©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chaperone Function of Calreticulin When Expressed in the Endoplasmic Reticulum as the Membrane-anchored and Soluble Forms (*)

(Received for publication, April 3, 1995; and in revised form, June 20, 1995)

Ikuo Wada (§) Shin-ichi Imai Masahiro Kai Fumio Sakane Hideo Kanoh

From the Department of Biochemistry, Sapporo Medical University School of Medicine, South 1, West 17, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane-anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with other calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations.


INTRODUCTION

Maturation of newly synthesized proteins in the ER (^1)is constantly monitored by the quality control apparatus, which is thought to operate to prevent externalization of immature proteins along the secretory pathway (Rose and Doms, 1988; Hurtley and Helenius, 1989). Although the molecular mechanism of such a machinery is unknown, the processing of N-linked oligosaccharides, particularly glucose trimming, has been known to play a role in this process (Fitting and Kabat, 1982; Gross et al., 1983; Peyrieras et al., 1983; Lodish and Kong, 1984; Rizzolo and Kornfeld, 1988; Rose and Doms, 1988). In 1989, Suh et al. found that the majority of misfolded G proteins of a vesicular stomatitis virus mutant (ts045) retained in the ER possessed mono-glucosylated oligosaccharides and that glucose was added post-translationally on the deglucosylated N-linked oligosaccharides. On the basis of these findings, they proposed a hypothesis that the ER has a mechanism that selectively transfers a single glucose onto the deglucosylated oligosaccharides of aberrant molecules, resulting in their retention in the ER (Suh et al., 1989). Although Parodi's group (Parodi et al., 1984; Trombetta et al., 1989; Ganan et al., 1991; Sousa et al., 1992; Trombetta and Parodi, 1992), conclusively showed that the majority of newly synthesized proteins are post-translationally glucosylated by the enzyme UDP-glucose:glycoprotein glucosyltransferase, which recognizes only denatured polypeptides, the molecular machinery responsible for their ER retention remained unknown. In 1994, Hammond et al. reported that calnexin, an ER membrane chaperone, formed a stable complex (lasting at least 1 h) with misfolded G protein of a vesicular stomatitis virus mutant (tsO45) at the permissive temperature and that treatment of the cells with the glucosidase inhibitors, CAS or 1-deoxynojirimycin, abolished the association. They therefore proposed that calnexin is a putative lectin molecule with a specificity for mono-glucosylated oligosaccharides (Hammond et al., 1994).

Calnexin, which was originally identified as a major calcium-binding protein of the ER membrane (Wada et al., 1991), has been known to function as a molecular chaperone involved in the normal folding process of nascent proteins (Bergeron et al., 1994). Its chaperone function was initially discovered by analysis of cross-linked products of nascent class I major histocompatibility antigen complex by Degen and Williams(1991). Later, several membrane and soluble proteins, most of which were N-glycosylated, were reported to transiently interact with calnexin during their early stages of maturation (Ahluwalia et al., 1992; Ou et al., 1993; Anderson and Cresswell, 1994; Hammond et al., 1994; Le et al., 1994; Lenter and Vestweber, 1994; Loo and Clarke, 1994; Pind et al., 1994; Wada et al., 1994; Kim and Arvan, 1995). In addition, nascent gp80, the major secretory protein in Madin-Darby canine kidney cells, was highly susceptible to proteinase K digestion when associated with calnexin, as compared to the nascent dissociated molecules (Wada et al., 1994). Furthermore, treatment of cultured cells with chemicals that disrupt protein folding resulted in the formation of relatively stable complexes with calnexin (Ou et al., 1993; Wada et al., 1994; Kim and Arvan, 1995). At the cellular level, unassembled multi-subunit proteins (Jackson et al., 1994; Rajagopalan et al., 1994) as well as the naturally occurring mutants, Delta Phe, cystic fibrosis transmembrane conductance regulator (Pind et al., 1994), or null variant of alpha1-antitrypsin (Le et al., 1994), were also shown to be retained in the ER by calnexin. Thus, the dissociation process is highly correlated to the progress of proper protein folding. However, the precise role of calnexin in the folding process is not clear. Hammond and Helenius(1994) recently proposed that calnexin may be the major chaperone for the folding of vesicular stomatitis virus G protein, since CAS inhibited G protein maturation as well as calnexin association. While this study demonstrated the importance of a CAS-sensitive chaperone in the folding process, the involvement of other chaperones that require glucose trimming for the association cannot be ruled out.

In the search for another chaperone whose function is coupled with glucose trimming, we focused on an abundant ER luminal protein, calreticulin (Michalak et al., 1992), which has a sequence similarity (33% identity in human) to calnexin including two unique sets of internal repeats (Wada et al., 1991; Hawn et al., 1993; Tjoelker et al., 1994). This protein is thought to be mostly responsible for calcium storage in the ER (MacLennan et al., 1972; Michalak et al., 1991; Baksh et al., 1992; Treves et al., 1992) and is one of the major ER luminal proteins, collectively called reticuloplasmins (Koch, 1987; Smith and Koch, 1989). Since nascent proteins would first come in contact with membrane proteins prior to luminal proteins, we designed a system to evaluate the maximum chaperone function of calreticulin. We constructed a membrane-anchored calreticulin by fusing a truncated calreticulin (residues 1-340) to the COOH-terminal portion of calnexin. The amino-terminal luminal domain of calnexin was confirmed to be essential for its chaperone function. We then studied the interaction of the calreticulin chimera with nascent secretory proteins in HepG2 cells. Finally, we tested if calreticulin indeed functions as a chaperone at its physiological location by expressing an internally tagged soluble calreticulin in HepG2 cells. Here, we demonstrate that calreticulin is another novel CAS-sensitive chaperone in the ER.


EXPERIMENTAL PROCEDURES

General Recombinant DNA Methods

Restriction enzymes and DNA modification enzymes were purchased from standard commercial sources and used according to the manufacturers' instructions. PCR was performed as described (Wada et al., 1991), except that equal units of Vent polymerase (New England Biolabs) and Taq polymerase were included to increase the fidelity of template amplification. The recombinant plasmids constructed as described below were confirmed by a combination of restriction enzyme mapping and DNA sequencing. The oligonucleotides used for plasmid construction are listed below.

Oligonucleotides (5`-3`)

The oligonucleotides used were as follows: CN1, TATCGTCCTCCTGTCGACTGGCAGTGC; HA1, TCGATATCCATATGATGTTCCAGATTATGC; HA2, TCGAGCATAATCTGGAACATCATATGGATA; PL, ATGATGCTAGCTTGTTGTTGTAGATGATTC; CN2, GGATCTAGAGGAGCGCCCGTGGCTC; CR1, TAACTCGAGCCATGCTCCTTTCGGTGCCG; CR2, GATGCTAGCATCTGCTTCTCTGCAGCCTTG; CR3, GTGGCCTCTACAGCTCATCC; CR4, GAAGGAAGAAGATGAGGTCGACGCCACTGGCCAAGCC; HA3, TCGAGTATCCATATGATGTTCCAGATTATGCTC; HA4, TCGAGAGCATAATCTGGAACATCATATGGATAC.

Plasmid Construction

Construction of pCN(HA)

A unique SalI site was created at the 3`-end of Thy (Wada et al., 1991) of dog calnexin cDNA subcloned into pBluescript SK vector (Stratagene) by an in vitro mutagenesis employing two consecutive PCR (Ito et al., 1991) with CN1 as a calnexin-specific oligonucleotide. A double-stranded oligonucleotide (HA1 and HA2) encoding YPYDVPDYA with TCGA overhanging at both ends was inserted into the SalI-digested mutant calnexin cDNA. The mutated calnexin insert was isolated by digestion with XhoI and NotI and subcloned into the compatible site of pRep8 (Invitrogen). The resultant expression vector was designated pCN(HA).

Construction of pPL/CN(HA)

Sheep prolactin cDNA in pBluescript SK vector was a generous gift of Dr. A. Wong (Harvard Medical School). The cDNA of preprolactin encoding amino acid residues -30-198 was isolated by a PCR amplification with a vector-specific T3 primer and PL primer including a NheI site at the 5`-end and subcloned into pBluescript SK. The calnexin cDNA fragment amplified by PCR with the T3 and CN2 primers encoded Glu-Glu including a Xba site at the 5`-end. This fragment was subsequently digested with XbaI and fused to the compatible NheI site of the prolactin cDNA. The cDNA of prolactin (residues -30-198) connected to calnexin(459-573) was digested with XhoI and NotI to isolate the chimeric construct and then cloned into the corresponding site of pRep8, which was designated pPL/CN(HA).

Construction of CR/CN(HA)

Calreticulin cDNA encoding amino acid residues -17-340 was obtained by PCR amplification with a pair of primers, CR1 and CR2, from cDNA reverse-transcribed from mRNA of rat liver as described (Wada et al., 1991). The calreticulin cDNA was isolated by restriction with XhoI and NheI, both sites being included at the 5`-ends of CR1 and CR2, respectively. The resultant calreticulin cDNA was ligated to a fragment of calnexin cDNA encoding Glu-Glu. The fused cDNA was isolated by restriction with XhoI and NotI and ligated to the corresponding sites of pRep8. The expression plasmid thus constructed was designated pCR/CN(HA).

Construction of pCR(HA)

The full-length calreticulin cDNA was obtained by a PCR amplification with a pair of primers, CR1 and CR3, and a unique SalI site was created at the 3`-end of Gly (Fliegel et al., 1989) of calreticulin cDNA by a mutation employing CR4 oligonucleotide as described above. A double-stranded oligonucleotide (HA3 and HA4) was inserted into the novel SalI site and subcloned into pRep8. The resultant expression vector was designated pCR(HA).

Cell Culture, Expression, and Immunoprecipitation

HepG2 or 293 cells were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) on 24-well plates (Falcon) unless otherwise indicated in the figure legends. The recombinant plasmids were introduced into the cells according to a modified calcium phosphate precipitation protocol. (^2)2 days after transfection, 4 mM histidinol was added to the medium to enrich the population expressing the HisD gene product encoded by pRep8 vector. The cells were subsequently cultured in the selection medium for 7-10 days. Pulse-chase experiments using [S]methionine, followed by immunoprecipitation, were carried out as previously described (Wada et al., 1994) except that 1% Triton X-100 instead of sodium cholate was used to prepare cell extracts. The HA tag antibody, 12CA5, was obtained from Boehringer Mannheim. Antibodies to alpha1-antitrypsin, alpha-fetoprotein, and transferrin were purchased from Wako Pure Chemicals (Tokyo). Antibodies against calreticulin (immunoblot grade) were obtained from Affinity BioReagents (Neshanic Station, NJ). Immune complexes were analyzed by SDS-PAGE (Garfin, 1990), and the radioactive bands were visualized and quantitated using a BAS2000 phosphorimage analyzer (Fuji) equipped with Pictrography.


RESULTS

We intended to determine if calreticulin could function as a chaperone when expressed in the same environment as calnexin (i.e. anchored to the luminal surface of the ER membrane). To test this, we made constructs consisting of calreticulin residues 1-340 fused to residues 459-573 of the carboxyl terminus of calnexin. This construct includes the membrane-spanning domain of calnexin as well as the cytosolic tail. To distinguish the chimeric proteins from cellular calnexin, we inserted an epitope tag YPYDVPDYA (influenza virus hemagglutinin Tyr-Ala) flanked by an arginine residue near the carboxyl terminus of calnexin (Fig. 1, top), since the carboxyl-terminal six residues of calnexin are reportedly required for its ER retention (Rajagopalan et al., 1994). The tagged calnexin was initially tested on 293 cells to confirm that the tag did not impair its chaperone function. Cells were labeled with [S]methionine and lysed with the buffer containing Triton X-100. Immunoprecipitation with anti-calnexin antibodies revealed that the endogenous calnexin transiently interacted with nascent cellular proteins, including a major 61-kDa polypeptide (Fig. 2, lanes1 and 2). When the tagged calnexin, CN(HA), was expressed in 293 cells, the HA tag monoclonal antibody 12CA5 precipitated the same set of nascent cellular proteins including the 61-kDa protein. This protein dissociated from CN(HA) after 60 min of chase (lanes3 and 4). These transiently associated proteins were not observed in the immunoprecipitates of pRep8-transfected cells using 12CA5 antibody (lane7). Thus, these experiments confirmed that the internal tag at the cytoplasmic tail was recognized as a distinctive epitope by 12CA5 antibody and that the tag insertion did not impair the apparent chaperone function of calnexin.


Figure 1: Schematic illustration of constructs. CN(HA), the amino acids of canine calnexin are numbered from the initiator methionine. HA epitope tag flanked with Arg was inserted at the COOH terminus of Ser. TM, transmembrane domain; CN, calnexin. PL/CN(HA), prolactin (residues -30-198) was connected to the carboxyl-terminal region of the tagged calnexin. Ligation of the two cDNA fragments resulted in generation of two additional amino acid residues, Lys and Leu at the joint. PL, prolactin. CR/CN(HA), calreticulin (residues -17-340) was fused to the tagged calnexin lacking the luminal domain. L was inserted at the joint as a result of ligation of the two cDNA fragments. CR, calreticulin. CR(HA), HA epitope tag flanked by a few amino acids as indicated was inserted at the COOH terminus of Glu. The arrows indicate the sites of cleavage of signal sequences.




Figure 2: The luminal domain is essential for the chaperone function of calnexin in 293 cells. Control vectors (pRep8) (lanes1, 2, and 7), pCN(HA) (lanes3 and 4), or pPL/CN(HA) (lanes5 and 6) were transfected into 293 cells. The cells were labeled for 30 min with [S]methionine and chased for 0 min (lanes1, 3, 5. and 7) or 60 min (lanes2, 4, and 6). Cell extracts were processed for immunoprecipitation using anti-calnexin antibodies plus 12CA5 antibody (lanes1 and 2) or 12CA5 alone (lanes3-7). Efficiency of the immunoprecipitation for the tagged molecule using 12CA5 was 90-100%. The immunoprecipitates were analyzed on a 9% SDS-gel. The bands corresponding to endogenous calnexin, CN(HA), and PL/CN(HA) are indicated by arrows. Arrowhead indicates a 61-kDa polypeptide, the major calnexin ligand in 293 cells. The numbers to the left indicate the positions of the molecular mass markers for beta-galactosidase, bovine serum albumin- and ovalbumin. Note that PL/CN(HA) (lanes5 and 6) gave a fuzzy band of 47 kDa due to N-glycosylation.



We next confirmed that the luminal portion of calnexin was essential for its interaction with nascent proteins. Prolactin, a well-characterized secretory protein, was fused to the truncated and tagged calnexin lacking the luminal portion (Fig. 1, PR/CN(HA)). Sheep prolactin used in the experiments contains one N-linked oligosaccharide (Li, 1976). When the prolactin chimera was immunoprecipitated from 293 cells transfected with pPL/CN(HA), a fuzzy band of 47 kDa corresponding to the glycosylated chimera was observed (Fig. 2, lanes5 and 6). Although the calculated molecular weight is 22,562, the prolactin chimera migrated abnormally on SDS-PAGE, presumably because of the extremely acidic charge of the calnexin cytoplasmic tail (Wada et al., 1991). However, no interaction of the chimera with nascent polypeptides, including the major 61-kDa calnexin-associated protein, was observed (lane5 of Fig. 2). Localization of PL/CN(HA) as well as CN(HA) or wild type CN to the peripheral network structure around the nucleus was confirmed by indirect immunofluorescence with 12CA5 antibody (data not shown). Therefore, we confirmed that the truncated calnexin lacking the luminal domain has no apparent chaperone function.

For subsequent experiments, we used HepG2 cells, since these hepatoma cells have a well developed secretion apparatus (Bouma et al., 1989) and since the chaperone function of calnexin has been well defined in these cells (Ou et al., 1993). We intended to confirm that the calnexin association with HepG2 secretory proteins is coupled to their glucose trimming. We pulse labeled HepG2 cells in the absence (Fig. 3, lanes1 and 3) or presence of 1 mM CAS (lanes2 and 4), followed by immunoprecipitation with anti-calnexin antibody (Fig. 3, lanes1 and 2) or anti-transferrin antibody (lanes3 and 4). Lane1 shows a set of nascent HepG2 polypeptides, which were coimmunoprecipitated with calnexin. As reported by Ou et al.(1993), the major bands of 80, 66, and 54 kDa corresponded to transferrin, alpha-fetoprotein, and alpha1-antitrypsin, respectively, as confirmed by sequential immunoprecipitations (data not shown). The band corresponding to alpha1-antichymotrypsin, which comigrated with alpha1-antitrypsin in the previous study (Ou et al., 1993) gave a slightly faster migration than the alpha1-antitrypsin band in our SDS-PAGE system, which used a 9% gel. Among the four major associated polypeptides, the identity of the 62-kDa polypeptide is not known. Association of these nascent polypeptides with calnexin was markedly reduced by CAS treatment of the cells (Fig. 3, lane2). The CAS treatment did not affect synthesis or core glycosylation of nascent transferrin but did produce a slightly slower migrating band, presumably reflecting inhibition of glucose trimming.


Figure 3: Effect of CAS on the association of newly synthesized proteins with calnexin in HepG2 cells. HepG2 cells were preincubated for 30 min with (lane2) or without (lane1) 1 mM CAS. The cells were subsequently labeled for 30 min with [S]methionine in the presence (lanes2 and 4) or absence (lanes1 and 3) of 1 mM CAS and then lysed with a buffer containing 1% Triton X-100. Endogenous calnexin was immunoprecipitated with anti-calnexin antibodies (lanes1 and 2). Cell extracts were also immunoprecipitated with anti-transferrin antibodies (lanes3 and 4). The immune complexes were resolved on a 9% SDS-gel and visualized by BAS 2000 equipped with Pictrography. The arrows indicate the positions of calnexin (CN, lanes1 and 2) and transferrin (TF, lanes3 and 4). Arrowheads, from top to bottom of gel, point to the major bands corresponding to transferrin, alpha-fetoprotein, a 62-kDa unidentified protein, and alpha1-antitrypsin, respectively. Numbers to the left show the positions of the molecular mass markers.



We next attempted to see if calreticulin could function as a chaperone when anchored to the ER membrane, where newly synthesized proteins are highly enriched. Since the carboxyl terminus region of calreticulin is composed of an ER retention signal (residues 396-399) and an acidic cluster(342-395), both of which are also found in the cytoplasmic tail of calnexin, we reasoned that calreticulin residues 1-341 might be sufficient to act as a chaperone. We therefore constructed a chimeric molecule (Fig. 1, CR/CNtc(HA)) by fusing calreticulin (residues -17-340) to the membrane/cytoplasmic domain of calnexin. We then expressed the chimera in HepG2 cells and lysed under denaturing conditions. The membrane-anchored calreticulin gave a major band of 67 kDa and a faint band of 75 kDa (Fig. 4A, lane3). The calculated molecular weight is 54,076, again showing an anomalous mobility on the SDS-PAGE gel. Both bands were also detected on immunoblots of cellular membranes that had been pre-washed with 0.1 M sodium carbonate, pH 11 (Morimoto et al., 1983; Wada et al., 1992), and probed with antibodies against calreticulin or calnexin (data not shown). The identity of the 75-kDa band, which was not clearly seen upon a shorter pulse (e.g.Fig. 4B, lanes1-3), is not known. A similar doublet was also observed for the intact calreticulin (Dupuis et al., 1993). (^3)Although rat calreticulin has one putative N-glycosylation site at Asn (Murthy et al., 1990), tunicamycin treatment did not change the mobility of the two bands, suggesting that they were not N-glycosylated. When HepG2 cells transfected with pCR/CN(HA) were lysed under non-denaturing conditions using Triton X-100, 12CA5 monoclonal antibody precipitated several nascent polypeptides (lane4 of Fig. 4A) including the four major bands. Essentially the same pattern was obtained when CN(HA) was analyzed (lane2 of Fig. 4A). The identity of the coprecipitates with CR/CN(HA) was confirmed by a series of sequential immunoprecipitations with 12CA5 antibody followed by individual antibodies against the secretory proteins, alpha1-antitrypsin (lane7), alpha-fetoprotein (lane8), and transferrin (lane9). Interaction of CR/CN(HA) with cellular calnexin or albumin, which is the major secretory protein of HepG2 cells, was not observed (data not shown).


Figure 4: Membrane-anchored calreticulin transiently interacts with nascent proteins. A, pCN(HA) (lanes1 and 2), pCR/CN(HA) (lanes3 and 4), or the control vector (pRep8) (lanes5 and 6) was transfected into HepG2 cells. The cells were lysed with a buffer containing SDS (lanes1, 3, and 5) or Triton X-100 (lanes2, 4, and 6) and immunoprecipitated with 12CA5 antibody. Four major bands coimmunoprecipitating with the tagged molecules are indicated by arrowheads. Arrows show the bands corresponding to CN(HA) (lanes1 and 2) or CR/CN(HA) (lanes3 and 4). For sequential immunoprecipitation, CR/CN(HA) was expressed in HepG2 cells cultured in a 35-mm dish. The cells were labeled for 30 min with [S]methionine, and the cell extract was prepared using 1% Triton X-100, followed by immunoprecipitation with 12CA5 antibody. The proteins that coprecipitated with CR/CN(HA) were extracted by incubating the immune complexes in 1% SDS at 65 °C for 10 min. This extract was then divided into three aliquots and re-immunoprecipitated with antibodies against alpha1-antitrypsin (lane7), alpha-fetoprotein (lane8), or transferrin (lane9). Asterisks indicate unknown polypeptides that were observed in the immunoprecipitates of 12CA5. B, HepG2 cells expressing CR/CN(HA) (lanes1-3) or the control vector (lanes4-7) were pulse labeled with [S]methionine for 15 min and chased for the indicated periods of time. The cell extracts were prepared using buffer containing 1% Triton X-100 and then immunoprecipitated with 12CA5 antibody (lanes1-4) or anti-calnexin antibodies (lanes5-7). Immune complexes were analyzed on a 9% gel. Arrowheads indicate the positions of the four major coprecipitates as described above. Asterisks indicate unknown polypeptides that were observed in the immunoprecipitates of 12CA5. C, CR/CN(HA)-transfected 293 cells were labeled for 30 min followed by immunoprecipitation with 12CA5 anti-tag antibody. Arrowhead indicates the 61-kDa major cellular protein associating with the calreticulin chimera.



We next tested whether the association of CR/CN(HA) with nascent proteins was transient. Pulse-chase experiments (Fig. 4B, lanes1-3) revealed that the majority of the polypeptides bound to the calreticulin chimera dissociated as the cells were chased. When quantitated by BAS2000, 55% of alpha-fetoprotein and 31% of transferrin dissociated from the membrane-anchored calreticulin at 15 min of chase and 80 and 62%, respectively, at 45 min. Comparable results were obtained for cellular calnexin (Fig. 4B (lanes5-7); alpha-fetoprotein, 48% at 15 min and 78% at 45 min; transferrin, 22% at 15 min and 65% at 45 min) as well as for CN(HA) (data not shown).

The transient interaction of calreticulin (residues 1-340) with nascent chains on the ER membrane was also confirmed in 293 cells. Immunoprecipitation from 293 cells transfected with pCR/CN(HA) using 12CA5 antibody showed that the 61-kDa polypeptide observed in Fig. 2was also the major protein in the immunoprecipitates and dissociated after 1 h of chase (Fig. 4C).

Nigam et al.(1994) reported that calreticulin as well as immunoglobulin heavy chain binding protein (BiP), calnexin, ERp72, Grp94, and protein-disulfide isomerase bound to urea-denatured secretory proteins immobilized on Sepharose beads and that all of them with the exception of calnexin were eluted from the beads by incubation with 1 mM ATP. Therefore, we tested if ATP could disrupt the nascent chain/membrane-anchored calreticulin complex. We isolated the immune complex by incubating 12CA5 monoclonal antibody with lysate from HepG2 cells expressing pCR/CNtc(HA), which had been pre-labeled with [S]methionine for 30 min. The complex was then incubated with 1 mM ATP on ice for 1 h or at 16 °C for 15 min. However, ATP treatment did not release the nascent chains from the complex (data not shown).

Next, we examined whether the chaperone function of calreticulin (residues 1-340) was dependent on glucose trimming. We treated the HepG2 cells expressing CR/CN(HA) with 1 mM CAS and then isolated the immune complex with 12CA5 antibody. As shown in Fig. 5, the set of polypeptides coisolated from non-treated cells including transferrin, alpha-fetoprotein, and alpha1-antitrypsin was not recovered in the immune complex of CR/CN(HA) isolated from the CAS-treated cells.


Figure 5: CAS abolishes the interaction of the membrane-anchored calreticulin with nascent proteins. CR/CN(HA)-transfected HepG2 cells were incubated for 30 min with (lane2) or without (lane1) 1 mM CAS and then labeled for 30 min with [S]methionine in the presence (lane2) or absence (lane1) of 1 mM CAS. The cell lysates were processed for immunoprecipitation with 12CA5 antibody. Arrowheads indicate the four major coprecipitates. Asterisks indicate unknown polypeptides that were observed in the immunoprecipitates of 12CA5.



Finally, we tested if the apparent chaperone function of calreticulin (residues 1-340) above described is expressed at its physiological location, i.e. the lumen of the ER. Since calreticulin contains an ER retention signal (KDEL) at the COOH terminus, which can function as an independent domain (Munro and Pelham, 1987), we placed a HA tag internally near the COOH terminus of calreticulin (Fig. 1, bottom) and expressed it in HepG2 cells. The expressed molecules were extractable from the cells by 0.1 M sodium carbonate, pH 11, confirmed by an immunoblot, and were shown to be confined to the perinuclear structure of HepG2 cells when tested by indirect immunofluorescence (data not shown). Immunoprecipitation with the tag antibody from the SDS-lysate of cells transfected with pCR(HA) yielded a single 58-kDa band (Fig. 6A, lane2), a molecular mass comparable to that previously reported (Michalak et al., 1992). When the expressed molecule of 58 kDa was isolated under non-denaturating conditions, two additional bands of 100 and 80 kDa were reproducibly observed in the immunoprecipitate (Fig. 6A, lane3). The prominent 47-kDa band in lane3 could not be observed in other repeated experiments because it comigrates with the band that was usually observed in control immunoprecipitations. Sequential immunoprecipitation (Fig. 6B, lane5) identified the 80-kDa band as transferrin. The identity of the 100-kDa band is not known. In contrast, three other major calnexin substrates (Fig. 6B, lane1) were recovered to a limited extent in the CR(HA)-immune complex (Fig. 6B, lanes2-4). The interactions of CR(HA)-bound proteins were also transient, and 20% of transferrin was released at 15 min of chase and 65% at 45 min (Fig. 6C, lanes2-4). As shown for the membrane-anchored calreticulin, CAS treatment abolished the transient association of nascent polypeptides with calreticulin (Fig. 6D, lane3), confirming that the apparent chaperone property of calreticulin at its physiological location is also coupled with glucose processing.


Figure 6: Soluble calreticulin interacts with nascent glycoproteins at varied efficiencies in a CAS-sensitive manner. A, HepG2 cells transfected with wild type vector (pRep8) or pCR(HA) were labeled for 30 min with [S]methionine and lysed with buffer containing 1% SDS (lane2) or 1% Triton X-100 (lanes1 and 3). Immunoprecipitation was carried out with 12CA5 antibody. Asterisks indicate unknown polypeptides directly bound to 12CA5. Two bands of 80 and 100 kDa in lane3 coisolated with CR(HA) are indicated by dots. B, HepG2 cells expressing CN(HA) (lane1) or CR(HA) (lane2) were labeled for 30 min and lysed with Triton X-100-containing buffer followed by immunoprecipitation with 12CA5. For sequential immunoprecipitations, CR(HA) was expressed in HepG2 cells plated in 35-mm dishes. Sequential immunoprecipitation with anti-alpha1-antitrypsin (lane3) or anti-alpha-fetoprotein (lane4) or anti-transferrin (lane5) antibodies was carried out as described in Fig. 4. The four major calnexin substrates are indicated by arrowheads. Asterisks and dots are as indicated above for panelA. C, HepG2 cells harboring pRep8 (lane1) or pCR(HA) (lanes2-4) were labeled for 15 min with [S]methionine and chased for 0 min (lanes1 and 2), 15 min (lane 3), and 45 min (lane4) by adding 5 mM methionine and 1 mM cysteine. The Triton X-100 extracts were processed for immunoprecipitation with 12CA5. Asterisks and dots are as indicated above for panelA. D, HepG2 cells harboring pRep8 (lane1) or pCR(HA) (lanes2 and 3) were labeled with [S]methionine for 30 min in the absence (lanes1 and 2) or presence (lane3) of 1 mM CAS after a 30-min preincubation period. Immunoprecipitation was carried out using 12CA5 antibody.




DISCUSSION

This study demonstrates that calreticulin (residues 1-340) transiently interacts with secretory glycoproteins of HepG2 cells when expressed as a membrane-anchored chimera and that the association was sensitive to CAS treatment. Although calnexin interacts with a selected set of proteins synthesized in the ER with various efficiencies (Margolese et al., 1993; Ou et al., 1993; Kim and Arvan, 1995; Arunachalam and Cresswell, 1995), the spectrum of proteins that calreticulin (residues 1-340) recognized on the membrane was remarkably similar to what was observed for calnexin. Therefore, we conclude that calreticulin per se is another novel chaperone that requires glucose processing of N-linked oligosaccharides to interact with nascent proteins. We further confirmed that calreticulin, expressed at its physiological location, indeed transiently associated with a set of nascent proteins in a CAS-inhibitable manner. Different from the membrane-anchored form, the soluble calreticulin appeared to interact preferentially with transferrin and only weakly with other major calnexin ligands (Fig. 6B). However, it is apparent that calnexin and calreticulin share a basically similar ligand specificity. These results clearly suggest that the ER possesses two kinds of molecular chaperones to fulfill its quality control function. At present, it is difficult to assess the quantitative contributions of the two chaperones in the folding process of secretory proteins. However, it is reasonable to assume that calnexin should play the major role in the maturation process, since the membrane rather than the ER lumen would be primarily enriched with nascent proteins. Such nascent proteins may have differential folding kinetics, and some of them, like transferrin as observed in this experiment, might have access to the luminal calreticulin without being trapped by calnexin. In this context, the soluble calreticulin may represent a ``back-up'' mechanism for the kinetically diverse process of nascent chain maturation.

Calreticulin was originally discovered as a ``high affinity calcium binding protein'' of skeletal muscle (MacLennan et al., 1972). Later, ER localization of this molecule was found in cells of non-muscular type from various species (see Michalak et al. (1992) and references therein). Several physiological functions have been ascribed to this abundant protein. At first, a role for calcium storage was proposed since calreticulin binds, per mole of protein, 20-50 mol of calcium at low affinity and 1 mol of calcium at high affinity. It was also known that the majority of calcium in the ER was found bound to proteins (MacLennan et al., 1972; Michalak et al., 1991; Baksh et al., 1992; Treves et al., 1992). At least three physiological phenomena have been reportedly correlated to this role. First, calreticulin expression was moderately induced upon activation of T lymphocytes (Burns et al., 1992). Second, calreticulin as well as calcium-ATPase sequestrated in vesicles were concentrated around the undigested particles when heat-killed yeasts were presented to neutrophils (Stendahl et al., 1994). This suggested that calreticulin may play a role in the regulation of calcium homeostasis upon phagocytosis. Finally, in cytolytic lymphocytes, calreticulin was found as the major constituent of lytic granules (Dupuis et al., 1993). In this case, calreticulin has been speculated to function as a calcium chelator in the granules, since an increase of free calcium led to perforin-mediated autolysis. Thus, it has been suggested that calreticulin plays a dynamic role in regulating calcium homeostasis. On the other hand, several studies revealed that calreticulin directly bound to polypeptides through the sequence KXFF(K/R)R, which is found in the DNA binding domain of nuclear hormone receptors as well as in the regulatory cytoplasmic domain of the integrin family (Burns et al.(1994), Dedhar et al.(1994), and reviewed by Dedhar(1994)). In fact, nuclear as well as cytoplasmic localization of calreticulin was also suggested (Rojiani, 1991; Michalak et al., 1992; Dedhar, 1994; Leung et al., 1994). Since calreticulin contains a cleavable signal sequence at the amino terminus, elucidation of a mechanism by which this protein escapes targeting by the signal recognition particle (Rapoport, 1992) to the ER would be of great interest. The chaperone function of calreticulin described in this study apparently differs from its association with polypeptides previously reported (Dedhar, 1994; Nigam et al., 1994) in that the association is limited to newly synthesized proteins and is inhibited by CAS (Fig. 5) or tunicamycin treatment (data not shown). Recently, Nauseef et al.(1995) reported that calreticulin bound to the lysosomal enzyme myeloperoxidase, being slowly dissociated over 2 h of chase. Since no lysosomal enzymes have so far been reported to interact with calnexin, it would be interesting to know if lysosomal enzymes preferentially interact with calreticulin and whether the interaction is also dependent on glucose trimming.

The luminal domain of calnexin was demonstrated to have a weak but specific affinity for the structure of Glc(1)Man(9)GlcNAc(2) itself (Ware et al., 1995). Remarkably, it was also found that a complex of calnexin with nascent class I major histocompatibility antigen molecules or alpha1-antitrypsin was resistant to endoglycosidase H treatment. Therefore, it was proposed that the recognition of Glc(1)Man(9)GlcNAc(2) triggers stable association of the polypeptide chain with calnexin. At present, the molecular basis for the proposed two-step mechanism is not clear. Given that calreticulin appears to have the same chaperone function as calnexin, it would be reasonable to assume that the two units of sequence repeats, PXXIPDPXAXKPEDWDE and PXXIPDPXAXKPEDWDE may be primarily responsible for the unique molecular properties common to both calnexin and calreticulin. These two sets of unique motifs are repeated four times in calnexin and three times in calreticulin. Furthermore, it should be pointed out that both molecules contain a short stretch of hydrophobic residues (Phe-Val, calnexin; Phe-Ile, calreticulin) despite the fact that the amino acid sequences in this region are not well conserved. Such a hydrophobic domain may play a role in binding to the immature polypeptides, as the hydrophobic patch is thought to be the common structure exposed on the surface of unfolded molecules. The tagged expression system described in this study may be suitable for mapping of the binding sites.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-11-611-2111 (ext. 2294); Fax: 81-11-642-8052 or 612-5861; wada{at}sapmed.ac.jp.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; CAS, castanospermine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

(^2)
H. Kimura, manuscript in preparation.

(^3)
I. Wada, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. H. Kimura (Research Center for Molecular Genetics, Hokkaido University) for comments on cDNA expression using pRep8 vector.


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