic oligosaccharides of [ 14 C]glucoselabeled HepG2 cells provided a mixture of [ 14 C]Glc 1 Man 5 GlcNAc and [ 14 C]Man 6 GlcNAc (27). The preparation of Glc 1 Man 4 GlcNAc was effected by submitting Glc 1 Man 9 GlcNAc (115,000 dpm) to acetolysis as described previously (13); the oligosaccharide was resolved from other fragmentation products by thin layer chromatography.
Glycopeptides containing a mixture of incompletely processed Nlinked oligosaccharides were obtained by Pronase digestion of the delipidated protein from thyroid slices labeled with [ 14 C]glucose during a 3-h incubation as described previously (22); by subsequent passage through a Dowex 1-X2 200 -400-mesh (acetate) column, the glycopeptides were enriched in glucosylated and deglucosylated polymannose carbohydrate units.
Affinity Chromatography of Oligosaccharides and Glycopeptides on Immobilized Calreticulin-A column (0.7 cm ϫ 6.5 cm) containing purified calreticulin (ϳ2.5 mg) linked to a tresyl-Sepharose matrix, according to the direction of the manufacturer (Pharmacia Biotech Inc.), was equilibrated at room temperature with a 50 mM NaMES, pH 6.8, buffer containing 60 mM NaCl and 40 M calcium acetate, prior to the application of 14 C-labeled oligosaccharides (2,000 -20,000 dpm) or glycopeptides (ϳ4 ϫ 10 5 dpm) in 300 l of this buffer. After addition to the column, the samples were allowed to interact with the immobilized calreticulin for 30 min prior to their elution with the equilibrating buffer at a flow rate of 6 ml/h, while 1-ml fractions were collected; aliquots were taken from each fraction for the determination of radioactivity by scintillation counting. For chromatography of the series of purified oligosaccharides, 3 H-labeled Glc 1 Man 9 GlcNAc, prepared from thyroid slices incubated with [2-3 H]mannose, was included with each sample to serve as an internal standard. For further study oligosaccharide fractions resolved by the calreticulin column were desalted, after titration to pH 7.8 with NH 4 OH, by passage through columns containing Dowex 50-X2, 200 -400-mesh (H ϩ ) overlaid with Dowex 1-X2, 200 -400-mesh (acetate). Glycopeptides were freed from salt by adsorption to Dowex 50-X2 resin and subsequent elution with 1.5 N NH 4 OH, which was then removed by lyophilization. When not in use, the calreticulinmatrix column was kept at 2°C in the presence of 0.02% sodium azide.
Structural and Analytical Procedures-Endomannosidase digestions of oligosaccharides were carried out in a manner similar to that previously reported (13) with rat liver Golgi membranes (25 g of protein) for 3 h at 37°C in the presence of 2 mM castanospermine, 10 mM EDTA, and 2 mM 1-deoxymannojirimycin, while treatment of glycopeptides with endo H (Genzyme) was performed as described previously (29). The products of these digestions were examined by thin layer chromatography after removal of salt and protein. Reduction of oligosaccharides was achieved with NaBH 4 under previously specified conditions (13). Protein was determined by the procedure of Peterson (30) using bovine serum albumin as a standard.
Thin Layer Chromatographic Procedures-Resolution of small oligosaccharides, including fragments from acetolysis treatment, was achieved on plastic sheets precoated with cellulose (0.1 mm thickness, Merck) in pyridine/ethyl acetate/water/acetic acid, 5:5:3:1 (Solvent A), whereas larger oligosaccharides were separated on plastic sheets coated with Silica Gel 60 (0.2 mm thickness, Merck) in 1-propanol/acetic acid/ water, 3:3:2 (Solvent System B). All chromatography was carried out with a wick of Whatman No. 3MM paper clamped to the top of the thin layer plate, and the components were detected by fluorography. For preparative purposes resolved oligosaccharides were eluted from the plates with water, and the resultant eluates, after extraction with peroxide-free ether to remove scintillants, were passed through small columns of Dowex 50 (H ϩ ) and Dowex 1 (acetate).
Radioactivity Measurements-Liquid scintillation counting was carried out with Ultrafluor with a Beckman LS 7500 instrument; double channel measurements were made when 14 C-and 3 H-labeled oligosaccharides were present together. Detection of radioactive components was accomplished with the use of X-Omatic AR film (Eastman Kodak) at Ϫ80°C either by autoradiography of immunoblots on nitrocellulose sheets or by fluorography of thin layer chromatographic plates that were sprayed with a scintillation mixture containing 2-methylnaphthalene (31).

Presence of Calreticulin in the Rat Liver Golgi Endomannosidase Preparation Obtained by Glc-Man-Affi-Gel
Chromatography-An examination of amino acid sequences occurring in the electrophoretically resolved protein components (60 and 56 kDa) of the ligand affinity-purified endomannosidase preparation ( Fig. 1) indicated quite unexpectedly that the higher molecular mass constituent was closely related to calreticulin, while the faster 56 kDa-band had no similarity to any previ-ously reported protein and was found to embody the enzyme activity. The sequences of two tryptic peptides derived from the 60 kDa-component, which were TWIHPEIDNPEYSPDANIY and SGTIFDNFLITNDEAYAEEFGNET, demonstrated a 100% homology to calreticulin, in which they corresponded to amino acid residues 271-289 and 306 -329, respectively, in the published sequences of this protein from several species (32). Immunoblotting confirmed the calreticulin identity of the slower moving band of the affinity chromatographically purified endomannosidase and also demonstrated that this protein is present in the unfractionated rat liver Golgi membranes (Fig. 1).
In order to determine if calreticulin by itself binds to the Glc-Man-Affi-Gel matrix, we chromatographed a sample of the purified protein on this column under the same conditions as employed for the Triton-solubilized Golgi membranes and observed on the basis of immunoblotting that this protein was indeed retained and eluted under the same conditions as the endomannosidase activity (Fig. 2). Moreover, when a Golgi extract was placed on the Affi-Gel column, the immunologically detected calreticulin and enzymatically monitored endomannosidase activity emerged in the same fractions (data not shown).
Immobilized Calreticulin Resolves Glc 1 Man 9 GlcNAc from Unglucosylated Man 9 GlcNAc-In view of our previously demonstrated understanding that endomannosidase has a high degree of specificity for monoglucosylated polymannose oligosaccharides, which is responsible for its retention on Glc-Man-Affi-Gel (12), we explored the possibility that the binding of calreticulin to this matrix has a similar basis. Indeed, when an incompletely resolved mixture of radiolabeled Man 9 GlcNAc and Glc 1 Man 9 GlcNAc was chromatographed on a calreticulin-Sepharose column, thin layer chromatographic examination of the resulting two peaks indicated that the glucosylated oligosaccharide was bound, while the unglucosylated polymannose component was unretained (Fig. 3).
Evaluation of the Binding Specificity of Calreticulin-The observation that Glc 1 Man 9 GlcNAc was bound by immobilized calreticulin prompted us to determine the specificity of this interaction with a series of purified oligosaccharides, which differed in the number of glucose residues and the size of their polymannose component. In the assay system employed, the FIG. 1. Immunochemical identification of calreticulin in the ligand affinity chromatographically purified rat liver endomannosidase preparation and the Golgi membranes from which it was obtained. Subsequent to polyacrylamide gel electrophoresis in SDS, the endomannosidase obtained by Glc-Man-Affi-Gel chromatography (0.3 g of protein, AG) as well as unfractionated rat liver Golgi membranes (50 g of protein, GOL) and calreticulin standard (0.7 g of protein, CRT) were immunoblotted with antiserum against rat calreticulin as described under "Experimental Procedures." The components were detected by autoradiography after reaction of the bound antibody with 125 I-labeled protein. For comparison the components of an aliquot of the endomannosidase preparation (1 g of protein) were visualized by silver staining (AG, Silver) after the electrophoresis. The designated molecular size markers expressed as kDa were Escherichia coli ␤-galactosidase (116,000), bovine serum albumin (66,000), hen ovalbumin (45,000), and bovine erythrocyte carbonic anhydrase (29,000).

FIG. 2. Immunochemical detection of calreticulin in the eluted fractions from a Glc-Man-Affi-Gel column.
Polyacrylamide gel electrophoresis in SDS, followed by immunoblotting with anti-calreticulin serum, was carried out on concentrated aliquots (300 l) of neutralized fractions eluted with the glycine HCl, pH 3.0, buffer from a GlcMan-Affi-Gel column (12), which had been loaded with 10 g of purified rat calreticulin. The numbers on top of the lanes refer to the fraction (4 ml) emerging from the column upon application of the glycine buffer as described previously (12). When Triton-solubilized Golgi membranes were chromatographed on this column, a similar immunoblot of the emerging fractions was obtained; the calreticulin and endomannosidase activity peaks (fraction 2) coincided. The detection of the components by autoradiography and the molecular size markers were the same as in Fig. 1. The lane designated as CRT contained a calreticulin standard.  C-labeled oligosaccharides were individually chromatographed on calreticulin-Sepharose along with 3 H-labeled Glc 1 Man 9 GlcNAc to serve as reference for their position of emergence from the column (Fig. 4). From these analyses it became evident that binding of Man 9 GlcNAc to calreticulin occurred only in its monoglucosylated state and, moreover, that effective although somewhat diminished interaction occurred with oligosaccharides in which the polymannose portion had been truncated (Figs. 4 and 5). However, it became apparent from the complete failure of Glc 1 Man 4 GlcNAc to be retained by the column that at a minimum a structure like Glc 1 Man 5 GlcNAc with its ␣136 branch point is required for the calreticulin binding (Fig. 5). Peptides containing the Glc 1 Man 9 GlcNAc 2 carbohydrate unit bound to about the same extent as unconjugated Glc 1 Man 9 GlcNAc (Fig. 5), indicating that the di-N-acetylchitobiose segment and the linkage amino acid had little influence on the interaction with calreticulin; indeed, no detectable difference was noted in the binding of Glc 1 Man 9 terminating with an N-acetylglucosamine residue or in a di-N-acetylchitobiose moiety (data not shown). A clear difference in the binding of Glc 1 Man 9 GlcNAc in the reduced and unreduced state was, however, noted (Figs. 4 and 5), and this unexpected finding was noted irrespectively of whether or not the oligosaccharides were 14 C-or 3 H-labeled.
Immobilized Calreticulin Can Effectively Separate Free and N-linked Monoglucosylated Polymannose Oligosaccharides from Mixtures of Processing Intermediates-Application of 14 Cradiolabeled glycopeptides from partially processed thyroid glycoproteins onto a calreticulin-Sepharose column resulted in a selective binding of Glc 1 Man 9 GlcNAc 2 -containing peptides to the immobilized matrix, as revealed by thin layer chromatographic examination of the oligosaccharides released by endo H digestion of the bound and unbound fractions (data not shown).
The immobilized calreticulin column also proved to be highly effective in removing Glc 1 Man 9 GlcNAc from complicated mixtures of polymannose intermediates even with the additional presence of the tri-and diglucosylated Man 9 GlcNAc components (Fig. 6). Thin layer chromatography indicated that the unbound material was specifically freed from the Glc 1 Man 9 GlcNAc oligosaccharide, which was recovered in the bound fraction (Fig. 6). The minor saccharide component migrating ahead of the Glc 1 Man 9 GlcNAc in the bound fractions was identified as Glc 1 Man 8 GlcNAc, which has been reported to occur as an N-linked processing intermediate (33).
The effectiveness of the calreticulin-Sepharose in resolving metabolic intermediates was further made apparent when the cytosolic heptasaccharide fraction from HepG2 cells, which has been reported to consist of Glc 1 Man 5 GlcNAc and Man 6 GlcNAc (27), was loaded onto the column (Fig. 7). Thin layer chroma- FIG. 4. Assay of the binding capacity of several monoglucosylated polymannose oligosaccharides to a column of immobilized calreticulin. Purified 14 C-labeled oligosaccharides (2,000 -20,000 dpm), together with a 3 H-labeled Glc 1 Man 9 GlcNAc internal standard, were chromatographed on a calreticulin-Sepharose column as described under "Experimental Procedures." The radioactivity in each 1-ml fraction was determined by double channel scintillation counting and is plotted for the 14 C-labeled (q) and 3 H-labeled (f) oligosaccharide. The scale refers only to the 14 C radioactivity; the 3 H-labeled Glc 1 Man 9 GlcNAc standard was present at approximately 3-fold greater radioactivity than the 14 C-labeled components. The abbreviations employed were as follows: G 3 M 9 , Glc 3 Man 9 GlcNAc; G 1 M 9 , Glc 1 Man 9 GlcNAc; G 1 M 8 , Glc 1 Man 8 GlcNAc (isomer B); G 1 M 5 , Glc 1 Man 5 GlcNAc; G 1 M 9 (Red), Glc 1 Man 9 GlcNAc reduced with NaBH 4 . tography after endomannosidase digestion did indeed reveal that the unabsorbed material consisted primarily of Man 6 GlcNAc, which was resistant to the action of this enzyme, while the bound oligosaccharide represented Glc 1 Man 5 GlcNAc, which was converted to Man 4 GlcNAc through the release of Glc 1 Man 1 (Fig. 7). DISCUSSION It is apparent from the present investigation that calreticulin cofractionates with endomannosidase during affinity chromatography of Triton-solubilized Golgi proteins on a Glc-Man-Affi-Gel column. This was surprising, particularly in view of the fact that only two polypeptide components in approximately equal amounts were retained by this matrix from a complex mixture of components, and alerted us to the possibility that calreticulin has a lectin-like binding capacity.
Studies carried out with immobilized calreticulin and an array of oligosaccharides derived from N-linked carbohydrate units revealed that protein-saccharide interactions were limited to monoglucosylated polymannose components. While the presence of a terminal mannose-linked glucose residue was clearly critical for the binding to take place, substantial interaction was retained after extensive trimming of the two unglucosylated chains of the polymannose unit. Indeed, even after truncation of Glc 1 Man 9 GlcNAc to Glc 1 Man 5 GlcNAc, about 65% of the initial binding capacity was still observed.
Preservation of the ␣136-inked mannose residue of the latter oligosaccharide was, however, essential to lectin recognition, as when this inner branch point was excised to yield Glc 1 Man 4 GlcNAc (Glc␣133Man␣132Man␣132Man␣13 3Man␤134GlcNAc) interaction with calreticulin could no longer be detected. On the other hand, the carbohydratepeptide linkage region appeared to have no discernible influence on binding, as monoglucosylated oligosaccharides in Nglycosidic linkage or in their unconjugated state terminating in either N-acetylglucosamine or di-N-acetylchitobiose interacted with the calreticulin to the same extent.
The selectivity of the immobilized calreticulin for monoglucosylated polymannose oligosaccharides or glycopeptides made it a highly effective tool for sorting out these components from complex mixtures of processing intermediates. Indeed, the high specificity of the calreticulin stands out in contrast to the mannose/glucose-binding lectins, such as concanavalin A (34), which cannot discriminate between glucosylated and unglucosylated polymannose oligosaccharides (35).
While calreticulin and endomannosidase are believed to function quite differently, namely as molecular chaperone (1,36,37) and processing enzyme (13,14), respectively, our study demonstrates some intriguing similarities that merit comment. The selective retention of these two proteins on a matrix containing Glc␣133Man substituents was an expression of a common specific interaction with monoglucosylated polymannose oligosaccharides. The inability of calreticulin to bind tri-and diglucosylated oligosaccharides was mirrored by the previously reported low in vitro reactivity of endomannosidase with such saccharide species. Also relevant was the finding that monoglucosylated oligosaccharides with extensively truncated mannose chains could still effectively interact with both the chaperone and the enzyme (14), particularly since this property stands in pronounced contrast to the specificity of glucosidase II, which is known to require the untrimmed mannose branches for interaction with its substrate (38).
Although calreticulin is generally believed to be primarily situated in the ER (32), our finding of this protein in the Golgi is consistent with reports indicating its presence at the cell surface (39,40) and other subcellular compartments (41)(42)(43). Indeed, it is apparent that calreticulin takes part in intracellular trafficking which accounts for this wide distribution (43)(44) and distinguishes it from calnexin, the other lectin-like chaperone, which is a membrane-bound ER-resident protein (45). FIG. 6. Selective retention of Glc 1 Man 9 GlcNAc on a calreticulin-Sepharose column from mixtures of oligosaccharide processing intermediates. A mixture of 14 C-labeled oligosaccharides consisting of similar amounts (ϳ8,000 dpm) of Glc 1-3 Man 9 GlcNAc and Man 5-9 GlcNAc components was chromatographed on a calreticulin-Sepharose column as described under "Experimental Procedures," and the radioactivity in 1-ml fractions was determined by scintillation counting (left panel). After desalting equal aliquots of the bound (BD) and unbound (UN) peaks as well as a portion of the initial mixture (IN) were chromatographed on a silica-coated plate in Solvent System B for 20 h, and the components were detected by fluorography (G 3 M 9 to M 5 , right panel). A mixture of Glc 1 Man 9 GlcNAc and Man [5][6][7][8][9] GlcNAc was similarly chromatographed on an immobilized calreticulin column, and the initial, bound, and unbound oligosaccharides were examined by thin layer chromatography (G 1 M 9 to M 5 , right panel). The abbreviations are the same as in Fig. 3, except for the following: G 3 , Glc 3 Man 9 GlcNAc; G 2 , Glc 2 Man 9 GlcNAc; G 1 , Glc 1 Man 9 GlcNAc; M 5 , Man 5 GlcNAc.
The presence of molecular chaperones with affinity for proteins with monoglucosylated N-linked oligosaccharides has provided the basis for a model (1) that accounts for their preferential association with glycoproteins at an early stage of processing and explains the observed accelerated protein degradation due to impaired folding or oligomerization during a glucosidase blockade (4,5). However, as this scheme also postulated that dissociation of glycoproteins from the chaperone is brought about by the action of glucosidase II, its relevance would be limited to the ER locale where this enzyme is situated (46). If deglucosylation is required to dissociate the calreticulinglycoprotein complexes in a more distal location, which would be either the Golgi itself or an ER-Golgi intermediate compartment, another mechanism is required. This has prompted us to propose a tentative modified model (Fig. 8) for such a more distal site in which removal of the glucose is achieved by endomannosidase through the excision of a Glc␣133Man disaccharide. This scheme would take into account the occurrence of calreticulin and endomannosidase in comparable amounts in this location and, more importantly, the fact that endomannosidase in marked contrast to glucosidase II has the capacity to interact with N-linked oligosaccharides in which the mannose chains have been trimmed. The latter characteristic is relevant, as glycoproteins that exit from the ER will have already undergone a substantial degree of processing though the action of ER-resident mannosidases (25,26). Although the ER may be the primary site for protein folding and oligomerization to take place, a number of instances have already been described in which such quality controlling events take place in more distal compartments (47,48).
The highly specific lectin-like interaction of molecular chaperones like calreticulin and calnexin with the N-linked oligosaccharides of glycoproteins represents an intriguing example of the biological role of saccharide chains and in particular extends the function of the polymannose-linked glucose residues beyond that of their well known involvement in the process of cotranslational N-glycosylation (49,50). Since it is quite likely, however, that the carbohydrate-protein interaction is only one manner in which the binding of chaperones to polypep-tide intermediates is mediated (10,45,51), definition of the mechanisms utilized by various cell types for their diverse secretory proteins will require extensive further investigation.