The chaperone and potential mannan-binding lectin (MBL)
co-receptor calreticulin interacts with MBL through the
binding site for MBL-associated serine proteases
Rasmus Pagh
1
, Karen Duus
1
, Inga Laursen
2
, Paul R. Hansen
3
, Julie Mangor
2
, Nicole Thielens
4
,
Ge
´
rard J. Arlaud
4
, Leif Kongerslev
5
, Peter Højrup
6
and Gunnar Houen
1
1 Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark
2 Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark
3 Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
4 Laboratoire d’Enzymologie Mole
´

October 2007, accepted 3 December 2007)
doi:10.1111/j.1742-4658.2007.06218.x
The chaperone calreticulin has been suggested to function as a C1q and
collectin receptor. The interaction of calreticulin with mannan-binding
lectin (MBL) was investigated by solid-phase binding assays. Calreticulin
showed saturable and time-dependent binding to recombinant MBL, pro-
vided that MBL was immobilized on a solid surface or bound to mannan
on a surface. The binding was non-covalent and biphasic with an initial
salt-sensitive phase followed by a more stable salt-insensitive interaction.
For plasma-derived MBL, known to be complexed with MBL-associated
serine proteases (MASPs), no binding was observed. Interaction of calreti-
culin with recombinant MBL was fully inhibited by recombinant MASP-2,
MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A
variants characterized by defective MBL binding ability. Furthermore,
MBL point mutants with impaired MASP binding showed no interaction
with calreticulin. Comparative analysis of MBL with complement compo-
nent C1q, its counterpart of the classical pathway, revealed that they
display similar binding characteristics for calreticulin, providing further
indication that calreticulin is a common co-receptor/chaperone for both
proteins. In conclusion, the potential MBL co-receptor calreticulin binds to
MBL at the MASP binding site and the interaction may involve a confor-
mational change in MBL.
Abbreviations
AP, alkaline phosphatase; CRD, carbohydrate recognition domain; pNPP, para-nitrophenyl phosphate; MAp19, MBL-associated protein of
19 kDa; MASP, MBL-associated serine protease; pMBL, plasma-derived MBL; rMBL, recombinant MBL; TTN, Tris-Tween-NaCl.
FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS 515
carbohydrates on the surface of micro-organisms, and
the binding avidity is correlated to the degree of oligo-
merization [1,3–6].
Upon binding to carbohydrate patterns, MBL acti-

the serine proteases C1r and C1s [32,33].
The function of C1q is similar to that of the collec-
tins, and the role of these molecules in the immune
system relies on their ability to bind to repeating
patterns of certain carbohydrate residues and other
components on the surface of micro-organisms and
apoptotic cells, as well as to antigen-bound immuno-
globulins. C1q recognizes IgG and IgM, bound to the
surface of invading pathogens, as well as blebs on the
surface of apoptotic cells, and MBL binds to patho-
gens and apoptotic cells [4,32–38] and changes confor-
mation upon binding [39]. Target recognition activates
the associated proteases (MASPs or C1r/C1s), which
subsequently activate the complement system by cleav-
ing C4 and C2 to form the C3-convertase. This leads
to the deposition of C3b on the target cell, formation
of the membrane attack complex and release of ana-
phylatoxins, thus killing pathogens and opsonizing
them for phagocytosis.
Several receptors are involved in opsonization and
phagocytosis (e.g. the C3b receptor). Receptors for
MBL and C1q are also assumed to play a role in opso-
nization and clearance and have been the subject of
intensive research. Several candidate receptors have
been suggested, including megalin, CD91 (a
2
-macro-
globulin receptor), CD35, CD93, gC1qR (hyaluronic
acid binding protein) and cC1qR (calreticulin)
[32,35,40–44].

binding to immobilized C1q. These included: (a) a
time- and concentration-dependent saturable binding
under conditions comprising a physiological salt con-
centration and a relatively high detergent concentra-
tion (25 mm Tris, 0.15 m NaCl, 0.5% Tween 20, pH
7.5), to avoid non-specific binding (Fig. 1A) and (b) an
initial salt-sensitive binding with maximal interaction
at physiological ionic strength, which is gradually
changed to a salt-insensitive binding during interaction
(Fig. 1B). The binding could be disrupted by exposure
to high concentrations of urea (8 m) or SDS (10%)
(results not shown), indicating that the interaction was
based on non-covalent forces. Binding experiments
between calreticulin and MBL were performed both in
Calreticulin MBL interaction R. Pagh et al.
516 FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS
the presence and absence of Ca
2+
ions (0–5 mm)as
well as in the presence of EDTA (5 mm), and no major
difference was observed except for a small stimulating
effect of 0.5–1 mm Ca
2+
(Fig. 2). A complication
related to these experiments was that Ca
2+
was not
compatible with 0.5% Tween 20, and experiments with
Ca
2+

This ruled out the possibility that the binding was an
artefact caused by biotinylation of calreticulin. The
calreticulin used to demonstrate binding was mono-
meric but binding of oligomeric calreticulin to rMBL
could also be observed (results not shown).
Preparations of rMBL and pMBL were analysed
by size-exclusion chromatography and showed nearly
identical elution profiles, as measured by absorbance
at 280 nm (Fig. 3). However, rMBL eluted slightly ear-
lier from the column than pMBL. SDS/PAGE analysis
of the fractions collected from the size-exclusion chro-
matography revealed that rMBL contained somewhat
higher oligomeric forms than pMBL when analyzed
under non-reducing conditions, whereas only pMBL
contained associated MASPs (appearing as a band of
70 kDa under reducing conditions), in agreement with
the different origins and modes of production of these
preparations (Fig. 4). The comparison of pMBL and
rMBL, with respect to oligomerization, is not straight-
forward because pMBL originates from a pool of
0
1
2
3
Time (min)
A 405 nm
C1q
rMBL
Control
0

of 1 lgÆmL
)1
in carbonate buffer pH 9.6. Wells were coated
and washed as described above followed by incubation with
0.33 lgÆmL
)1
biotin-labelled calreticulin diluted in TTN for different
time intervals, prior to the addition of 0.5
M NaCl to the wells.
EDTA
(m
M)
CaCl
2
(mM)
0
1
2
3
5 0 0.5 1 2 5
A 405 nm
Fig. 2. Influence of calcium ions and EDTA on MBL calreticulin
interaction. Biotin-labelled calreticulin was incubated in rMBL-
coated plates. The interaction took place in incubation buffer
(25 m
M Tris, 0.15 M NaCl, pH 7.5) with addition of 0–5 mM of CaCl
2
or 5 mM EDTA. The interaction was quantified by incubation with
AP-conjugated streptavidin and pNPP.
R. Pagh et al. Calreticulin MBL interaction

dently of the degree of oligomerization (Fig. 3B). This
hypothesis was further investigated by performing vari-
ous inhibition and binding assays. Binding of calreticu-
lin to rMBL could be inhibited by co-incubation with
recombinant MASP-2, whereas a MASP-2 variant
(D105G), defective in MBL binding ability [56],
showed a decreased inhibitory activity (Fig. 6A). When
the immobilized rMBL was first pre-incubated with
MASP-2 in the presence of calcium ions, complete
inhibition was observed (Fig. 6B). Calreticulin binding
was also strongly inhibited by co-incubation with
recombinant MASP-3 (Fig. 6C) and the inhibitory effi-
ciency increased as a function of the MASP-3 concen-
tration used (Fig. 6D). MAp19 was also inhibitory,
whereas the Y59A MAp19 mutant, characterized by a
reduced MBL binding activity [57] showed a signifi-
cantly decreased inhibitory potential (Fig. 6C). In line
with these data, two MBL point mutants (K55A and
K55E) with defective MASP-binding capability [31]
showed no detectable interaction with calreticulin
(Fig. 6E).
Further experiments were conducted using a
synthetic peptide, GLRGLQGPOGKLGPOG-NH
2
(where O = hydroxyproline), spanning the putative
MASP-binding region of MBL [29]. As shown in
Fig. 7, this peptide was found to inhibit interaction of
calreticulin with MBL to an extent of approximately
50%. The binding of calreticulin to MBL as well as to
C1q was also shown to be inhibited by fucoidan, a sul-

14
0
1
2
A
B
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
(mL)
0
2
4
6
8
10
12
14
0
1
2
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
mAu
mAu
A
405 nm
A
405 nm
(mL)
Fig. 3. Elution profiles from size-exclusion chromatography of (A)
rMBL and (B) pMBL. Hatched bars represent results from ELISA
analysis of the collected fractions for binding of biotin-labelled cal-

rMBL
b-calreticulin
AP-strep.
Mannan
pMBL
b-calreticulin
AP-strep.
Mannan
–
b-calreticulin
AP-strep.
rMBL
–
b-calreticulin
AP-strep.
pMBL
–
b-calreticulin
AP-strep.
Fig. 5. Interaction of calreticulin with MBL bound to immobilized
mannan. Wells were coated as indicated with rMBL and pMBL
(1 lgÆmL
)1
), or with mannan (1 mgÆmL
)1
) followed by incubation
with rMBL and pMBL. Subsequently, wells were incubated with
biotin-labelled calreticulin (0.33 lgÆmL
)1
) in TTN followed by incuba-

100
75
60
25
20
15
37
kDa
250
150
100
75
60
37
25
20
15
33 34 35 36 37 38 39 40 41 42 43 44 45 33 34 35 36 37 38 39 40 41 42 43 44 45
Fraction number
Fig. 4. SDS/PAGE analysis of peak fractions
from size exclusion chromatography of
rMBL (A1–A2) and pMBL (B1–B2) as shown
in Fig. 2. (A1, B1) SDS/PAGE under reducing
conditions. (A2, B2) Non-reducing SDS/
PAGE. Gels (4–12%) were stained with
Coomassie Brilliant Blue. *MASP-derived
bands.
R. Pagh et al. Calreticulin MBL interaction
FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS 519
by a short synthetic peptide mapping to the MASP-2

1
2
3
Positive
control
rMASP-2
(D105G) mutant
rMASP-2
A 405 mm
75
50
150
1
A
B
C
D
E
23
0
1
2
3
NONE rMASP-2
Inhibitor
A 405 nm
0
1
2
Negative

2
Positive control
(rMBL)
rMBL (K55E) rMBL (K55A) Negative control
(Ovalbumin)
A 405 nm
1234
150
75
50
25
37
Fig. 6. (A) Inhibition of calreticulin binding to rMBL by wild-type and
mutant (D105G) MASP-2. Wells were coated at 4 °C for 24 h with
100 lL of rMBL (1 lgÆmL
)1
in carbonate buffer, pH 9.6). The wells
were then washed for 3 · 1 min in TTN and incubated with 100 lL
of supernatants from either non-transfected cells (positive control),
HEK293 cells containing wild-type MASP-2 or the D105G mutant,
together with the addition of 1 lgÆ mL
)1
of biotinylated calreticulin,
thereby obtaining a 100-fold molar excess of the MASPs. Control
experiments with anti-MASP-2 and anti-MBL sera confirmed the
presence of rMBL and MASP-2, respectively, in the wells (not
shown). The results are presented as the mean ± SD of duplicate
absorbance readings at 405 nm. The presence and integrity of
MASP-2 in the used supernatant were confirmed by immunoblot:
lane 1, MASP-2; lane 2, rMASP-2 D105G; lane 3, control superna-

conformational change may take place in calreticulin
upon binding to immobilized MBL, resulting in a non-
covalent biphasic binding in terms of salt sensitivity.
Although, it cannot be ruled out that the immobiliza-
tion of MBL may simply increase the number of bind-
ing sites or that further conformational changes may
also occur in MBL, these characteristics are strikingly
similar to those reported for the interaction between
calreticulin and C1q [53]. Conformational changes in
calreticulin have previously been reported to occur in
conjunction with Ca
2+
deprivation or removal of the
C-domain, and these changes induced a polypeptide-
receptive state of calreticulin [59,60].
Calreticulin is a multi-functional chaperone which
has been shown to possess Ca
2+
binding, lectin-like
and polypeptide binding properties [61–67]. Calreticu-
lin has been reported to be a candidate co-receptor for
the collectins and C1q and to be present on cell sur-
faces in complex with CD91 [48–52]. This implies that
calreticulin is capable of associating with CD91 using
one site, and interacting with the collectins or C1q
through another site. To determine which part of cal-
reticulin participates in the interaction with MBL, we
performed preliminary inhibition studies with recombi-
nant calreticulin N- and P-domains, which both
showed some inhibitory activity (results not shown).

ciates with calreticulin, which may occur on the cell
surface in complex with CD91 [48–52]. The obvious
advantage of this process is that the target would be
opsonized for binding to CD91, whether or not the
a
2
-macroglobulin/MASP complex remains bound to
MBL or dissociates. In general, it may be anticipated
that the process of infectious target/apoptotic cell rec-
ognition depends on multiple factors and ligands, and
that it has an inherent redundancy, in order to achieve
maximal specificity and safety in self/non-self discrimi-
nation. Thus, the MBL/MASP/a
2
-macroglobulin/
calreticulin/CD91 system only constitutes a part of the
phagocytic scavenging system.
In conclusion, the potential MBL co-receptor/chap-
erone calreticulin interacts with MBL at its MASP-
binding site. The interaction of calreticulin with MBL
is similar to that observed for C1q, indicating that
pathogenic targets, activating the lectin or classical
complement pathways, might be eliminated through
interaction with the calreticulin/CD91 complex.
Experimental procedures
Reagents
Amino acids, ovalbumin, p-nitrophenyl-phosphate (pNPP)
substrate tablets, 5-Br-4-Cl-3-indolylphosphate/nitrobluetet-
razolium substrate tablets, urea, dimethylsulfoxide, glycerol,
0

and blocking for 1 h in TTN. The inhibitors dissolved in dimethylsulf-
oxide (10 mgÆmL
)1
) were diluted 1 : 100 in TTN and added together
with biotin-labelled calreticulin (0.33 lgÆmL
)1
). Subsequently, wells
were incubated with AP-labelled streptavidin and developed with
pNPP. Results are presented as the mean ± SD of duplicate absor-
bance readings at 405 nm.
R. Pagh et al. Calreticulin MBL interaction
FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS 521
dithiothreitol, sodium carbonate, Tris, Tris-hydrochloride,
N-hydroxy-succinimidobiotin, BSA, hemoglobin, lysozyme,
C1q, rabbit C1q antiserum and fucoidan from Fucus vesicu-
losus were obtained from Sigma (St Louis, MO, USA).
Acetonitrile, N,N-dimethyl formamide, MgCl
2
and
Tween 20 were obtained from Merck (Darmstadt, Ger-
many). NaCl was from Unikem (Copenhagen, Denmark).
Alkaline phosphatase (AP)-conjugated streptavidin was
from DakoCytomation (Glostrup, Denmark). MaxiSorp
microtitre plates were from Nunc (Roskilde, Denmark).
Q-Sepharose, Superose 6 and Sephacryl S-100 HR were
from Amersham Biosciences/GE Healthcare (Uppsala, Swe-
den). Recombinant MBL was from NatImmune (Copen-
hagen, Denmark). NaCl and Na
2
HPO

sulfate (337 gÆL
)1
) was added to the water phase and the
precipitated proteins removed by centrifugation. The super-
natant was then ultradiafiltered and chromatographed on a
Q-Sepharose ion-exchange column. Eluted calreticulin was
further purified by size-exclusion chromatography on a
Sephacryl S-100 HR column. The purified protein showed a
single band of apparent molecular mass 60 kDa by SDS/
PAGE.
Biotinylation of calreticulin
The purified calreticulin was dialysed against 0.1 m
NaHCO
3
, pH 9.0, at 4 °C, followed by addition of
N-hydroxysuccinimidobiotin in N,N-dimethyl formamide
(10 mgÆmL
)1
) to a final concentration of 4 mgÆmg
)1
calreti-
culin. The solution was incubated for 2 h at room tempera-
ture with end-over-end agitation, and then dialysed against
NaCl/Pi (0.15 m NaCl, 10 mm NaH
2
PO
4
/Na
2
HPO

cine gels. After running of the gels, the protein bands were
stained with Coomassie Brilliant Blue (GelCODE blue stain
reagent) and then with silver as described previously [77].
Immunoblotting
Gels were electroblotted overnight to nitrocellulose
membranes using a semidry apparatus (Bio-Rad) and a
current of 200 mA for 1 h and 20 mA overnight. The
membrane was then washed in 50 mm Tris, pH 7.5,
0.3 m NaCl, 1% Tween 20 for 30 min. All subsequent
incubations and washings were in the same buffer. The
primary rabbit antiserum directed against the C-terminus
of MASP-2 [54] was diluted 1 : 1000 and the membrane
was incubated with this for 1 h followed by three 5-min
washes. Next, the membrane was incubated with
AP-conjugated goat immunoglobulins against rabbit
immunoglobulins diluted 1 : 1000. After washing three
times for 5 min, the bound antibodies were visualized by
incubation in staining solution (5-Br-4-Cl-3-indolylphos-
phate/nitrobluetetrazolium).
Calreticulin MBL interaction R. Pagh et al.
522 FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS
Binding assays
Binding assays were carried out in polystyrene microtitre
plates. Unless otherwise stated, incubations and washings
were performed at room temperature on a shaking table by
adding 100 lL per well of TTN buffer (25 mm Tris, 0.15 m
NaCl, 0.5% Tween 20, pH 7.5). For blocking, 200 lL per
well of TTN was used. Proteins (rMBL, pMBL, C1q, cal-
reticulin) were immobilized using 0.05 m sodium carbonate,
pH 9.6, as the coating buffer. Control wells only received

represented as the mean ± SD of single experiments.
Acknowledgements
We thank Kirsten Beth Hansen, Dorthe Tange Olsen,
Inger Christiansen and Jette Petersen for their excellent
technical work and the Novo Nordisk Foundation for
a grant to P. Højrup and a scholarship grant to
K. Duus. Steffen Thiel, Institute of Medical Microbiol-
ogy, University of Aarhus, Denmark is thanked for
providing recombinant proteins.
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