©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conformational Changes Induced in the Endoplasmic Reticulum Luminal Domain of Calnexin by Mg-ATP and Ca(*)

(Received for publication, April 20, 1995; and in revised form, May 12, 1995)

Wei-Jia Ou (1) (2) John J. M. Bergeron (2)(§) Yan Li (4)(¶) C. Yong Kang (4)(¶) David Y. Thomas (1) (2)(§) (3)(**)

From the  (1)Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada, the (2)Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada, the (3)Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada, and the (4)Departments of Zoology and of Microbiology and Immunology, Faculty of Medicine, The University of Western Ontario, London, Ontario N6A 5B7, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The type I membrane protein calnexin functions as a molecular chaperone for secretory glycoproteins in the endoplasmic reticulum with ATP and Ca as two of the cofactors involved in substrate binding. Protease protection experiments with intact canine rough microsomes showed that amino acid residues 1-462 of calnexin are located within the lumen of the endoplasmic reticulum. Expression using the baculovirus Sf9 insect cell system of a recombinant truncated calnexin corresponding to residues 1-462 (calnexinTMC) revealed an association in vivo with a coexpressed secretory glycoprotein substrate, human immunodeficiency virus type I gp120. For the in vitro characterization of calnexinTMC, we purified this secreted form to homogeneity from the medium of Sf9 cells. We demonstrate that the properties of the purified calnexinTMC correspond to those of full-length calnexin in canine microsomes with at least one intramolecular disulfide bond and binding to Ca. CalnexinTMC underwent a marked and reversible conformational change following Ca binding as measured by its resistance to proteinase K digestion of a 60-kDa fragment and also by the change from an oligomeric form of calnexinTMC to a monomeric form. We also found that calnexin bound Mg-ATP leading to a conformational change from a monomeric to an oligomeric form that coincided as with markedly increased proteinase sensitivity. Our results identify the luminal domain of calnexin as responsible for binding substrates, Ca, and Mg-ATP. Because Ca and ATP are required in vivo for the maintenance of calnexin-substrate interactions, conformational changes in the luminal domain of calnexin induced by Ca and Mg-ATP are relevant to the in vivo function of calnexin as a molecular chaperone.


INTRODUCTION

Calnexin is an integral membrane protein of the endoplasmic reticulum (ER)()that functions as a molecular chaperone and as a constituent of the quality control apparatus of the ER (reviewed in Bergeron et al.(1994)). Calnexin was originally identified as a major integral membrane phosphoprotein of the ER (Wada et al., 1991). Cloning of the cDNA and sequencing predicted that calnexin is a type I membrane protein with the predicted luminal domain revealing regions of sequence similarity with the major Ca-binding protein of the ER lumen, calreticulin. Based on the binding of Ca to ER membrane proteins as well as the isolated protein, it was shown that calnexin is one of the two major Ca-binding resident proteins of the membrane of canine rough ER (Wada et al., 1991; Cala et al., 1993).

The function of calnexin as a molecular chaperone initially arose from the demonstration that it is associated in the ER with newly synthesized major histocompatibility complex class I heavy chain and other cell surface receptors (see Bergeron et al.(1994) for review). Subsequently the repertoire of membrane protein substrates for calnexin was expanded to soluble monomeric secretory glycoproteins (Ou et al., 1993; Le et al., 1994; Wada et al., 1994). It was observed that there is a requirement for glycosylation for proteins to bind to calnexin in vivo, because the N-linked glycosylation inhibitor tunicamycin prevented the association of newly synthesized, incompletely folded substrates with calnexin (Ou et al., 1993). In addition, deoxynojirimycin and castanospermine, inhibitors of glucosidase I and II, also abrogated the association of newly synthesized, incompletely folded viral membrane glycoproteins with calnexin. These observations led to the proposal that calnexin was a lectin with specificity for monoglucosylated N-linked oligosaccharides (Hammond et al., 1994). Support for this hypothesis has come from the recent demonstrations of direct binding between a recombinant soluble calnexin and the free oligosaccharide GlcManGlcNAc (Ware et al., 1995) and of binding of glucosylated thyroglobulin and soybean agglutinin in a cell free system.()

However, in vivo calnexin can also bind non-glycosylated proteins (Rajagopalan et al., 1994; Loo and Clarke, 1994; Kim and Arvan, 1995). The N-linked oligosaccharide can also be removed by endoglycosidase H from coimmunoprecipitates of substrates bound to calnexin without disrupting their association (Ware et al., 1995). These studies indicate that the N-linked oligosaccharide may be preferred for the initial interaction of newly synthesized glycoproteins with calnexin but is not necessary to maintain the interaction.

There are several different regions of calnexin that have been argued to be responsible for binding substrates. The transmembrane and immediate juxtamembrane regions of newly synthesized substrate membrane proteins have been indicated to be required for binding to calnexin (Margolese et al., 1993). A role for cytosolically oriented phosphorylation of calnexin has also been proposed in the association of different allotypes of major histocompatibility complex class I heavy chains with calnexin (Capps and Ziga, 1994). Furthermore, an ATP requirement has been demonstrated for the association of the newly synthesized soluble glycoprotein gp80 with calnexin in Madin-Darby canine kidney cells (Wada et al., 1994). However, it is clear that secretory glycoproteins can only interact directly with the luminal domain of calnexin and that membrane substrates also have the opportunity for this interaction. Taken together, these studies indicate multiple mechanisms of presentation and binding of substrates to calnexin (Bergeron et al., 1994).

In order to establish the mechanism of association of calnexin with its substrates and to determine the cofactors involved, we have expressed a soluble recombinant derivative of calnexin in the baculovirus-infected Sf9 expression system. We demonstrate here that soluble calnexin can associate with the newly synthesized soluble glycoprotein substrate HIV-1 gp120 when they are coexpressed in Sf9 cells. We further show that soluble calnexin purified from the extracellular medium of recombinant baculovirus-infected Sf9 cells undergoes conformational changes as revealed by the formation of a Ca-dependent proteinase-resistant core and a Ca-dependent change from an oligomeric to a monomeric state, whereas Mg-ATP promoted a change from a monomeric to an oligomeric state that coincided with increased proteinase sensitivity. Finally, we show that secreted soluble calnexin is at least partially disulfide-bonded intramolecularly and can bind ATP directly but does not have detectable ATPase activity. These findings extend the functional significance of calnexin as an ATP-binding molecular chaperone with properties distinguishable from those of the ER luminal Ca-binding and ATP-binding chaperone, BiP.


EXPERIMENTAL PROCEDURES

Recombinant Baculovirus and Infection of Sf9 Cells

cDNA encoding a truncated canine calnexin (amino acid residues 1-462) followed immediately by an in-frame stop codon was generated by polymerase chain reaction and subsequently cloned into the transfer vector pETL1 at the BamHI site filled in with Klenow polymerase. The recombinant baculovirus was isolated by the -galactosidase screening and used to infect Sf9 cells that were grown in Sf9000 II serum-free medium as described by Vialard et al.(1990). The sequence of the calnexin construct in the baculovirus vector was confirmed, and the following differences were observed with the published sequence (Wada et al., 1991). Nucleotides 199 and 201 are determined to be A and A and not T and C, respectively, as previously determined. This changes the codon for residue 67 from TCC to ACA, and the corresponding amino acid change is Ser to Thr. There is also a threonine at this position in human calnexin (David et al., 1993). The sequence of a peptide of canine calnexin from this region also predicted a threonine at this position. Thus we conclude that the original canine calnexin nucleotide sequence is incorrect in this region. We also found one other difference with the published canine calnexin nucleotide sequence at nucleotide 201, which is a change of the originally predicted C to an A; this does not change the amino acid sequence. Expression of HIV-1 gp120 in Sf9 cells by recombinant baculovirus has been described previously (Li et al., 1994).

Purification of CalnexinTMC

Sf9 cells were infected with recombinant baculovirus expressing calnexinTMC and grown for 72 h. PMSF and p-hydroxymercuriphenylsulfonic acid (final concentrations, 1 mM and 0.1 mM, respectively) were added to the medium. All of the buffers used for purification of calnexin contained 1 mM CaCl. Ammonium sulfate (70% saturation) was added to the medium, and the supernatant was passed through a phenyl-Sepharose column (2.7 5 cm). The flow-through was desalted using a G-25 Sephadex column and loaded onto a DEAE-Sepharose column (2.7 10 cm) (Pharmacia Biotech Inc.) equilibrated with 10 mM Tris-HCl (pH 7.5), 50 mM NaCl. The column was washed with the same buffer, and the proteins were eluted with a linear gradient of 50-600 mM NaCl. The calnexinTMC-containing fractions were pooled, concentrated with a Centriprep 30 (Pharmacia Biotech Inc.), and further purified on a fast protein liquid chromatography Mono-Q column (10/10) equilibrated with 10 mM Tris-HCl (pH 7.5), 200 mM NaCl. The proteins were eluted with a gradient of 200-800 mM NaCl. CalnexinTMC was detected by immunoblotting with anti-CN1, a rabbit polyclonal antibody made against a synthetic peptide encompassing residues 30-48 of the NH terminus of calnexin. This antibody recognizes mammalian calnexin but not insect calnexin.

Proteinase K Digestion

Purified calnexinTMC (1 mg/ml) was incubated with increasing concentrations of proteinase K (Boehringer Mannheim) in 20 mM Hepes (pH 7.4), 50 mM NaCl buffer at 30 °C for 30 min. Protease digestion of native calnexin was carried out using solubilized canine pancreatic ER membranes (2 mg/ml) in the presence of 1% CHAPS (Sigma). After the addition of 1 mM PMSF to the proteinase K incubations, proteins were separated on SDS-PAGE (5%-15% gel) followed by Coomassie Blue staining for the purified calnexinTMC (see Fig. 6A) or by immunoblotting with anti-CN1 for the ER membranes (see Fig. 6B).


Figure 6: Protease digestion of calnexin. A, purified calnexinTMC (1 mg/ml) was incubated with the indicated concentrations of proteinase K in the presence of 2 mM CaCl (lanes 1-7) or 5 mM EGTA (lanes 8-13), or calnexinTMC was preincubated with 5 mM EGTA for 5 min and subsequently treated with 10 mM CaCl for 5 min. The treated calnexinTMC was then incubated with proteinase K (lanes 14-19). The proteinase K digestion was terminated by the addition of 1 mM PMSF, and the mixtures were analyzed on SDS-PAGE (5-15% gel) followed by Coomassie Blue staining. The significance of the fragments at 30 kDa in lanes 7, 13, and 19 are uncertain. B, canine pancreatic ER membranes (2 mg/ml) were incubated with increasing concentrations of proteinase K in the presence of 1% CHAPS under the same conditions as described in A. The proteins were separated on SDS-PAGE followed by immunoblotting with anti-CN1. Lane 20 in B is the fragment of calnexinTMC produced by 100 µg/ml proteinase K treatment in the presence of CaCl (lane 6 in A). The fragment matches exactly the fragment of native calnexin produced by the same amount of proteinase K. CN, calnexinTMC; CN, native calnexin. C, purified calnexinTMC (lane 1) was incubated with proteinase K (PK, lanes 2 and 3) or trypsin (lanes 4 and 5) in the presence of 1 mM CaCl at 30 °C for 30 min. After PMSF was added to stop the reactions, proteins were analyzed on SDS-PAGE followed by Coomassie Blue staining. PK, proteinase K; CN, calnexinTMC.



ATP Binding Assay

Purified calnexinTMC (0.1 mg/ml) was incubated with 20 µCi/ml (25 µM) [-P]ATP (800 Ci/mmol; DuPont Canada, Mississauga, Ontario) in 20 mM Hepes-NaOH (pH 7.5) buffer containing 50 mM NaCl, 10 mM MgCl, 2 mM CaCl at 20 °C for 10 min and then exposed to UV radiation for 5 min. For inhibition experiments, reagents were incubated with calnexin prior to the addition of [-P]ATP. The P-labeled protein was separated on SDS-PAGE and stained with Coomassie Blue followed by radioautography.

Metabolic Labeling and Immunoprecipitation

24 h after infection with recombinant baculoviruses, Sf9 cells were labeled with 100 µCi/ml [S]methionine (1000 Ci/mmol, Amersham Corp.) for 15 min at 25 °C. The labeled cells were washed with cold phosphate-buffered saline and lysed in 50 mM Hepes-NaOH buffer (pH 7.4) containing 200 mM NaCl, 2% CHAPS, 1 mM CaCl, 1 mM PMSF, and 5 µg/ml each leupeptin and aprotinin. The cell lysate was precleared with protein A-Sepharose and immuno-precipitated with anti-CN1 or anti-gp120 antibodies as described previously (Ou et al., 1993).

Analytical Methods

Protein concentration was determined as described by Bradford(1976). The Ca overlay assay was as described in Wada et al.(1991). SDS-PAGE was performed as described by Laemmli(1970). Nondenaturing gel electrophoresis was carried out in the absence of SDS using the Laemmli system. Protein sequencing of calnexinTMC or its protease-cleaved fragments was determined using an Applied Biosystems 470A protein sequencer equipped with an on-line phenylthiohydantoin amino acid analyzer.


RESULTS

Orientation of Calnexin in Vivo

From its primary sequence calnexin is predicted to be a type I integral membrane protein of the ER (Wada et al., 1991). In this study we confirm the orientation of calnexin in the ER membrane by using proteinase K digestion of canine microsomes and antibodies to peptides in different regions of calnexin (Fig. 1). Digestion of intact microsomes revealed a 70-kDa proteinase-resistant fragment, indicating that the majority of the protein was intraluminal (Fig. 1A). The result is consistent with the demonstration that in dog cardiac sarcoplasmic reticulum vesicles a 70-kDa fragment of calnexin is protected from digestion by proteases (Cala et al., 1993). Both the native and protease-resistant fragment of calnexin were recognized by antibodies to residues 30-48 of calnexin (anti-CN1). Antibodies to residues 555-573 (anti-CN4) only recognized full-length calnexin in intact microsomes, and proteinase treatment abolished this recognition (Fig. 1B). These results confirm that in vivo calnexin is a type I membrane protein and that residues 1-462 of calnexin are luminally oriented followed by a hydrophobic membrane-spanning domain and a cytosolically oriented COOH-terminal tail (Fig. 1C).


Figure 1: A, protease sensitivity of calnexin. 10 µl of 0.5 M KCl-washed canine microsomal membranes (1 mg/ml) were incubated with the indicated concentrations of proteinase K in 0.25 M sucrose, 1 mM dithiothreitol, 50 mM KCl, 50 mM Tris-HCl (pH 7.5) at 20 °C for 30 min in the presence (lane 9) or absence (lanes 1-8) of 0.5% CHAPS. In all cases Ca was omitted from the incubations. The reaction was terminated by the addition of 1 ml of 1% PMSF and processed for immunoblotting with antibody made to residues -8 to +268 of bacterially expressed calnexin as described in Ahluwalia et al.(1992). The mobility of intact calnexin and the proteinase resistant fragment is indicated. B, membrane orientation of calnexin. Dog pancreatic stripped rough microsomes (25 µg) were incubated with trypsin (200 µg/ml) at 20 °C for 30 min either in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 0.2% Triton X-100. The reaction was terminated by the addition of 1 mM PMSF. Lanes 1 and 4 are control samples incubated in the absence of trypsin or detergent. Samples were subjected to SDS-PAGE followed by immunoblotting with anti-CN1 (NH terminus; lanes 1-3) or anti-CN4 (COOH terminus; lanes 4-6). C, deduced orientation of canine calnexin (modified from Fig. 10of Wada et al.(1991)). Shown are the positions of the residues to which anti-CN1 (-calnexin 1) and anti-CN4 (-calnexin 4) were raised, the predicted transmembrane segment, and the luminal region of calnexin with the four regions (A-D) of high sequence similarity to calreticulin.




Figure 10: Direct binding of ATP to calnexinTMC. CalnexinTMC (lanes 1-4) or bovine serum albumin (lanes 5) was incubated with [-P]ATP (20 µCi/ml, 25 µM) in the presence of 10 mM MgCl (lanes 1 and 4), 2 mM CaCl (lanes 2), or 10 mM EDTA (lanes 3) for 10 min at room temperature. The mixtures were irradiated with UV radiation (lanes 2-5) for 5 min or not exposed (lanes 1). The proteins were analyzed on SDS-PAGE and stained with Coomassie Blue (A) followed by radioautography (B). C.B., Coomassie Blue. C, ATP binding assays were carried out as described under ``Experimental Procedures'' except that, prior to the addition of [-P]ATP, calnexinTMC was preincubated with the indicated reagents for 5 min. Proteins were analyzed on SDS-PAGE followed by radioautography. The bands corresponding to calnexinTMC were excised from the gels, and their radioactive content was determined by liquid scintillation counting. The values are expressed as percentages of the control. DTT, dithiothreitol; IAA, iodoacetamide; NEM, N-ethylmaleimide.



Heterologous Coexpression and Association of a Truncated Soluble Calnexin with Newly Synthesized HIV-1 gp120

Because calnexin can transiently bind both membrane and soluble secretory proteins, the luminal domain is predicted to be responsible for this interaction. With type I membrane proteins removal of the membrane spanning domain can lead to their secretion from cells. Introduction of a stop codon at residue 463 (i.e. immediately preceding the predicted transmembrane domain of calnexin) (Fig. 1C) led to the secretion of calnexinTMC. 24 h after recombinant baculovirus infection (Fig. 2, lanes 3 and 4), intracellular calnexinTMC with a mobility of 70 kDa was recognized by the anti-CN1 antibody with increasing amounts of the protein being secreted at 48, 72, and 96 h postinfection (Fig. 2, lanes 5-10). Whereas two polypeptides of 70- and 69-kDa were observed for intracellular calnexinTMC (Fig. 2, lanes 5, 7, and 9), extracellularly only the 70-kDa form was observed (Fig. 2, lanes 6, 8, and 10).


Figure 2: Secretion of calnexinTMC from Sf9 cells. Sf9 cells were infected with recombinant baculovirus expressing calnexinTMC as described under ``Experimental Procedures.'' At the indicated times postinfection, cells (C) and medium (M) were separately harvested and analyzed on SDS-PAGE followed by immunoblotting with anti-CN1. CN, calnexinTMC.



We devised an experiment that clearly shows that calnexinTMC can interact in vivo with a viral glycoprotein. We compared the association of calnexinTMC and that of full-length calnexin with a calnexin substrate, HIV-1 gp120.()HIV-1 gp120 is normally processed from the precursor gp160 by proteolytic cleavage, generating soluble gp120 and the COOH-terminal membrane attached gp41; but in this experiment gp120 alone was expressed by inserting a termination codon at the site of cleavage. As shown in Fig. 3, 24 h after infection of Sf9 cells with recombinant baculovirus containing calnexinTMC (lane 1), HIV-1 gp120 (lane 2), or full-length calnexin (lane 5), they could be detected by immunoprecipitation of pulse-labeled cells with either anti-CN1 (lanes 1 and 5) or anti-HIV-1 IgG (Fig. 3, lane 2). Coinfection with baculoviruses containing either calnexinTMC (Fig. 3, lane 3) or full-length calnexin (Fig. 3, lane 6) with HIV-1 gp120 revealed their association because they could be coimmunoprecipitated with anti-CN1. The lack of radiolabeled calnexin in the immunoprecipitates with HIV-1 IgG (Fig. 3, lanes 4 and 7) is due to the large pool of pre-existing calnexin (Ou et al. 1993). The gp120 molecules that were associated with calnexin (Fig. 3, lanes 3 and 6) were incompletely folded, because they were not recognized by the receptor CD4, as has been recently shown for full-length calnexin and was also shown for gp120 in association with calnexinTMC (data not shown). Thus, full-length calnexin and calnexinTMC appear to be equally capable of interacting with a soluble glycoprotein substrate.


Figure 3: Expression and association of calnexinTMC or full-length canine calnexin with HIV-1 gp120. Sf9 cells were infected with recombinant baculovirus expressing either calnexinTMC (lane 1) or HIV-1 gp120 (lane 2) or coinfected with recombinant baculovirus containing calnexinTMC and gp120 (lanes 3 and 4) or viruses containing full-length calnexin alone (lane 5) or coinfection with recombinant baculoviruses containing full-length calnexin and gp120 (lanes 6 and 7). 24 h after infection, the cells were labeled with 100 µCi/ml [S]methionine for 15 min, and the labeled cells were lysed and immunoprecipitated with anti-CN1 (lanes 1, 3, 5, and 6) or anti-gp120 (lanes 2, 4, and 7) antibodies. The immunoprecipitates were analyzed on SDS-PAGE followed by fluorography. CN, calnexinTMC; CN, full-length calnexin.



Purification of Soluble Secreted CalnexinTMC

The association of intracellular soluble calnexinTMC with gp120 suggested that the luminal domain alone interacts with substrates. Thus we used this expression system to purify calnexinTMC for in vitro characterization. At 72 h postinfection, calnexinTMC was about 1% of the total protein in the medium from Sf9 cells, and it was purified to homogeneity (see ``Experimental Procedures''). The final preparation showed a single band for calnexinTMC with an apparent molecular mass of 70 kDa on SDS-PAGE (Fig. 4A). We confirmed the NH-terminal sequence of the first 20 residues of the purified protein as HEGHD(D)(D)MID(I)ED(D)LD(D)VIE (residues in parentheses indicate the inherent difficulty in identifying consecutive residues), which is identical to that for calnexin purified from microsomes (Wada et al., 1991).


Figure 4: Purification of calnexinTMC. Purification of calnexin TMC from the culture medium of infected Sf9 cells was carried out as described under ``Experimental Procedures.'' The proteins from each purification step were separated on SDS-PAGE followed by Coomassie Blue (C. B.)staining (A) or by immunoblotting with anti-CN1 (B). Lane 1, culture supernatant; lane 2, supernatant of 70% saturated ammonium sulfate; lane 3, phenyl-Sepharose fraction; lane 4, DEAE-Sepharose fraction; lane 5, Mono-Q fraction. CN, calnexinTMC.



Characterization of Purified CalnexinTMC

Calcium Binding

The luminal domain of calnexin shows 4 regions of sequence similarity with calreticulin, the major ER luminal calcium-binding protein (Fig. 1C and Wada et al. (1991)). The results in Fig. 5show that the detergent phase of Triton X-114-extracted canine pancreatic ER membranes contains a major calcium-binding protein corresponding to calnexin (90 kDa) as well as the other major calcium-binding ER membrane protein pgp35 (35 kDa) (Fig. 5, lane 1). We were also able to show that purified soluble calnexinTMC can bind calcium using the Ca overlay technique, whereas in a control experiment bovine serum albumin did not (Fig. 5, lane 3). Thus both full-length calnexin from ER membranes and purified calnexinTMC show similar behavior in their ability to bind calcium.


Figure 5: Ca binding to calnexinTMC. Triton X-114 detergent phase extracted from 50 µg of canine pancreatic ER membrane (lanes 1), 2 µg of purified calnexinTMC (lanes 2), or 5 µg of bovine serum albumin (lanes 3) were separated on SDS-PAGE followed by Coomassie Blue (C. B.) staining (A) or by electrotransfer to a nitrocellulose membrane. The membrane was then incubated with Ca followed by radioautography as described under ``Experimental Procedures'' (B). CN, calnexinTMC. The 90- and 35-kDa proteins that displayed calcium binding in lane 1 are native calnexin and pgp35, respectively (Wada et al., 1991).



Proteinase Sensitivity

Sensitivity to proteinase digestion was used to evaluate protein domain structure. When calnexinTMC was incubated with low concentrations (1-10 µg/ml) of proteinase K in the presence of calcium, a 65-kDa fragment was generated (Fig. 6A, lanes 2-4). With increasing concentrations of the proteinase (100-300 µg/ml), a fragment of 60 kDa was generated (Fig. 6A, lanes 6 and 7). The proteinase digestion of native calnexin in the solubilized ER membrane resulted in a similar profile (Fig. 6, compare A, lanes 2-7, with B, lanes 2-7). Low (1-10 µg/ml) and high (100-300 µg/ml) concentrations of the proteinase-produced fragments of 65 and 60 kDa, respectively (Fig. 6, compare A and B, lanes 19 and 20). Sequence analysis of the 60-kDa fragment revealed the major sequence to be KSKPDTSAPTSP, and therefore it includes the sequence used to generate the anti-CN1 antibody (residues 30-48). Digestion of calnexinTMC with trypsin in the presence of calcium also generated the 60-kDa fragment (Fig. 6C, lanes 4 and 5); but the sequence was SKPDTSAPTSP, just one residue downstream from the proteinase K cleavage site. Our data indicate that proteinase-sensitive sites exist at both the carboxyl and the amino termini of calnexin. But there is a proteinase-resistant core structure that requires calcium to maintain resistance, because, in the presence of EGTA, both calnexinTMC and native calnexin became highly sensitive to a low concentration (10 µg/ml) of proteinase K (Fig. 6, A and B, lanes 8-13). To determine if the proteinase-resistant and -sensitive forms of calnexin are reversible, calnexinTMC was first incubated with EGTA, leading to the proteinase-sensitive conformation, and then excess calcium was added to the protein followed by the addition of proteinase K. This procedure restored the proteinase-resistant conformation (Fig. 6A, lanes 14-19). Full-length calnexin also displayed the same reversible conformational change of calnexinTMC (Fig. 6B, lanes 14-19). Thus both full-length calnexin from ER membranes and purified calnexinTMC can be digested with protease to generate the same resistant core fragment.

Disulfide Bonded Calnexin

Calnexin has four cysteine residues (161, 195, 361, and 367 for canine calnexin), which are conserved in calnexins from all species (Fig. 7). To investigate the redox state of secreted purified calnexin, we compared the mobility of the protein on SDS-PAGE under reducing and nonreducing conditions (Fig. 8). Usually monomeric proteins with intramolecular disulfide bonds migrate faster on nonreducing gels than on reducing gels. Under nonreducing conditions, the mobility of both calnexinTMC and native calnexin in isolated ER membranes revealed an increased mobility (Fig. 8, compare lanes 1 and 2 and compare lanes 3 and 4), indicating that calnexin contains at least one internal disulfide bond. As a positive control, we used albumin, which contains 17 disulfide bonds (Simon and Dugaiczyk, 1981), and detected a larger increase in mobility under nonreducing conditions (Fig. 8, lanes 5 and 6). Because calnexin can only contain at the most two disulfide bonds, the mobility shift found was correspondingly less. Hence, at least one and perhaps both potential disulfide bonds in calnexin are formed intramolecularly.


Figure 7: Alignment of four cysteine residues among calnexins from various sources. Four conserved cysteines (indicated by arrows) and their surrounding sequences are shown. Identical residues are boxed. On the right side are the amino acid residue numbers from the NH termini.




Figure 8: Disulfide bonding of calnexinTMC. Purified calnexinTMC (lanes 1 and 2), canine pancreatic ER membranes (lanes 3 and 4), or bovine serum albumin (BSA, lanes 5 and 6) were separated on SDS-PAGE under reducing (lanes 1, 3, and 5) or nonreducing (lanes 2, 4, and 6) conditions followed by immunoblotting with anti-CN1 (lanes 1-4) or staining with Coomassie Blue (C. B.) (lanes 5 and 6). Small differences in mobility were observed for disulfide bonded (ox) and reduced (red) forms of calnexinTMC (CN) or native calnexin (CN).



Calcium- and ATP-dependent Conformational Change of CalnexinTMC

Purified calnexinTMC was analyzed on native gels to investigate its state of oligomerization (Fig. 9, A and B). In the presence of calcium, calnexinTMC was a monomer (Fig. 9, A and B, lanes 2). However, after incubation with EGTA, dimeric and oligomeric calnexinTMC were observed (Fig. 9, A and B, lanes 3). These oligomers were also visualized by immunoblotting (Fig. 9B, lane 3) with anti-CN1, which reacted more strongly with oligomeric calnexin than with monomeric calnexin. Remarkably, ATP also induced the formation of calnexin oligomers, suggesting the direct interaction of the nucleotide with calnexinTMC (Fig. 9, A and B, lanes 4). The stronger binding of anti-CN1 antibody to ATP-induced oligomers of calnexin is also consistent with a conformational change exposing the epitope. When native calnexin was analyzed on nondenaturing gels, the protein migrated as large aggregates on the top part of the gels (data not shown).


Figure 9: Calcium- and ATP-dependent conformational change of calnexinTMC. Purified calnexinTMC (lanes 1) was treated with 1 mM CaCl (lanes 2), 10 mM EGTA (lanes 3), or 1 mM Mg ATP (lanes 4) at 25 °C for 10 min. The protein was analyzed on 5-15% nondenaturing polyacrylamide gels followed by Coomassie Blue (C. B.) staining (A) or immunoblotting with anti-CN1 (B). The oligomeric states of calnexinTMC are indicated on the right. M, monomer; D, dimer; T, tetramer. The preferential reaction of anti-CN1 with oligomeric calnexin is revealed in B. C, purified calnexinTMC was incubated at 30 °C for 30 min with 20 µg/ml (lanes 2, 4, 6, 8, and 10) or 100 µg/ml (lanes 3, 5, 7, 9, and 11) of proteinase K (PK) in the presence of 5 mM CaCl (lanes 2, 3, 8, and 9), 10 mM EGTA (lanes 4, 5, 10, and 11), or 1 mM ATP (10 mM MgCl) (lanes 6-11). After 1 mM PMSF was added to terminate the reactions, the proteins were separated on 10% SDS-PAGE followed by Coomassie Blue staining. Lane 1 is purified calnexinTMC without protease digestion.



Because both EGTA and ATP induced oligomerization of calnexinTMC, we tested the protease sensitivity of calnexinTMC in the presence of ATP. When purified calnexinTMC was treated first with Mg-ATP and then with proteinase K (Fig. 9C, lanes 6 and 7), calnexinTMC was completely digested. Addition of excess calcium to the ATP-treated calnexinTMC partially restored proteinase K resistance (Fig. 9C, lanes 8 and 9), but, compared with calcium alone (Fig. 9C, lanes 2 and 3), calnexinTMC was much more sensitive to proteinase K digestion. Because 10 mM MgCl was present in the reaction mixtures, the possibility that ATP effect is as a chelator can be excluded. Hence the Mg-ATP effect on oligomerization can occur in the presence of calcium.

ATP Binding

Several molecular chaperones bind ATP (Gething and Sambrook, 1992), and their function is regulated by ATP (Munro and Pelham, 1986; Flynn et al., 1989). In vivo in Madin-Darby canine kidney cells, ATP is required to maintain the interaction of the newly synthesized soluble glycoprotein gp80 with calnexin (Wada et al., 1994). We established that purified calnexinTMC can bind ATP. Purified calnexinTMC was incubated with [-P]ATP in the absence or presence of magnesium or calcium, and the samples were analyzed by SDS-PAGE followed by radioautography. Binding of ATP was Mg-dependent and required UV-enhanced cross-linking (Fig. 10B, lane 4), suggesting that ATP does not form a covalent interaction with the protein and that UV radiation promoted the cross-linking of ATP and the protein. The nucleotides, ATP, ADP, and AMP were all able to compete for binding (Fig. 10C). ATPS was the most potent competitor for ATP binding. GTP also showed strong inhibition of ATP binding, which has also been observed for other chaperone proteins (Csermely and Kahn, 1991). The reducing reagent, dithiothreitol, strongly inhibited ATP binding, whereas sulfydryl reagents, such as iodoacetamide and N-ethylmaleimide, had little effect on the binding activity, suggesting that the oxidized disulfide bonds in calnexin are required for ATP binding. Using a variety of conditions, which included the presence of denatured proteins, we were unable to detect any ATPase activity in purified calnexinTMC.


DISCUSSION

We have shown that calnexin is a type I membrane protein as predicted from its sequence, that the luminal domain can bind in vivo to a secretory glycoprotein, and that the luminal domain and full-length calnexin share several properties. When calnexin was originally identified, it was apparent that it was related by sequence to the major Ca-binding protein of the ER lumen, calreticulin (Wada et al., 1991). The generation of soluble calnexinTMC further extends the similarity to calreticulin (32% identical); we suggest that this is because of the high degree of sequence conservation between calnexinTMC and calreticulin that our observations on calnexinTMC are also directly applicable to calreticulin.

There is evidence from in vivo experiments that calnexin can transiently interact with newly synthesized secretory glycoproteins and membrane proteins during their maturation in the ER. Although there are indications that membrane proteins such as major histocompatibility complex class I can interact with residues within or near the transmembrane domain or even perhaps with the cytosolically directed COOH-terminal domain (Margolese et al., 1993; Capps and Ziga, 1994), it is clear that secretory glycoproteins can only interact with the luminal domain. The orientation of the protein was experimentally determined by peptide-specific antibodies and protease digestion of rough microsomes, confirming that the NH-terminal domain is in the ER lumen and would interact with substrates. Thus it is probable that this is the region that is responsible for the recognition and molecular chaperone activity for all substrate proteins. We were able to demonstrate by coexpressing the two genes in Sf9 insect cells that, in vivo, this region does interact with the secretory glycoprotein HIV-1 gp120. This was tested by expressing soluble truncated calnexin deleted for the transmembrane and cytosolic domains, calnexinTMC, and assessing its interaction with the soluble glycoprotein HIV-1 gp120, a known substrate for full-length calnexin. By the criterion of coimmunoprecipitation, newly synthesized gp120 was found to be associated with soluble calnexin. The choice of the Sf9 cell to assess expression of both proteins was especially appropriate because our anti-CN1 antibody did not recognize insect cell calnexin. Hence, the coimmunoprecipitation studies indicated a direct interaction in vivo between calnexinTMC and HIV-1 gp120. Although models exist for the interaction of calnexin with its substrates via recognition features in its transmembrane or cytosolic domains (Margolese et al., 1993; Capps and Ziga, 1994), our in vivo data with calnexinTMC and HIV-1 gp120 indicate that substrate recognition is luminal as predicted from the studies of Ou et al.(1993), Wada et al.(1994), and Le et al.(1994). Our studies define the luminal domain as the region responsible for the molecular chaperone activity of calnexin.

The secretion from insect cells of calnexinTMC and its subsequent ease of purification enabled us to examine that this form of calnexin, which functions as a molecular chaperone, shares other properties with the full-length calnexin. Soluble calnexinTMC, when purified from the extracellular medium, was found to be monomeric but to contain at least one intramolecular disulfide bond. All members of the calnexin family from humans to plants reveal sequence conservation about the 4 invariant cysteine residues (Fig. 7). Based on the different mobilities of calnexin in reducing and nonreducing gels, we conclude that at least one potential disulfide bond is formed internally.

In the presence of Ca, both native calnexin and calnexinTMC formed a core structure resistant to proteases. Calcium was required to maintain the core structure because it was destroyed by EGTA. All substrates evaluated by Ou et al.(1993), Wada et al.(1994), and Le et al.(1994) required Ca for their association with calnexin. Soluble calnexinTMC bound Ca, and Ca addition led to the conversion of oligomeric calnexinTMC to monomeric calnexinTMC. Remarkably, Mg ATP led to the conversion of monomeric calnexinTMC to oligomeric calnexinTMC. Oligomerization was accompanied by a change in the conformation of individual calnexin molecules as observed by an increased sensitivity to proteinases. The opposite effects of Mg-ATP and Ca in regulating calnexin oligomerization is reminiscent of the inverse effects of these same agents in promoting the association (Ca) or dissociation (ATP) of incompletely folded substrate proteins with BiP (Suzuki et al., 1991; Gaut and Hendershot, 1993). Wada et al.(1994) demonstrated that ATP is required to maintain the association of the substrate gp80 with calnexin in vivo in Madin-Darby canine kidney cells. This observation may now be explained by our demonstration of ATP binding to calnexin and the ATP-dependent conformational change in calnexin. The direct binding of ATP to calnexin in vitro was specific because adenosine nucleotides (e.g. ATP, ADP, AMP, and the ATP analogue ATPS) were able to compete for binding, binding was Mg-dependent, and binding was inhibited by dithiothreitol. However, sulfydryl reagents that reacted with free sulfydryl groups did not affect the ATP binding activity. Hence, we conclude that it is the disulfide bond(s) that act to maintain a specific protein conformation in calnexin competent for binding ATP, and it is not free sulfydryl groups that are involved in ATP binding.

The detail in conformational changes of calnexin as related to its substrate binding remains to be studied. However, it is clear that these conformational changes induced by Ca and ATP are relevant to its chaperone function in vivo. The luminal content of Ca in the ER has been estimated to be at concentration of approximately 5 mM (Somlyo et al., 1985), and the translocation of ATP into the ER and its binding to ER proteins have also been documented (Clairmont et al., 1992). The requirement of both calcium and ATP for the binding of substrate proteins to calnexin suggests that the binding of substrate proteins may accompany conformational changes in calnexin. Another molecular chaperone of the ER, BiP also binds Mg-ATP. Mg-ATP and Ca are also regulatory to BiP function and effect conformational changes in BiP (Suzuki et al., 1991; Blond-Elguindi et al., 1993; Brot et al., 1994). BiP is a potent ATPase during the step of substrate release from the chaperone (Suzuki et al., 1991). Although we have been unable to detect ATPase activity thus far of the purified calnexinTMC, this is likely regulated by substrate binding to calnexin and/or an associated protein. Direct binding of purified calnexinTMC in vitro with peptides or denatured proteins has only been demonstrated when they are glucosylated with UDP-Glc:glycoprotein glucosyltransferase on ManN-linked glycosylation (Parodi et al., 1984). However, ATPase activity can be detected in immunoprecipitates of full-length calnexin with associated secretory glycoproteins.()Hence, the possibility still exists that the presence of a substrate-calnexin interaction is required for ATPase activity or perhaps, more probably, that a calnexin-associated protein may be required for ATPase activity. Good candidates for these protein are the ER membrane proteins that we originally found associated with calnexin (Wada et al., 1991).

Current models for the actions of several chaperones including the GroEL family and BiP involve a cyclical association and dissociation of protein-folding intermediates with the chaperones correlated with changes in the chaperone quaternary structures regulated by ATP binding and hydrolysis (Freiden et al., 1992; Blond-Elguindi et al. 1993; Todd et al., 1994; Weissman et al. 1994). One model for chaperone function of calnexin (Hammond et al., 1994) proposes that glycoprotein-folding intermediates associated with calnexin undergo a cyclical reglucosylation as the mechanism of cyclical substrate presentation to calnexin. Calnexin and BiP have recently been proposed to act sequentially and cooperate in the folding of newly synthesized glycoproteins in vivo (Hammond and Helenius, 1994; Kim and Arvan, 1995). ATP binding and hydrolysis are likely to regulate the cooperation between calnexin/calreticulin and BiP in protein folding in the ER lumen in vivo.


FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X53616[GenBank Link].

§
Supported by the Medical Research Council of Canada and Glaxo Canada Inc.

Supported by the National Health Research Development Program/Medical Research Council Joint Program on AIDS.

**
To whom correspondence should be addressed: Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6155; Fax: 514-496-6213.

The abbreviations used are: ER, endoplasmic reticulum; PMSF, phenylmethylsulfonyl fluoride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HIV-1, human immunodeficiency virus type I; ATPS, adenosine 5`-O-(3-thiotriphosphate); BiP, binding protein.

W.-J. Ou, J. J. M. Bergeron, A. Parodi, and D. Y. Thomas, submitted for publication.

Y. Li, J. J. M. Bergeron, W.-J. Ou, C. Y. Kang, and D. Y. Thomas, submitted for publication.

W.-J. Ou, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Ikuo Wada (Sapporo, Japan) and Pamela Cameron (McGill) for participating in the experiment of Fig. 1. We also thank Dr. Alexander Bell (McGill) for constructive criticism of the manuscript and France Dumas for protein sequencing. We thank Roseanne Tom and Dr. Amine Kamen for the insect cell culture service at the Biotechnology Research Institute, Daniel Tessier for establishing the time course of baculovirus infection, and Daniel Dignard for confirming the sequence of calnexinTMC in the recombinant baculovirus. Human anti-HIV-1 immunoglobulin was obtained from Dr. Alfred Prince through the AIDS Research and Reagent Program, NIAID, NIH.


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