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J. Biol. Chem., Vol. 275, Issue 35, 27177-27185, September 1, 2000

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The Conformation of Calreticulin Is Influenced by the Endoplasmic Reticulum Luminal Environment^*

Elaine F. Corbett^§, Karolina M. Michalak, Kim Oikawa^¶, Steve Johnson, Iain D. Campbell, Paul Eggleton, Cyril Kay^¶, and Marek Michalak^**

From the  Canadian Institutes of Health Research Group in Molecular Biology of Membranes, the ^¶ Canadian Institutes of Health Research Group in Protein Structure and Function, Protein Engineering Network of Centers of Excellence and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and  Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, OX1 3QU Oxford, United Kingdom

Received for publication, March 13, 2000, and in revised form, May 12, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca²⁺, Zn²⁺, and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca²⁺, Zn²⁺, and ATP. At low Ca²⁺ concentration (<100 µM), calreticulin is rapidly and fully degraded by trypsin, indicating that under these conditions the protein is in a highly trypsin-susceptible conformation. Increasing Ca²⁺ concentration up to 500 µM or 1 mM resulted in protection of the full-length calreticulin and in generation of the 27-kDa fragment highly resistant to trypsin digestion. The 27-kDa protease-resistant core of the protein represented the NH₂-terminal half of calreticulin and was identified by its reactivity with specific antibodies and by NH₂-terminal amino acid sequence analysis. Ca²⁺-dependent changes in calreticulin's sensitivity to proteolysis indicate that agonist-induced fluctuation in the free ER luminal Ca²⁺ concentration may affect the protein conformation and function. Trypsin digestion of calreticulin in the presence of Zn²⁺ resulted in the formation of a 17-kDa central protease-resistant core in the protein corresponding to the central region of the protein, indicating that under these conditions the N- and C-domains of the protein are in an extended conformation. Here we also show that calreticulin is an ATP-binding protein but that it does not contain detectable ATPase activity. Digestion of the protein with trypsin in the presence of Mg²⁺-ATP protects the full-length protein. These results indicate that calreticulin may undergo frequent, ion-induced conformation changes, which may affect its function and its ability to interact with other proteins in the lumen of the ER.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The endoplasmic reticulum (ER)¹ plays an essential role in a variety of cellular processes, including the synthesis, post-translational modification, and folding of membrane-associated and secreted proteins (1-6). The lumen of the ER is a dynamic environment perfectly designed for the task of protein folding. It contains a high concentration of Ca²⁺ binding chaperones and an optimal concentration of ions and nucleotides for protein folding, modification, and assembly.

Several studies have indicated that changes in the concentration of Ca²⁺ in the ER lumen ([Ca²⁺]_ER) affect many ER functions, including the synthesis, folding, post-translational modification, and secretion of proteins; protein conformation; and chaperone-chaperone or chaperone-substrate interactions (7-15). The ER lumen also contains ATP, which is required to support the correct folding and formation of disulfide bonds in many proteins (16-18). An ATP transporter has been identified in the ER (19, 20), and the ER proteins PDI and BiP are known to bind/utilize ATP in their chaperone action (21-25).

Calreticulin is one of the major Ca²⁺-binding proteins in the lumen of the ER. The protein can be divided into three main structural regions (26). The highly conserved N-domain binds Zn²⁺ with high capacity (27), whereas the proline-rich P-domain binds Ca²⁺ with high affinity (28). The C-domain, which may play a role in the Ca²⁺ storage function of calreticulin, binds Ca²⁺ with high capacity (28). In the lumen of the ER, the protein functions as a chaperone and a modulator of Ca²⁺ homeostasis (26). Calreticulin binds monoglucosylated oligosaccharides (4) and misfolded proteins (29, 30). Importantly, calreticulin responds to fluctuating Ca²⁺ levels in the lumen of the ER by modulating its ability to interact with other ER proteins and substrates (15). We have suggested that the binding of Ca²⁺ to the C-domain of calreticulin may allow it to function as a "Ca²⁺ sensor" for other ER chaperones (15). However, the precise effects of ER constituents such as Ca²⁺, Zn²⁺, nucleotides, and other proteins on the structure and function of calreticulin remain to be determined.

In this study, we have investigated the effect of changes in the ER luminal environment (specifically Ca²⁺, Zn²⁺, and ATP) on conformational changes in calreticulin. We show that Ca²⁺, Zn²⁺, and ATP affect the tertiary structure of calreticulin, as indicated by changes in its sensitivity to protease digestion. Furthermore, we show directly that calreticulin is an ATP-binding protein but does not contain detectable ATPase activity. Our results indicate that dynamic changes within the lumen of the ER can have profound effects on calreticulin's structure, stability, and function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- ATP-agarose, CL-2B-agarose, bovine IgG, Mg²⁺-ATP, ADP, AMP, GTP, GDP, trypsin, V8 protease, cathepsin G, and elastase were from Sigma. The Pichia expression system was from Invitrogen. SDS-PAGE reagents and molecular weight markers were from Bio-Rad. Peroxidase-conjugated rabbit anti-goat and goat anti-rabbit IgGs were from Jackson ImmunoResearch Laboratories. The ECL detection kit for Western blotting was from Amersham Pharmacia Biotech. All chemicals were of the highest grade available.

Expression and Purification of Recombinant Calreticulin in Pichia Protein Expression System-- The cDNA of full-length rabbit calreticulin was cloned into pPIC-9 and transformed into the Pichia KM71 strain as recommended by the manufacturer. The expression of calreticulin was induced with 0.5% methanol. The protein was purified by DEAE-Sepharose and Resource Q FPLC column chromatography.² The NH₂-terminal amino acid sequence of full-length calreticulin expressed in Pichia was NH₂-EAYVEF¹EPVVYFL-, where the underlined residues correspond to the amino acid sequence of mature calreticulin. Biochemical and biophysical analysis of the recombinant calreticulin using mass spectroscopy, CD analysis, disulfide mapping, Ca²⁺ binding measurements, and antibody reactivity revealed that it was identical to the native, tissue-purified protein.²

Proteolytic Digestions-- Purified, recombinant calreticulin expressed in Pichia was incubated with trypsin at 1:100 (trypsin/protein; w/w) at 37 °C and in the presence or absence of varying concentrations of Ca²⁺, Zn²⁺, and Mg-ATP (as indicated in the figure legends). Aliquots were taken at the indicated time points, and the reaction was stopped by the addition of the Laemmli sample buffer (31). Calreticulin (15 µg) was also digested in the absence (5 mM EDTA) or in the presence of Ca²⁺ (2 mM Ca²⁺) with elastase, V8 protease, and cathepsin G at a calreticulin/enzyme ratio of 20:1 or 320:1 (w/w) for 2 h at 37 °C at pH 6.4 (elastase) or pH 7.4 (all others). The proteins were separated by 10 or 15% SDS-PAGE (31) and either stained with Coomassie Blue or transferred onto a nitrocellulose membrane (32) and probed with the indicated anti-calreticulin antibodies.

Antibodies-- Goat anti-calreticulin antibodies were extensively characterized (33). This antibody recognizes the C-domain but not the N-domain of calreticulin. Antibodies against the N-terminal synthetic peptide of calreticulin were a generous gift of Dr. K.-H. Krause (University of Geneva). This antibody recognizes the first 12 amino acids of mature calreticulin and has also been extensively characterized (34-36). Antibody binding was detected with appropriate peroxidase-conjugated secondary antibodies diluted 1:10,000, followed by an ECL reaction and exposure to x-ray film. Images of the Coomassie Blue-stained gels and x-ray films were scanned and processed for publication using Adobe Photoshop version 5.5 software.

ATP-Agarose Chromatography-- ATP affinity chromatography was carried out using ATP-agarose and CL-2B-agarose as the control. Calreticulin (5 µg) was incubated at 37 °C for 60 min. with ATP-agarose or CL-2B agarose beads (0.7 g/ml) in a binding buffer containing 10 mM Mops, pH 7.0, 100 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, and 2 mg of bovine IgG (to block nonspecific binding). Beads were then placed in a glass column followed by extensive washing with a binding buffer to remove unbound proteins. ATP bound proteins were eluted with a binding buffer containing 5 mM Mg²⁺-ATP. Proteins associated nonspecifically with the ATP-agarose were subsequently eluted with 2 M NaCl. Eluted proteins were precipitated with acetone at 20 °C for 16 h, separated on 15% SDS-PAGE, and transferred onto a nitrocellulose membrane. The membranes were probed with anti-calreticulin antibodies as described above.

Circular Dichroism and Equilibrium Sedimentation Measurements-- CD analysis was performed at 25 °C using a Jasco J-720 spectropolarimeter (Jasco Inc., Easton, MD), interfaced to an Epson Equity 386/25 computer and controlled by Jasco software as described previously (15). The instrument was calibrated with ammonium d-(+)-10 camphor sulfonate at 290.5 and 192 nm. Each sample was scanned 10 times, and noise reduction was applied to remove the high frequency before calculating molar ellipticities. The cell path length used was 0.02 cm, and the protein concentration was 0.6 mg/ml in the far ultraviolet. Molar ellipticities were calculated from the equation,

(Eq. 1)

where _obs is in millidegrees, l represents the path length in cm, and c represents the concentration in mol/liter × number of amino acids in the sequence. The unit for molar ellipticity is degree cm²/dmol. The CD spectra were analyzed for secondary structure elements by the Contin ridge regression analysis program of Provencher and Glöckner (37).

Sedimentation Velocity Measurements-- Sedimentation velocity experiments were carried out at 20 °C and 50,000 rpm using the XLI Analytical Ultracentrifuge and Interference optic following the procedures outlined in the instruction manual (Spinco Business Center of Beckman Instruments, Inc. Palo Alto, CA). 400 µl of sample solution and 400 µl of dialysate were loaded into two sector CFE centerpiece sample cells containing sapphire windows. Runs were performed for 4 h, during which a minimum of 30 scans were taken. The sedimentation velocity data were analyzed using the Second Moment Method contained in the Beckman Analysis Program (Optima XL-A/XL-I Data Analysis Software, version 4.0; Spinco Business Center of Beckman Instruments, Inc.) to determine the sedimentation coefficient from the following relationship,

(Eq. 2)

where S represents the observed sedimentation coefficient;  represents angular velocity; r represents radial position of the solvent/protein boundary; and t represents the time in seconds. By measuring the movement of the boundary over time and plotting ln r versus ²t, a straight line should be obtained with the slope = S.

The program Sednterp (Sedimentation Interpretation Program, version 1.01, created by David B. Haynes (Magdelan College), Tom Laue (University of New Hampshire), and John Philo (Amgen)) was then used to calculate S_20,w according to the following relationship,

(Eq. 3)

where S_20,w represents the sedimentation coefficient corrected to water at 20 °C; s represents the observed sedimentation coefficient; _t represents the viscosity of water at run temperature; ₂₀ represents the viscosity of water at 20 °C; _sol represents the viscosity of sample solution at known temperature t'; _w represents the viscosity of water at t' degrees; _20,w represents the density of water at 20 °C; _t, sol represents the density of sample solution at run temperature; and  represents the partial volume of the solute.

Miscellaneous Methods-- ATP hydrolysis was carried out using a coupled enzyme system (38). Protein concentration was determined by the procedure of Bradford (39). NH₂-terminal sequence analysis of native and recombinant proteins was carried out on samples, which were electroblotted to Immobilon (polyvinylidene difluoride) membranes (40). Free Ca²⁺ concentrations were calculated using Max Chelator, Winmaxc version 1.70.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺-dependent Conformation Changes in Calreticulin-- In response to agonist stimulation of cells, the [Ca²⁺]_ER undergoes dynamic changes (41). In a steady-state situation, the [Ca²⁺]_ER is in excess of 400 µM. After agonist-evoked Ca²⁺ release from the ER, the [Ca²⁺]_ER is reduced to <50 µM (42). Therefore, upon agonist stimulation, Ca²⁺-binding proteins in the lumen of the ER, including calreticulin, experience frequent changes in Ca²⁺ concentration. To evaluate the importance of [Ca²⁺]_ER in calreticulin's function, we investigated the effect of Ca²⁺ on calreticulin's sensitivity to protease digestion. Large quantities of calreticulin were expressed in Pichia and purified.² The purified protein was digested with trypsin (1:100, trypsin/calreticulin) over a range of physiologically relevant Ca²⁺ concentrations, from low (0-100 µM, representing empty Ca²⁺ stores) to high (500-1000 µM, representing full Ca²⁺ stores) (41). Fig. 1 shows that in the absence of Ca²⁺ (A) or in the presence of 100 µM Ca²⁺ (B) calreticulin was almost completely degraded by trypsin within 2 min of incubation. This indicates that at low concentrations of Ca²⁺ calreticulin was highly susceptible to digestion by trypsin. In contrast, at Ca²⁺ concentrations of 500 µM (C) or 1 mM (D) calreticulin exhibited resistance to trypsin digestion, and when digestion occurred it resulted in the generation of a 27-kDa protein, which was highly resistant to further trypsin digestion (Fig. 1, C and D). At high Ca²⁺ concentration, the proteolytic activity of trypsin is highly increased (43, 44), yet the 27-kDa fragment was extremely resistant to trypsin digestion. Indeed, in the presence of 1 mM Ca²⁺, the 27-kDa fragment was resistant to trypsin digestion for a period greater than 6 h at 37 °C (not shown). Similar results were obtained when calreticulin was digested with elastase, V8 protease, and cathepsin G (Fig. 2).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Trypsin digestion of calreticulin. Purified calreticulin was incubated with trypsin at 1:100 (trypsin/protein; w/w) at 37 °C and in the presence or absence of a varying concentration of Ca2+ as indicated. Aliquots were taken at the time points indicated, and the proteins were separated by SDS-PAGE and stained with Coomassie Blue as described under "Experimental Procedures." The arrow indicates the 27-kDa protease-resistant fragment of calreticulin. The positions of Bio-Rad molecular weight marker proteins are indicated.

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Protease digestion of calreticulin. Purified calreticulin (lane 1) was digested with V8 protease (A), elastase (B), and cathepsin G (C) as described under "Experimental Procedures." Digestion was carried out in the presence of 5 mM EDTA or in the presence of 2 mM Ca2+. Lane 2, digestion in the presence of 2 mM Ca2+ at a calreticulin/enzyme ratio of 320:1 (w/w); lane 3, digestion in the presence of 5 mM EDTA at a calreticulin/enzyme ratio of 320:1 (w/w); lane 4, digestion in the presence of 2 mM Ca2+ at a calreticulin/enzyme ratio of 20:1 (w/w); lane 5, digestion in the presence of 5 mM EDTA at a calreticulin/enzyme ratio of 20:1 (w/w). The arrow indicates the 27-kDa protease-resistant fragment of calreticulin. The positions of the molecular weight marker proteins are indicated.

To identify the Ca²⁺-sensitive, trypsin-resistant 27-kDa proteolytic fragment of calreticulin, we carried out immunological analysis and NH₂-terminal amino acid sequence analysis. Calreticulin was digested with trypsin in the presence of 1 mM Ca²⁺ followed by Western blot analysis. First, we used a rabbit anti-synthetic peptide antibody, which recognizes the first 12 amino acids of mature calreticulin (34, 35). Fig. 3A shows that these antibodies reacted with full-length calreticulin, as expected, and with the 27-kDa tryptic fragment of the protein. This indicates that proteolysis proceeded from the C-terminal region of the protein, leaving the amino-terminal region intact. Next, we used a goat anti-calreticulin antibody, which recognizes the C-domain of calreticulin (28). As expected, this antibody recognized the full-length protein (Fig. 3B). However, the 27-kDa fragment was not recognized by this antibody (Fig. 3B), indicating that it did not include the C-domain of the protein. A short, C-domain fragment of calreticulin (~17-kDa) was detected by the goat antibody during the first 5 min of tryptic digestion, but this fragment was completely digested after 10 min of incubation with trypsin (Fig. 3B). The identity of the trypsin-resistant 27-kDa fragment was further confirmed by NH₂-terminal amino acid sequence analysis. The amino acid sequence we obtained (NH₂-EAYVEF¹EPVVYFKEQF-) corresponded to the NH₂-terminal amino acid sequence of full-length recombinant calreticulin expressed in Pichia. Identical NH₃-terminal amino acid sequence (NH₂-EAYVEF¹EPVVYFKEQF-) was also obtained for a 27-kDa fragment generated with elastase, V8 protease, or cathepsin (Fig. 2), indicating that it was identical to the 27-kDa fragment generated with trypsin.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Immunological identification of protease-resistant fragment of calreticulin. Purified calreticulin was digested with trypsin (1:100; trypsin/protein; w/w) in the presence of 1 mM Ca2+ for the time indicated. Proteins were separated by SDS-PAGE, transferred to nitrocelluose membrane, and probed with antibodies against the N terminus of calreticulin (A) or the C terminus of the protein (B). The arrow indicates the 27-kDa protease-resistant fragment. The positions of Bio-Rad molecular weight marker proteins are indicated.

These results indicate that calreticulin's susceptibility to tryptic digestion, particularly in the NH₂-terminal region, is altered by the binding of Ca²⁺. This probably results from Ca²⁺-dependent conformational changes in the protein. It appears that at Ca²⁺ concentrations approximating those in full ER Ca²⁺ stores, the N-domain and P-domain region of calreticulin form a 27-kDa, protease-resistant core (Fig. 3). Under these conditions, the C-domain of the protein is in an extended conformation, hence accessible to digestion with the protease.

Zn²⁺-dependent Conformational Changes in Calreticulin-- Zn²⁺ plays an important regulatory role in intracellular signal transduction, and it is an important structural and functional element of many proteins including calreticulin (45). Zn²⁺ binds to the N-domain of calreticulin, affecting its interaction with other proteins and inducing conformational changes, as determined by CD spectra analysis (15, 46). Therefore, we examined the effect of Zn²⁺ on calreticulin's protease resistance. Calreticulin was digested in the presence of 200 µM Zn²⁺, a concentration that also produces the largest conformational change in the protein (15, 46).

Fig. 4 shows that during the first 10 min of trypsin digestion, in the presence of 200 µM Zn²⁺, two major proteolytic fragments were generated. One was a 48-kDa fragment that reacted with anti-N-terminal calreticulin antibodies, and the other was a 19-kDa fragment that did not react (data not shown). During prolonged digestion, the 48-kDa fragment disappeared, and the 19-kDa fragment was digested further, with the removal of an additional 40-50 NH₂-terminal residues, producing a 17-kDa trypsin-resistant fragment (Fig. 4, 90-180 min). Neither the 19-kDa nor the 17-kDa fragment reacted with antibodies against the N- and C-terminal regions of calreticulin, suggesting that they represented an internal region of the protein. Amino acid sequence analysis of each fragment revealed two NH₂-terminal sequences, indicating that they are actually doublets. The N-terminal amino acid sequences of the 19-kDa fragments were NH₂-⁷¹GQPL- and NH₂-⁷⁸TVKHEQNI-. The N-terminal amino acid sequences of the 17-kDa fragments were NH₂-¹¹⁸DSXYXIM- and NH₂-¹⁵⁸IVRP-. Zn²⁺ has been reported to have either no effect on trypsin activity (47) or to increase its proteolytic activity (43, 44), yet a 17-kDa fragment was relatively resistant to trypsin digestion in the presence of 200 µM Zn²⁺.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Zn2+-dependent proteolysis of calreticulin. Purified calreticulin was incubated with trypsin (1:100; trypsin/protein; w/w) at 37 °C in the presence of 200 µM Zn2+. Digestion was stopped at time points indicated, and the proteins were separated by SDS-PAGE followed by staining with Coomassie Blue as described under "Experimental Procedures." The arrows indicate the positions of 48-, 19-, and 17-kDa protease-resistant fragments of calreticulin. The positions of Bio-Rad molecular weight marker proteins are indicated.

Previous studies have shown that Zn²⁺ binding to calreticulin induces conformational changes in the protein (15, 46). Here we show that these changes lead to the formation of a central, protease-resistant core, which encompasses most of the P-domain of calreticulin. At the same time, the N terminus and C terminus of calreticulin may be in an extended conformation, which would explain their accessibility to digestion by the protease (Fig. 4). Trypsin digestion of calreticulin in the presence of both 1 mM Ca²⁺ and 100 µM Zn²⁺ resulted in the generation of proteolytic fragments identical to those observed in the presence of 200 µM Zn²⁺ alone (data not shown).

ATP-dependent Conformational Changes in Calreticulin-- Protein folding in the lumen of the ER requires ATP (16-18). In order to investigate possible roles for ATP in the function of calreticulin, we carried out limited proteolytic digestion of calreticulin in the presence and absence of Mg²⁺-ATP. Mg²⁺ and ATP have no effect on trypsin proteolytic activity (44, 48). Fig. 5 shows that in the presence of 1 mM Mg²⁺-ATP full-length calreticulin was quite resistant to trypsin digestion and was protected for the first 45 min of incubation. The identity of the "protected" full-length calreticulin was confirmed by NH₂-terminal amino acid sequence analysis (data not shown). Mg²⁺ alone had no protective effect (data not shown). ADP, AMP, GTP, and GDP also did not have any protective effect (data not shown), indicating that they did not bind to calreticulin. After 5 min of incubation with trypsin, in addition to the full-length protein, a 30-kDa major proteolytic fragment was observed (Fig. 5B). The 30-kDa fragment was rapidly digested to small peptides. It appears that calreticulin binds Mg²⁺-ATP and that this significantly reduces its susceptibility to proteolytic digestion, probably via a conformational change. A striking difference in the protective effects of Ca²⁺, Zn²⁺, and ATP against tryptic digestion of calreticulin is that only ATP protected the C-domain of the protein.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   ATP-dependent proteolysis of calreticulin. Purified calreticulin was incubated with trypsin (1:100; trypsin/protein; w/w) at 37 °C in the presence or absence of 1 mM Mg2+-ATP. Digestion was stopped at the time points indicated, and the proteins were separated by SDS-PAGE and stained with Coomassie Blue as described under "Experimental Procedures." The arrow indicates the position of full-length calreticulin (60-kDa). The positions of Bio-Rad molecular weight marker proteins are indicated.

Next, to mimic a more physiological environment, we investigated the combined effect of Ca²⁺, Zn²⁺, and Mg²⁺-ATP on calreticulin's sensitivity to trypsin. Fig. 6 shows the effect of ATP on the conformation of calreticulin, in the presence of 200 µM Zn²⁺ (A) or 1 mM Ca²⁺ (B). In the presence of Zn²⁺ and Mg²⁺-ATP, the digestion of calreticulin by trypsin produced a 55-kDa trypsin-resistant fragment (Fig. 6A). Under these conditions, the full-length calreticulin also showed significant resistance (more than 2 h) to proteolysis. The NH₂-terminal amino acid sequence of the 55-kDa fragment was NH₂-⁷⁸TVKHEQNID-, which is identical to the NH₂-terminal amino acid sequence of the 19-kDa tryptic fragment produced in the presence of Zn²⁺ alone (Fig. 4). The difference between these two fragments was that the 19-kDa fragment was missing the C-domain of calreticulin. This supports our previous observation that Mg²⁺-ATP protects the C-domain of calreticulin from tryptic digestion.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Proteolysis of calreticulin. Purified calreticulin was incubated with trypsin (1:100; trypsin/protein; w/w) at 37 °C in the presence of 1 mM Mg2+-ATP and 200 µM Zn2+ (A) or in the presence of 1 mM Mg2+-ATP and 1 mM Ca2+ (B). The proteins were separated by SDS-PAGE and stained with Coomassie Blue. The arrows indicate the positions of full-length calreticulin (60 kDa) and 58-, 55-, and 27-kDa protease-resistant fragments of the protein. The positions of Bio-Rad molecular weight marker proteins are indicated.

Fig. 6B shows that tryptic digestion of calreticulin in the presence of Mg²⁺-ATP and Ca²⁺ resulted in the generation of three major fragments, one 58, one 55, and one 27 kDa. The NH₂-terminal amino acid sequence of the 58-kDa fragment was NH₂-EAYVEF¹EPVVYFK-, and it corresponded to a full-length recombinant calreticulin. This indicated that under these conditions proteolysis of calreticulin proceeded from the C-terminal region of the protein. The NH₂-terminal amino acid sequence of the 27-kDa fragment was also NH₂-EAYVEF¹EPVVYFKEQF-. This sequence, which corresponds to the amino-terminal sequence of full-length, mature calreticulin, is identical to the NH₂-terminal amino acid sequence of the 27-kDa trypsin-resistant fragment obtained in the presence of 500 µM Ca²⁺ (Figs. 1-3). The 27-kDa tryptic fragment obtained in the presence of Mg²⁺-ATP and Ca²⁺, as expected, interacted with the anti-N-terminal antibody but not with the anti-C-terminal antibody (data not shown).

Biophysical Analysis of ATP-dependent Conformational Changes in Calreticulin-- To examine ATP-dependent conformational changes in calreticulin, far UV CD analysis of the protein was carried out in the absence and presence of Mg²⁺-ATP, Mg²⁺-ATP plus Zn²⁺, Mg²⁺-ATP plus Ca²⁺, and Mg²⁺-ATP plus Ca²⁺ and Zn²⁺. Control experiments were carried out in the absence and presence of 1 mM Ca²⁺, 1 mM Zn²⁺, and 1 mM Mg²⁺. The CD spectra were analyzed for secondary structural elements by the Contin ridge regression analysis program (37), and representative data are shown Table I. The apoprotein has ~10% -helix and 66% -sheet--turn, similar to values published earlier (15). The addition of Mg²⁺-ATP alone and of Mg²⁺-ATP with Zn²⁺ and/or Ca²⁺, resulted in small (just outwith experimental error) changes in secondary structural elements. Since CD changes are averaged over the entire molecule, they are often not a very sensitive measure of small conformational changes.

The binding of ATP to calnexin causes its ER luminal region to dimerize (49). Therefore, we used sedimentation velocity analysis to explore possible effects of ATP on the oligomerization of calreticulin. Table II indicates that S_20,w values of calreticulin, at a concentration of 3.07 mg/ml, are indistinguishable under all conditions examined. Apparently, the binding of ATP to calreticulin does not induce it to form oligomers.

Calreticulin Binds ATP-- ATP-dependent conformational changes in calreticulin, as measured by its altered sensitivity to proteolysis, suggest that the protein binds ATP. In order to demonstrate directly that calreticulin binds ATP, we carried out ATP affinity chromatography of the purified protein (Fig. 7A). CL-2B agarose chromatography of the protein was carried out as a control (Fig. 7B). Calreticulin was applied to the column. This was followed by extensive washing with the binding buffer and then elution of ATP binding proteins with 5 mM ATP. Importantly, a significant amount of calreticulin was bound to the ATP-agarose and was specifically eluted with 5 mM ATP (Fig. 7A, 5 mM ATP lane). In contrast, calreticulin did not bind to the CL-2B agarose control column (Fig. 7B). Subsequent application of 2 M NaCl eluted no further calreticulin, indicating that all calreticulin bound to the column had been eluted by the ATP (Fig. 7A, 2 M NaCl lane). These experiments demonstrate that calreticulin binds ATP.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Direct binding of ATP to calreticulin. Calreticulin was incubated at 37 °C with ATP-agarose or control agarose (CL-2B Agarose) beads in a binding buffer as described under "Experimental Procedures." ATP-bound proteins were eluted with a binding buffer containing 5 mM Mg2+-ATP (5 mM ATP lane) followed by a wash with 2 M NaCl (2 M NaCl lane). Eluted proteins were separated on SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were probed with anti-C terminus calreticulin antibodies as described under "Experimental Procedures." The positions of Bio-Rad molecular weight marker proteins are indicated. CRT, calreticulin.

Chaperones localized in the lumen of the ER are also known to hydrolyze ATP (21-25). Therefore, we tested calreticulin's ability to hydrolyze ATP, in the presence and absence of 100 µM and 1 mM Ca²⁺, using a coupled enzyme system (38). Microsomal vesicles isolated from HEK293 cells were used as a control (Table III) (38). We found that calreticulin did not hydrolyze ATP under these conditions (Table III).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we show that environmental conditions in the lumen of the ER can affect calreticulin's conformation as documented by its sensitivity to proteolytic with trypsin, elastase, V8 protease, and cathepsin G. Specifically, the binding of Ca²⁺, Zn²⁺, and Mg²⁺-ATP to calreticulin appears to induce conformational changes in the protein, which lead to the formation of a protease-resistant core structure. The protease-resistant core always encompasses the P-domain of the protein. Ca²⁺ binding protects the N-terminal (N-domain and P-domain) region of calreticulin, Zn²⁺ protects its P-domain, and Mg²⁺-ATP protects the full-length protein. These results suggest that the N- and C-domains of calreticulin are flexible and may undergo frequent, ion-induced changes in conformation. These conformational changes may explain how Ca²⁺ and Zn²⁺ may alter the function of calreticulin. In this study, we also show that calreticulin binds ATP. Despite this, the protein does not hydrolyze Mg²⁺-ATP, and ATP does not induce formation of calreticulin oligomers. We conclude that changes in the concentration of Ca²⁺, Zn²⁺, and Mg²⁺-ATP in the lumen of the ER significantly affect the conformation, stability, and function of ER luminal chaperones.

ATP is required to support correct folding and correct disulfide bond formation in many proteins, and it is an important component of the ER luminal environment (16-20). For example, ATP is required in the lumen of the ER for protein phosphorylation (50-57), protein degradation (58), and protein folding and post-translational modification (16, 17, 22-25). Many chaperones residing in the ER bind and use ATP. For example, BiP and PDI both bind and hydrolyze ATP (21-25). Grp94 also binds ATP (19), although neither ATP binding nor hydrolysis is essential for its peptide binding activity (59). Calreticulin and other ER chaperones (Grp170, PDI, BiP, ERp72, and Grp94) form Ca²⁺-dependent and ATP-sensitive complexes with misfolded proteins (60, 61). Here we provide the first direct evidence that calreticulin binds ATP, although we found no evidence that it hydrolyzes the ATP. We also show that ATP significantly reduces calreticulin's susceptibility to tryptic digestion, indicating that ATP stabilizes specific conformation of the protein. Importantly, calreticulin does not bind GTP, GDP, ADP, and AMP as determined by its sensitivity to proteolysis.

Fig. 8 shows a proposed model for conformational changes of calreticulin in the ER lumen. The most distinctive feature of ATP binding to calreticulin is that it protected the full-length protein from tryptic digestion (Fig. 8, 1). This is in contrast to Ca²⁺ and Zn²⁺. For example, Ca²⁺ binding to the low affinity and high capacity C-domain results in conformational changes in the C-terminal region of calreticulin, making it susceptible to proteolysis (Fig. 8, 2 and 3). In the presence of Zn²⁺, the N- and C-domain regions of calreticulin also appear to take an extended conformation that is susceptible to proteolysis (Fig. 8, 2). While there is no obvious nucleotide-binding site in calreticulin, the protection of the C-domain by ATP is suggestive that its binding involves the C-domain. This is further supported by the ATP-dependent protection of the C-domain in the presence of Zn²⁺ (Fig. 8, 4). On the other hand, ATP has less effect on the C-domain of calreticulin in the presence of Ca²⁺ (Fig. 8, 5), indicating that Ca²⁺ may compete with ATP for binding to the C-domain. ATP binding could play a role in protection of this region of the protein or in regulation of its activity. Furthermore, ATP causes a slight enhancement in oligosaccharide binding to the P-domain of calreticulin, whereas Ca²⁺ enhances this binding significantly (14). Ca²⁺- and ATP-dependent conformational changes of calreticulin described here may play an important role in the regulation of this chaperone function of calreticulin. Recently, Saito et al. (29) reported that ATP affects interaction between calreticulin and misfolded proteins. Via these interactions, the chaperone activity of calreticulin may be modified or controlled by changes in the concentration of Ca²⁺ and ATP in the lumen of the ER. Other recent observations support this suggestion. Similarly, ATP-induced conformational changes in BiP, another ER chaperone, are necessary for it to release bound peptide (23, 24). ATP binding to calreticulin results in dramatic changes in the structure of the protein (Fig. 8, 1), and it may play a role in the regulation of calreticulin chaperone activity by increasing its surface hydrophobicity and enhancing its ability to suppress aggregation of both glycosylated and nonglycosylated unfolded proteins.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   A hypothetical model of protease sensitivity of calreticulin. 1, effects of ATP on the proteolysis of calreticulin. In the presence of ATP, full-length calreticulin is resistant to digestion with trypsin. Ca2+ binding to the C-domain of calreticulin in the presence of ATP (5) results in conformational changes in the protein, leading to unmasking of the C-domain of the protein. 4 shows that in the presence of ATP and Zn2+, the N-domain of the protein is an extended conformation. 2, Zn2+ binding to the N-domain of calreticulin results in a conformational change, leading to unmasking of the N- and C-domains of the protein and to the formation of a protease-resistant core consisting of the P-domain and a part of the N-domain of the protein. Ca2+ binding to the C-domain of calreticulin (3) induces conformational change, resulting in proteolysis of the C-domain and formation of a protease-resistant core encompassing the N- and P-domains of the protein.

Mg²⁺-ATP binds to calnexin, induces protein oligomerization, and increases calnexin's sensitivity to proteolysis (49). In contrast, we found that ATP binding to calreticulin does not promote its oligomerization; however, it protects calreticulin from proteolysis. These findings indicate that, while ATP may play an important role in both proteins, its effects are significantly different. Importantly, calreticulin and calnexin interact with very different substrates, and the effects of ATP and Ca²⁺ may be responsible for these differences.

Calreticulin binds Zn²⁺, and Zn²⁺ is found in intracellular compartments known to contain calreticulin such as the ER, sarcoplasmic reticulum, synaptic vesicles, and secretory granules (62-65). This binding involves highly conserved histidine residues in the N-domain of the protein (27), and it induces conformational changes as measured by CD spectroscopy and fluorescence techniques (15, 27, 29). Zn²⁺ also affects interactions between calreticulin and other ER chaperones and between calreticulin and misfolded proteins (15, 27, 29). In this study, we show that Zn²⁺-dependent conformational changes in calreticulin result in the formation of a protease-resistant central core (Fig. 8, 2). The N-domain and the C-domain of the protein are unprotected and therefore may be in an extended conformation (Fig. 8, 2). A recent study reports that Zn²⁺ enhances calreticulin's ability to reduce the aggregation of newly synthesized proteins (29). The N- and C-domains of calreticulin may be involved in this, via interaction with misfolded proteins (not glycoproteins) (Fig. 8, 2). Indeed, CD spectra analysis shows that Zn²⁺ binding increases the hydrophobicity of calreticulin, which could enhance its interaction with hydrophobic regions of misfolded proteins.

The ER is one of the most important sources of intracellular Ca²⁺. Although much Ca²⁺ in the lumen of the ER is stored by binding to chaperones, a significant portion of the Ca²⁺ is free (41). In response to external stimuli, the concentration of this Ca²⁺ (free [Ca²⁺]_ER) fluctuates (41, 42). The free [Ca²⁺]_ER changes from as high as 500 µM to as low as 1 µM (42). In this study, we have shown for the first time that Ca²⁺ binding also alters calreticulin's conformation as monitored by its susceptibility to tryptic digestion. At low Ca²⁺ concentrations (>100 µM, empty Ca²⁺ stores), Ca²⁺ binds only to high affinity sites located in the P-domain of calreticulin (28). Under these conditions, calreticulin appears to exist in a conformation that is "relaxed" (exposed and highly susceptible to proteolysis). When the concentration of Ca²⁺ is increased to >500 µM (corresponding to full Ca²⁺ stores) Ca²⁺ also binds to the C-domain of calreticulin. When this happens, the protein appears to undergo a conformational change, forming a core that is protease-resistant and that encompasses the highly conserved N-domain and the lectin-like P-domain of calreticulin, while the C-domain is in an extended conformation able to bind Ca²⁺ (Fig. 8, 3). Most importantly, it is low affinity Ca²⁺ binding in the C-domain that is responsible for these changes and not high affinity binding in the P-domain. It is likely that these Ca²⁺-induced conformational changes in calreticulin are responsible for the Ca²⁺-dependent interaction of calreticulin with PDI and ERp57 (Fig. 8, 3) (15, 46). Furthermore, Ca²⁺ binding to the ER luminal region of calnexin induces conformational changes in calnexin as measured by its resistance to proteinase K digestion (49). Chaperone function of both calreticulin and the ER luminal region of calnexin may be regulated by Ca²⁺-dependent changes in their conformation. Our results also indicate that the [Ca²⁺]_ER can significantly affect the conformation and stability of calreticulin. For example, the degradation of both PDI and calreticulin by ERp72 is Ca²⁺-dependent (66). Specifically, the degradation of PDI is enhanced at 1 mM Ca²⁺, whereas the degradation of calreticulin is inhibited (66).

The formation of the Ca²⁺-dependent protease-resistant 27-kDa fragment of calreticulin may have important physiological and pathophysiological implications. For example, autoantibodies against calreticulin are present in the sera of a number of autoimmune conditions (67). The NH₂-terminal region of calreticulin, corresponding to the Ca²⁺-resistant 27-kDa fragment, contains most of the antigenic sites (68). The same protease-resistant fragment of calreticulin is produced in the VERO76 cells infected with a wild type Rubella virus.³ It is tempting to speculate that viral infection or other cellular stresses may induce release of calreticulin and expose the protein to proteolytic enzymes in the presence of high Ca²⁺ concentration. This would result in the production of high levels of protease-resistant calreticulin fragment with a highly antigenic conformation. A similar NH₂-terminal fragment of calreticulin has been isolated from an Epstein-Barr virus-immortalized cell line and shown to inhibit angiogenesis and endothelial cell proliferation (69). In this study, conformational changes in the ER chaperone calreticulin have been defined in the presence of Ca²⁺, Zn²⁺, and ATP. It will be important to determine precisely how such conformational changes regulate the physiological function of the protein intracellularly. Moreover, proteolytic fragments of calreticulin released from cells undergoing stress have been observed to interact with proteins and cells of the immune system. Knowledge of these events will also help us understand the pathological significance of calreticulin released into the extracellular environment.

	ACKNOWLEDGEMENT

The superb technical assistance of Monika Dabrowska is greatly appreciated.

	FOOTNOTES

^* This work was supported by grants from the Canadian Institutes of Health Research and from the Heart and Stroke Foundations of Alberta (to M. M.), a grant from the Protein Engineering Network of Centers of Excellence (to C. M. K.), and Arthritis Research Campaign Grant E0521 (to P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a studentship from the Alberta Heritage Foundation for Medical Research and from the Heart and Stroke Foundation of Canada.

^** Canadian Institutes of Health Research Senior Scientist and Medical Scientist of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: CIHR Group in Molecular Biology of Membranes, Dept. of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-2256; Fax: 780-492-0886; E-mail: marek.michalak@ualberta.ca.

Published, JBC Papers in Press, June 6, 2000, DOI 10.1074/jbc.M002049200

² Andrin, C., Corbett, E. F., Johnson, S., Dabrowska, M., Campbell, I. D., Eggleton, P., Opas, M., and Michalak, M. (2000) Protein Purif. Exp., in press.

³ H. Nakhasi, personal communication.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; [Ca²⁺]_ER, ER lumen Ca²⁺ concentration; PDI, protein-disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; Mops, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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