Báo cáo khoa học: Dimerization and oligomerization of the chaperone calreticulin pptx

Dimerization and oligomerization of the chaperone calreticulin Charlotte S. Jørgensen 1 , L. Rebekka Ryder 1 , Anne Steinø 1 , Peter Højrup 2 , Jesper Hansen 2 , N. Helena Beyer 3 , Niels H. H. Heegaard 3 and Gunnar Houen 1 1 Department of Research and Development, Statens Serum Institut, Copenhagen, Denmark; 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 3 Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark The chaperone calreticulin is a highly conserved eukaryotic protein mainly located in the endoplasmic reticulum. It contains a free cysteine SH group but does not form disul- fide-bridged dimers under physiological conditions, indica- ting that the SH group may not be fully accessible in the native protein. Using PAGE, urea gradient gel electro- phoresis, capillary electrophoresis and MS, we show that dimerization through the SH group can be induced by lowering the pH to 5–6, heating, or under conditions that favour partial unfolding such as urea concentrations above 2.6 M or SDS concentrations above 0.025%. Moreover, we show that calreticulin also has the ability to self-oligomerize through noncovalent interactions at urea concentrations above 2.6 M at pH below 4.6 or above pH 10, at tempera- tures above 40 °C, or in the presence of high concentrations of organic solvents (25%), conditions that favour partial unfolding or an intramolecular local conformational change that allows oligomerization, resulting in a heterogeneous mixture of oligomers consisting of up to 10 calreticulin monomers. The oligomeric calreticulin was very stable, but oligomerization was partially reversed by addition of 8 M urea or 1% SDS, and heat-induced oligomerization could be inhibited by 8 M urea or 1% SDS when present during heating. Comparison of the binding properties of mono- meric and oligomeric calreticulin in solid-phase assays showed increased binding to peptides and denatured pro- teins when calreticulin was oligomerized. Thus, calreticulin shares the ability to self-oligomerize with other important chaperones such as GRP94 and HSP90, a property possibly associated with their chaperone activity. Keywords: calreticulin; chaperone; dimerization; heat shock protein; oligomerization. Calreticulin is a highly conserved ubiquitous protein, mainly located in the endoplasmic reticulum [1,2]. It has been found to be involved in many cellular processes, including calcium storage and chaperone function, and it has been reported to possess carbohydrate and peptide binding properties, and to play a role in assembly of the MHC I loading complex [1–9]. The crystal structure of the lumenal domain of the homologous membrane-bound chaperone calnexin has revealed a protein with a compact globular N domain with homology to legume lectins, composed of two antiparallel b-sheets and a long P domain b hairpin arm stretching away from the globular domain (Fig. 1) [10,11]. A calreticulin model has been proposed based on the calnexin structure, suggesting a globular N domain consisting of a concave and a convex b-sheet, a P domain composed of two antiparallel b-strands shown by NMR to form an extended hairpin fold, and a C domain with b-sheet and a-helical structure in the first part, proposed to shield the hydrophobic regions of the convex b-sheet, and random-coil structure in the second half [7,12–15]. In accordance with the calreticulin model, proteolytic mapping studies of calreticulin have shown that proteolytic cleavage with various proteases generates a truncated form lacking a major part of the C domain, confirming the presence of a looser structure in the second half of the C domain [15–18]. Previous studies ([15,17]; C. S. Jørgensen, C. Trandum, L. R. Ryder, M. Gajhede, L. K. Skov, P. Højrup, V. Bakholt & G. Houen, unpublished results) have shown that calreticulin has a rather low T m , which is surprising as it is a heat shock protein. Furthermore, it was found to be a conformationally flexible protein. These properties may be related to its function as a chaperone and stress protein, and therefore we decided to investigate the protein further in response to various forms of physical stress. We subjected it to high and low pH, elevated temperatures, or high concentrations of urea, detergent, or organic solvent, and recorded the behaviour of the protein using PAGE, MS, and capillary electrophoresis. Calreticulin responded by dimerizing and oligomerizing. Oligomerization is a property that it shares with other important chaperones such as GRP94 and HSP90 [19–21], and it is possibly associated with its chaperone function. Materials and methods Materials Glycine, Tris, Bistris, dithiothreitol, formaldehyde, silver nitrate, Triton X-114, Triton X-100, glycerol, urea, EDTA, diethanolamine, p-nitrophenyl phosphate, bromophenol Correspondence to G. Houen, Department of Research and Devel- opment, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. Fax: + 45 32683149, Tel.: + 45 32683276, E-mail: gh@ssi.dk (Received 29 April 2003, revised 1 August 2003, accepted 28 August 2003) Eur. J. Biochem. 270, 4140–4148 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03808.x blue, MgCl 2 , ZnCl 2 , fucose, galactose, b-lactoglobulin, ovalbumin, alkaline phosphatase-conjugated goat immuno- globulins against rabbit/mouse immunoglobulins, and sinnapinic acid were from Sigma (St Louis, MO, USA). NaCl, CaCl 2 , acetic acid, NaHCO 3 , Na 2 CO 3 (NH 4 ) 2 SO 4 , sodium thiosulfate, glucose, dimethylformamide, dimethyl sulfoxide, and Tween 20 were from Merck (Darmstadt, Germany). Acetonitrile and trifluoroacetic acid were from Rathburn (Walkerburn, Scotland, UK). Mouse monoclo- nal antibody against calreticulin was from Stressgen (Vic- toria, British Columbia, Canada). Mannose was from Fluka (Buchs, Switzerland). Tris/glycine gels (4–20/4– 12%) were from Novex (San Diego, CA, USA). Ethanol was from Danisco (Aalborg, Denmark). SDS was from BDH (Poole, Dorset, UK). Acrylamide and bisacrylamide were from SSI Diagnostika (Hillerød, Denmark). Q Seph- arose Fast Flow and Sephacryl S-100 were from Pharmacia (Uppsala, Sweden). Poros 50 R1 was from Applied Biosystems (Foster City, CA, USA). Milli Q water equip- ment, 10-kDa ultrafilters, and Centriprep centrifuge tubes (10-kDa cutoff) were from Millipore (Bedford, MA, USA). Maxisorp microtiter ELISA plates were from Nunc (Ros- kilde, Denmark). Rabbit antisera against calreticulin were prepared as described previously [22]. Purification of human placental calreticulin Human placental calreticulin was purified and identified using minor modifications of a well-established procedure [18]: 20 m M Bistris, pH 7.2, was used as buffer instead of sodium phosphate; the second ammonium sulfate precipi- tation was not performed, but instead an ultradiafiltration against 20 m M Tris/HCl, pH 7.5, followed by Q Sepharose ion-exchange chromatography using 20 m M Tris/HCl, pH 7.5, as buffer with stepwise elution using increasing concentrations of NaCl in the same buffer. Fractions containing calreticulin were identified by SDS/PAGE and ELISA using antisera that recognize the N-termini and C-termini of calreticulin [22], pooled and concentrated by ultradiafiltration against 20 m M Tris/HCl, pH 7.5, followed by size-exclusion chromatography on a Sephacryl S-100 HR column. The protein showed a single band of apparent molecular mass 60 kDa on SDS/PAGE and a single band of pI 4.6 on isoelectric focusing. Native PAGE Samples were mixed with an equal volume of sample buffer (0.2 M Tris/HCl, pH 8.8, 10% glycerol, 0.005% bromo- phenol blue), and loaded on 4–20% or 4–12% Tris/glycine gels (Novex). Electrophoresis was carried out at 150 V for 75 min using 25 m M Tris/192 m M glycine, pH 8.5, as electrophoresis buffer. After the electrophoresis, the gels were silver stained using the procedure described by Blum et al. [23]. Urea gradient PAGE Electrophoretic analysis of protein folding across a trans- verse urea gradient was carried out as described by Creighton [24–26] using 11% polyacrylamide gels. A Novex gel-moulding cassette was modified to facilitate casting of the gels. The urea gradients (0–8 M or 1–7 M )weremadein 50 m M Tris/HCl (pH 8.8)/11% acrylamide/0.3% bisacryl- amide, with two chambers connected. The mixing chamber was stirred with a magnetic bar, and a peristaltic pump was used to fill the gel cassettes. Samples were incubated at room temperature for 1 h in the presence or absence of 8 M urea and 5 m M dithiothreitol. Glycerol and bromophenol blue were then added to final concentrations of 10% and 0.1%. The gels were run at 4 °C overnight at 40 V using 50 m M Tris/HCl, pH 8.0, as buffer. Digestion of heat-denatured ovalbumin Denaturation and proteolytic digestion of heat-denatured ovalbumin was performed as described in Jørgensen et al. [6]. ELISA A proteinase K digest of heat-denatured ovalbumin, a peptide (GYVIIKPLVWV [6]), or ovalbumin (1 mgÆmL )1 ) was diluted 1 : 10/1 : 500/1 : 1000 followed by overnight incubation at 5 °C; 100 lL per well using 50 m M Na 2 CO 3 , pH 9.6, with or without the addition of 8 M urea/50 m M dithiothreitol as coating buffer. All subsequent incubations and washing steps were in 25 m M Tris/HCl (pH 7.5)/0.15 M NaCl/0.5% Tween 20. The plate was washed three times for 1 min, followed by a 30-min blocking step using the same buffer. The wells were incubated for 2 h with calreticulin (0.25 mgÆmL )1 , diluted 1 : 200) or heat-treated calreticulin (1 h at 57 °C; diluted 1 : 200). After being washed (3 · 1 min), the plate was incubated for 1 h with monoclo- nal antibody against calreticulin, washed again (3 · 1min) and incubated for 1 h with alkaline phosphatase-conjugated goat immunoglobulins against mouse immunoglobulins. After another three washes, bound conjugate was quantified using a p-nitrophenyl phosphate solution (1 mg p-nitro- phenyl phosphate per mL of 1 M diethanolamine, pH 9.8, 0.5 m M MgCl 2 ). The plate was read on a VERSAmax turnable microplate reader (Molecular Devises, Sunnyvale, CA, USA) at 405 nm using background subtraction at 690 nm. Fig. 1. Crystal structure of the lumenal domain of calnexin. 1JHN in The Protein Data Bank found at http://www.rcsb.org/pdb/ [10,39]. Ó FEBS 2003 Dimerization and oligomerization of calreticulin (Eur. J. Biochem. 270) 4141 MALDI-TOF-MS Protein micropurification and sample application was performed as described previously [27], using Poros 50 R1 for the micropurification. Samples were eluted with matrix (20 lgÆlL )1 sinnapinic acid in 70% acetonitrile/0.1% trifluoroacetic acid) directly on to the first matrix layer (20 lgÆlL )1 sinnapinic acid in 100% acetone) on the target plate [Scout 384 massive (aluminium) from Bruker Dalton- ics, Bremen, Germany]. Delayed extraction MALDI-TOF MS was carried out on a Bruker ultraflex MALDI reflector time-of-flight mass spectrometer (Bruker Daltonics) equipped with a nitrogen laser (k ¼ 337 nm). All mass spectra were collected in the linear positive ion mode. External calibration was carried out with protein standard II from Bruker (Bruker Dalton- ics). Data analysis was carried out using either the M/Z software package ( M / Z -Freeware edition, 2001-08-14; Pro- teometrics Inc., New York, NY, USA) or XTOF 1.5 (Bruker Daltonics). Capillary electrophoresis Capillary electrophoresis was performed on a Beckman P/ACE 2050 instrument using UV detection at 200 nm. Electrophoresis buffer was 0.1 M phosphate, pH 7.4. A 50-lm internal diameter uncoated fused silica capillary with 50 cm to the detector window and of 57 cm total length was used. Separations were carried out at a constant current of 80 lA (corresponding to voltages of  18 kV). The capil- lary was thermostatically controlled at 20 °C. Data were collected and processed by the Beckman system Gold software. The capillary was rinsed after electrophoresis for 1minwith0.1 M NaOHand1minwithwaterandthenfor 2 min with electrophoresis buffer. Samples for the heating experiments consisted of calreticulin at 0.20 mgÆmL )1 in NaCl/P i mixed with a peptide marker (Ac-Pro-Ser-Lys-Asp- OH)at0.1 mgÆmL )1 in a final volume of 50 lL. Then 30 lL of the sample was heated at 48 °C in an Eppendorf thermomixer (500 r.p.m.). At 20, 70 and 110 min, 10 lL aliquots were withdrawn and kept at )20 °C until analysed by capillary electrophoresis. The capillary electrophoresis analysis of the aliquots subsequently took place after dilution with 5 lL water and injection for 6 s corresponding to  5 nL sample volumes. Results Dimerization of calreticulin Calreticulin contains three cysteines, of which the first two (Cys88, Cys120) form a disulfide bridge whereas the third (Cys146) is free [18]. Initially we evaluated the accessibility of the cysteine side chains in calreticulin to thiol-specific reagents using MS. As expected, Cys146 reacted readily with the small molecule iodoacetic acid, whereas Cys88 and Cys120 were not derivatized (results not shown). This confirms that calreticulin has a free SH group on Cys146. In purified human placenta calreticulin, dimers were absent but we found that dimerization could be induced experiment- ally. Lowering the pH from 7 to 6 or 5 resulted in the appearance of a higher-molecular-mass band in native PAGE, with a mobility corresponding to a calreticulin dimer (Fig. 2). The band was identified as calreticulin by immunoblotting using a rabbit antiserum against the C-terminus of calreticulin, and as a covalently linked dimer from the molecular mass determined by MS analysis (results not shown). Dimerization was also seen in native PAGE after exposure of calreticulin to urea (above 2.6 M )orto SDS (at or above 0.025%). Moreover, urea also induced a small amount of oligomerization of calreticulin, which will be addressed in the next section. Dimerization by exposure to urea was further demonstrated by electrophoretic ana- lysis of calreticulin unfolding and refolding across a gradient of urea in polyacrylamide gels (Creighton gels; Fig. 3). When calreticulin was applied in native form and subjected to electrophoresis in the urea gradient gel, the occurrence of a dimer, formed at urea concentrations  3 M , could be seen to correlate roughly with the occurrence of the unfolded form of the protein. When calreticulin was applied in 8 M urea, the dimer was present throughout the gel. These results show that the free SH group on Cys146 in calreticulin is incapable of dimerization in the native conformation of calreticulin, but that it becomes exposed and capable of dimerization under conditions favouring partial or complete unfolding of the protein. Oligomerization of calreticulin As mentioned above, besides induction of dimerization, 2.7 M urea also induced a small degree of oligomerization of calreticulin. This effect was also seen at higher urea concentrations and was maximal at 5–6 M urea. At higher urea concentrations (7–8 M ), the larger oligomers were absent but trimers and tetramers were present in addition to the dimer (results not shown). Recombinant calreticulin has been reported to oligomerize/polymerize at 37–45 °C[28], and in agreement with this we could also demonstrate a temperature-dependent oligomerization of purified human placenta calreticulin. As seen in Fig. 4A, oligomerization was observed when the temperature was raised from 37 °C to 47 °C, and even more pronounced at 57 °Cand67°C. Fig. 2. Silver-stained native PAGE analysis (4–12 Tris/glycine gel) of pH-induced dimerization of calreticulin. Calreticulin was dialysed against 20 m M Tris/HCl, pH 5, 6, 7, or 8, as indicated below the gel. The calreticulin preparation used shows one major and two minor calreticulin bands just above and below the major band. Immuno- blotting experiments and MS analysis confirmed that all three bands contained calreticulin (results not shown). 4142 C. S. Jørgensen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 The oligomerization temperature, defined as the tempera- ture at which higher-molecular-mass bands began to appear upon native PAGE, was determined to be  40 °C. A new result was the finding that lowering the pH below 4.6 or increasing pH above 10 also induced oligomerization (Fig. 4B), as did the presence of 25% organic solvents (dimethylsulfoxide, dimethylformamide, ethanol or meth- anol) and nonionic detergent (Tween 20) (data not shown). Investigation of the temperature-dependent oligomerization at 47 °C showed that it was a relatively fast reaction, with oligomers observed after 10 min and maximal oligomeriza- tion after 1–2 h (results not shown). Most of the oligomers appeared, by visual inspection of native polyacrylamide gels, to consist of dimers to octamers, but larger oligomers were also observed. The identity of the higher-molecular- mass bands was confirmed by immunoblotting using an antibody against the C-terminal part of calreticulin (results not shown). Visual inspection of the silver-stained PAGE gel confirmed that the calreticulin band (monomer) was actually decreasing in intensity as the higher-molecular- mass bands appeared. Control experiments were performed with other proteins with low pI values (human serum albumin, pI 4.9; ovalbumin, pI 5.2; b-lactoglobulin, pI 5.2). These were tested for their ability to oligomerize at low pH or elevated temperatures, but none oligomerized, confirm- ing that oligomerization induced by pH or temperature is not a general property of proteins with low pI values, but a specific feature of selected proteins including calreticulin. Raising the pH to 7 after pH-induced oligomerization, or lowering the temperature after temperature-induced oligo- merization did not reverse the effect, indicating that once formed the calreticulin oligomers were stable (results not shown). Capillary electrophoresis of heat-treated calreticulin compared with nontreated calreticulin confirmed that the peak of the monomeric calreticulin (detected at  18 min) was reduced on heating: the longer the heating time or the higher the temperature, the smaller the peak became (Fig. 5). As capillary electrophoresis separates molecules according to their mass/charge ratios, the oligomers do not show up individually in the electropherogram, but form a broad peak detected with about the same migration time as the monomer. As a control, a marker peptide (detected at  10 min) was added to the calreticulin sample, and the size of this peak remained unchanged throughout the experi- ment, confirming that the reduction in the monomeric calreticulin band was specific to calreticulin, and not an experimentally induced artefact. MALDI-TOF MS analysis (Fig. 6) of heat-treated cal- reticulin showed peaks corresponding in mass up to at least pentameric calreticulin, confirming that the heat-treated Fig. 3. Creighton gels showing urea-induced unfolding and dimerization of calreticulin. Urea gradient (0–8/1–7 M ) PAGE of calreticulin (1 mgÆmL )1 ) folding and unfolding in 20 m M Tris/HCl, pH 7.5. (A) Calreticulin loaded on the gel in native form. (B) Calreticulin loadedafter1hofincubationin8 M urea. Gels were stained with Coomassie Brilliant Blue. Fig. 4. Oligomerization of calreticulin analysed by native PAGE with silver staining (4–12% Tris/glycine gels). (A) Heat-induced; calreticulin was incubated for 60 min at 37 °C, 47 °C, 57 °C, or 67 °C. (B) pH- induced; calreticulin was incubated for 90 min at pH values between 4 and 12, as indicated. Ó FEBS 2003 Dimerization and oligomerization of calreticulin (Eur. J. Biochem. 270) 4143 calreticulin consisted of assemblies of integer numbers of calreticulin molecules. However, the oligomerization may be partly induced by the experimental conditions in the sample sandwich on the MALDI target. Reduced SDS/PAGE of trypsin-treated (2 h at 37 °C) monomeric and oligomeric calreticulin indicated that the oligomeric calreticulin was more sensitive to trypsin diges- tion (Fig. 7). MALDI-TOF MS analysis of the resulting bands with lower-molecular-mass confirmed that cleavage had taken place exclusively from the C-terminus of calreti- culin (main fragments identified: 1–334, 1–261, and 1–205). The temperature-dependent oligomerization was also investigated using a truncated form of calreticulin (residues 1–334 [18]). The truncated calreticulin retained the ability to oligomerize, indicating that the C-terminus of calreticulin was not essential for oligomerization (Fig. 8). When oligomerization was induced by heat, pH, or dimethyl sulfoxide in the presence of 5 m M dithiothreitol, the disulfide-bridged dimer was not observed on native PAGE, but the larger oligomers appeared (Fig. 9A). This shows that a disulfide bridge mediates the formation of a dimer whereas the larger oligomers are formed by a mechanism not involving disulfide bridges. Oligomerization in the presence of dithiothreitol must involve formation of a noncovalent dimer, to which further monomers are added, but apparently this dimer further oligomerizes. From this it follows that two mechanisms of dimerization are possible in the absence of dithiothreitol, one disulfide bridge-mediated, and one involving only noncovalent interactions. It is conceivable that the disulfide-bridged dimer can further oligomerize, but from these results, it appears that the disulfide-bridged dimer does not oligomerize as easily as the noncovalent dimer. Exposure of the oligomers to highly denaturing conditions by addition of 8 M urea or 1% SDS after oligomerization of calreticulin resulted in partial reversal of the oligomerization; the largest oligomers disappeared but the smaller oligomers and the dimer were still present (Fig. 9B). In denaturing, reducing SDS/PAGE analysis of heat-treated calreticulin, oligomeric calreticulin was reduced to monomeric calreticulin, showing that heating in the presence of SDS and dithiothreitol could reverse the dimerization and oligomerization (data not shown). The presence of either 8 M urea or 1% SDS during heat treatment also completely inhibited the oligomerization of calreticulin, and only the calreticulin dimer was observed on native PAGE, consistent with the observation that urea and SDS induces dimerization (Fig. 9B). The oligomeriza- tion was further investigated in the presence of additives with the potential to stabilize or destabilize the protein (12 m M CaCl 2 , MgCl 2 , ZnCl 2 , EDTA, fucose, mannose, glucose, or galactose), and none of these prevented the oligomerization of calreticulin (results not shown). Obser- vations by Li et al. [17], who by CD analysis showed that Ca 2+ acted as a stabilizing ion, increasing thermal stability [from T m ¼ 40.2 °CtoT m (Ca 2+ ) ¼ 44.3–46.4 °C, increas- ing with increasing Ca 2+ concentration], whereas Zn 2+ acted as a destabilizing ion decreasing thermal stability [T m (Zn 2+ ) ¼ 29.9–36.7 °C, decreasing with increasing Fig. 6. MALDI-TOF MS of calreticulin demonstrating oligomerization of calreticulin, heated for 30 min at 50 °C. (A) Calreticulin monomer, dimer and trimer with molecular masses of 46 477, 93 113 and 139 393 Da, respectively. (B) Calreticulin trimer, tetramer and pen- tamer with molecular masses of 138 544, 184 409 and 229 709 Da, respectively. Fig. 5. Time course of changes in heat-treated calreticulin as monitored by capillary electrophoresis. Four separate analyses are shown of samples of calreticulin mixed with a peptide marker and exposed to either room temperature (upper trace) or increasing times at 48 °Cas indicated. Whereas the peptide marker at  10 min is unchanged, there are marked changes in the calreticulin peak at 18 min upon heating of the sample. 4144 C. S. Jørgensen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Zn 2+ concentration] led us to investigate these two ions further. Heat treatment of calreticulin with or without the addition of 7.5 m M CaCl 2 or ZnCl 2 , for 1 h at 37 °C, 40 °C or 47 °C, followed by native PAGE analysis, confirmed that calreticulin oligomerized already at 37 °C in the presence of Zn 2+ , at 40 °C if no additives were present, whereas in the presence of Ca 2+ temperatures above 40 °C were required for oligomerization to occur (Fig. 9C). In earlier studies, we showed that calreticulin interacts better with unfolded proteins than with the native proteins, and that it interacts even more strongly with certain peptides [6]. It was therefore possible that oligomerization would result in a higher affinity for denatured proteins and peptides. We investigated this hypothesis in solid-phase binding assays, and found that oligomerization indeed resulted in greater binding of calreticulin to denatured proteins and peptides; binding to proteinase K-digested heat-denatured ovalbumin increased 16 times, and binding to a peptide was increased six times on heat-induced oligomerization (Fig. 10). A similar increase in binding was observed for oligomerization induced by pH and dimethyl- sulfoxide (results not shown). Discussion Dimerization Our results show that calreticulin can dimerize through the free SH group on Cys146 at pH 5–6 or under conditions that favour unfolding of calreticulin (heating, urea concen- trations above 2.6 M , SDS concentration above 0.025%). From these results, it can be concluded that, under physiological conditions, the free SH group is shielded in the N domain but located close to the surface. It can be exposed under mildly denaturing or more denaturing conditions as may occur in vivo under heat shock conditions. The disulfide bridge in human placental calreticulin has been mapped to the first two cysteines, in contrast with bovine calreticulin where it has been mapped to the last two cysteines [18,29]. This raises the question whether the disulfide bridge may be prone to reduction or isomerization and whether this may contribute to the chaperone action of calreticulin. Calreticulin has been found to interact with both glycosylated and nonglycosylated unfolded proteins [6,28,30–33]. It has also been found to interact with protein disulfide isomerase and ERp57 through its N and P domains [33–35]. As the disulfide bridge and the free cysteine in calreticulin are located in the N domain, it is a possibility that the concerted actions of these folding catalysts may involve disulfide reshuffling in the substrates, the isomerases, and the chaperones. Oligomerization The physiologically most important property of calreticulin described here would seem to be the ability to oligomerize noncovalently under physical stress and in particular upon heat shock. The ability to self-oligomerize has been described for other chaperones including GRP94 and HSP90 [19–21], and the homologous endoplasmic reticulum protein calnexin [36–38]. Furthermore, Mancino et al. [28] have shown that recombinant calreticulin can oligomerize at temperatures above 37 °C. Here, we have shown that purified human placental calreticulin has the ability to oligomerize at temperatures above 40 °C, pH below 4.6, pH above 10, in the presence of 25% organic solvent or nonionic detergent, or at urea concentrations above 2.6 M . In conclusion, these conditions must favour local unfolding or conformational change leading to oligomerization. The oligomerization was not due to ionic interactions, and was not dependent on the presence of weakly bound Ca 2+ . In accordance with the findings of Li et al. [17], we found a lower oligomerization Fig. 8. Silver-stained polyacrylamide gel (4–20%) of 3.1 lg heat-trea- ted (1 h at 57 °C) calreticulin (lane 1) and estimated 0.2 lg heat-treated truncated calreticulin (lane 2). Fig. 7. Silver-stained reduced SDS/PAGE (4–12%) of calreticulin (with or without heat treatment for 1 h at 57 °C) with or without trypsin digestion (0.05 lg). All mixtures were incubated for 3 h at 37 °Cbefore the analysis. Lane 1, 3.6 lg calreticulin; lane 2, 3.6 lg trypsin-treated calreticulin; lane 3, 3.6 lg trypsin-treated heat-induced oligomeric calreticulin; lane 4, 3.6 lg heat-induced oligomeric calreticulin. Ó FEBS 2003 Dimerization and oligomerization of calreticulin (Eur. J. Biochem. 270) 4145 temperature in the presence of Zn 2+ , and a higher oligomerization temperature in the presence of Ca 2+ , indicating a looser structure in the presence of Zn 2+ , and a tighter structure in the presence of Ca 2+ .Theformationof disulfide bridges did not appear to be necessary for the oligomerization process, but addition of dithiothreitol inhibited dimer appearance, confirming that one dimer form was dependent on disulfide bridge formation. This does not rule out the formation of noncovalent dimers during the oligomerization process, parallel to the disulfide- dependent dimer, but this dimer is not seen to any major extent because of further oligomerization. Oligomerization could be partially reversed by 8 M urea or 1% SDS, leaving only the smaller oligomers. Moreover, oligomerization could be completely inhibited by 8 M urea or 1% SDS when present during the heat treatment, probably because of unfolding of the globular structure of the protein. These results show that the formation of higher oligomers involves only noncovalent interactions but does not rule out the possibility that the disulfide-bridged dimer can participate in oligomerization. Kapoor et al. [7] recently reported that, in contrast with the legume lectins, calreticulin does not form oligomers, presumably because of shielding of the hydrophobic regions on the convex b-sheet by an a-helix, thereby preventing interactions between the convex sheets of monomers. This explains why calreticulin in our experiments needs a local conformational change in order to oligomerize, e.g. unfold- ing of the a-helix or a movement of the helix, exposing the hydrophobic regions on the convex b-sheet. The MS analysis of trypsin-treated oligomeric calreticulin showed that it was exclusively cleaved in the C-terminus. As monomeric calreticulin was relatively insensitive to trypsin digestion, this indicated that calreticulin, upon oligomeri- zation, changed conformation thereby exposing specific sites in the C domain for trypsin cleavage. As self-oligomerization has been observed for various chaperones, it is an appealing possibility that chaperones/ heat shock proteins in general possess the ability to oligomerize. Heat shock proteins are characterized by their ability to withstand elevated temperatures, and it is also likely that the oligomerization of the proteins plays a role in chaperone activity under these conditions. As chaperones are involved in the folding process of other proteins, it seems likely that they should be structurally flexible proteins to be able to bind to the different kinds of polypeptides. Further- more, we have shown that oligomerization of calreticulin correlated with increased binding to denatured proteins and Fig. 9. Silver-stained polyacrylamide gels (4–12%). (A) Calreticulin heated in the absence or presence of 5 m M dithiothreitol. Lane 1, 1.6 lg calreticulin incubated for 1 h at 57 °C; lane 2, 1.6 lg calreticulin incubated for 1 h at 57 °C in the presence of 5 m M dithiothreitol. (B) Calreticulin heated with or without addition of urea to the sample before or after heating. Lane 1, 2.5 lg calreticulin in 8 M urea followed by 1 h incubation at 57 °C; lane 2, 2.5 lg calreticulin incubated for 1 h at 57 °C, followed by the addition of 8 M urea; lane 3, 2.5 lg calreticulin incubated for 1 h at 57 °C without urea (control). (C) Calreticulin heat-treated (37 °C lane 1–3, 40 °C lane 4–6, or 47 °C lane 7–9) with or without the addition of CaCl 2 or ZnCl 2 . Lanes 1, 4, 7, 2.5 lg calreticulin; lanes 2, 5, 8, 2.5 lg calreticulin + 7.5 m M CaCl 2 ; lanes 3, 6, 9, 2.5 lg calreticulin + 7.5 m M ZnCl 2 . Fig. 10. Investigation of binding of monomeric and heat-induced (1 h at 57 °C) oligomeric calreticulin, in solid-phase assay, to: denatured oval- bumin (1 mgÆmL -1 ) coated with 8 M urea/50 m M dithiothreitol, peptide (1 mgÆmL -1 ; GYVIIKPLVWV), or proteinase K digest of heat-dena- tured ovalbumin (1 mgÆmL -1 ). 4146 C. S. Jørgensen et al.(Eur. J. Biochem. 270) Ó FEBS 2003 peptides. The simplest explanation of this is increased avidity of the oligomer, i.e. the presence of multiple binding sites on the oligomeric entities. Consistent with our obser- vations, Yonehara et al. [19] found that heating of HSP90, another heat shock protein, induced a conformational change leading to oligomerization and greater binding to substrates. Together, our results indicate that oligomeriza- tion of heat shock proteins, which must be expected in the cell under heat shock conditions, could be an important property of these proteins. After heat shock, a higher proportion of denatured proteins will be present in the cell, and oligomerization of heat shock proteins and the resulting increased avidity may be a way in which the cell avoids aggregation and keeps these denatured proteins in solution. The polypeptide-binding site in calreticulin has been locali- zed to the globular N domain, and the P domain has been shown to be important for full chaperone activity [33]. For this reason, Ca 2+ bound to the C domain is not expected to have a direct effect on the polypeptide binding. However, as shown here, Ca 2+ may indirectly affect polypeptide binding by stabilizing the protein and increasing the oligomerization temperature at which the oligomers acquire increased avidity for polypeptide substrates. 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Oligomerization of calreticulin As mentioned above, besides induction of dimerization, 2.7 M urea also induced a small degree of oligomerization of