Heegaard3and Gunnar Houen1 1 Department of Research and Development, Statens Serum Institut, Copenhagen, Denmark; 2 Department of Biochemistry and Molecular Biology, University of Southe
Trang 1Dimerization and oligomerization of the chaperone calreticulin
Charlotte S Jørgensen1, L Rebekka Ryder1, Anne Steinø1, Peter Højrup2, Jesper Hansen2, N Helena Beyer3, Niels H H Heegaard3and Gunnar Houen1
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
2.6Mor SDS concentrations above 0.025% Moreover,we
show that calreticulin also has the ability to self-oligomerize
through noncovalent interactions at urea concentrations
above 2.6Mat pH below 4.6 or above pH 10,at
tempera-tures above 40C,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 8M urea or 1% SDS,and heat-induced oligomerization could
be inhibited by 8Murea or 1% SDS when present during heating Comparison of the binding properties of mono-meric and oligomono-meric 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 Tm,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)
Trang 2ovalbumin,alkaline phosphatase-conjugated goat
immuno-globulins against rabbit/mouse immunoimmuno-globulins,and
sinnapinic acid were from Sigma (St Louis,MO,USA)
NaCl,CaCl2,acetic acid,NaHCO3,Na2CO3(NH4)2SO4,
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 mM 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 mMTris/HCl,pH 7.5,followed by Q Sepharose
ion-exchange chromatography using 20 mM 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 mMTris/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 (0.2M 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 mM Tris/192 mM 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 (0–8Mor 1–7M) were made in
50 mMTris/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 8Murea and 5 mM dithiothreitol Glycerol and bromophenol blue were then added to final concentrations of 10% and 0.1% The gels were run at 4C overnight at 40 V using 50 mM 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 5C; 100 lL per well using 50 mMNa2CO3,
pH 9.6,with or without the addition of 8Murea/50 mM dithiothreitol as coating buffer All subsequent incubations and washing steps were in 25 mMTris/HCl (pH 7.5)/0.15M 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 57C; 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· 1 min) 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 1Mdiethanolamine,pH 9.8, 0.5 mM MgCl2) 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].
Trang 3Protein 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)1sinnapinic 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 (k¼ 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) orXTOF1.5 (Bruker
Daltonics)
Capillary electrophoresis
Capillary electrophoresis was performed on a Beckman
P/ACE 2050 instrument using UV detection at 200 nm
Electrophoresis buffer was 0.1M 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 20C Data were
collected and processed by the Beckman system Gold
software The capillary was rinsed after electrophoresis for
1 min with 0.1MNaOH and 1 min with water and then for
2 min with electrophoresis buffer Samples for the heating
experiments consisted of calreticulin at 0.20 mgÆmL)1 in
NaCl/Pimixed with a peptide marker
(Ac-Pro-Ser-Lys-Asp-OH) at 0.1 mgÆmL)1in a final volume of 50 lL Then 30 lL
of the sample was heated at 48C 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 2.6M) or to 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 3M,could be seen
to correlate roughly with the occurrence of the unfolded form of the protein When calreticulin was applied in 8M 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, 2.7Murea also induced a small degree of oligomerization
of calreticulin This effect was also seen at higher urea concentrations and was maximal at 5–6Murea At higher urea concentrations (7–8M),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 37–45C [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 37C
to 47C,and even more pronounced at 57 C and 67 C
Fig 2 Silver-stained native PAGEanalysis (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).
Trang 4The 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 47C 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
loaded after 1 h of incubation in 8 M urea Gels were stained with
Coomassie Brilliant Blue.
Fig 4 Oligomerization of calreticulin analysed by native PAGEwith 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.
Trang 5calreticulin 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 37C)
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 mMdithiothreitol,
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 8Murea 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 8Murea 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 mM CaCl2,MgCl2,ZnCl2,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
Ca2+acted as a stabilizing ion,increasing thermal stability [from Tm¼ 40.2 C to Tm(Ca2+)¼ 44.3–46.4 C,increas-ing with increasC,increas-ing Ca2+ concentration],whereas Zn2+ acted as a destabilizing ion decreasing thermal stability [T (Zn2+)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 C as
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.
Trang 6Zn2+ concentration] led us to investigate these two ions
further Heat treatment of calreticulin with or without the
addition of 7.5 mMCaCl2or ZnCl2,for 1 h at 37C,40 C
or 47C,followed by native PAGE analysis,confirmed that
calreticulin oligomerized already at 37C in the presence of
Zn2+,at 40C if no additives were present,whereas in the
presence of Ca2+temperatures above 40C 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 2.6M,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 37C Here,we have shown that purified human placental calreticulin has the ability to oligomerize at temperatures above 40C,pH below 4.6,pH above 10,in the presence of 25% organic solvent or nonionic detergent,
or at urea concentrations above 2.6M 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 Ca2+ 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 estimaheat-trea-ted 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 C before
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.
Trang 7temperature in the presence of Zn2+,and a higher
oligomerization temperature in the presence of Ca2+,
indicating a looser structure in the presence of Zn2+,and
a tighter structure in the presence of Ca2+ The formation of
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 8Murea or 1% SDS,leaving
only the smaller oligomers Moreover,oligomerization
could be completely inhibited by 8M 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,exposunfold-ing 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).
Trang 8peptides 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,Ca2+bound to the C domain is not expected to
have a direct effect on the polypeptide binding However,as
shown here,Ca2+may indirectly affect polypeptide binding
by stabilizing the protein and increasing the oligomerization
temperature at which the oligomers acquire increased
avidity for polypeptide substrates Thus,in a nonstimulated
cell,with Ca2+concentrated in the endoplasmic reticulum,
calreticulin will be less prone to oligomerization in response
to heat shock compared with a stimulated cell with lower
Ca2+concentration in the endoplasmic reticulum Further
studies on oligomerization of calreticulin in vivo in response
to heat shock and other kinds of stress,including Ca2+
deprivation,should be conducted
Acknowledgements
Kirsten Beth Hansen is thanked for excellent technical assistance.
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