Báo cáo khoa học: The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases pdf

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
´

culaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France
5 NatImmune, Copenhagen, Denmark
6 Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
Mannan-binding lectin (MBL) is an important compo-
nent of the mammalian innate immune system and a
member of the collectin family, which, among others,
also includes lung surfactant proteins A and D [1–6].
MBL is a homopolymer composed of 26-kDa polypep-
tides. The protomers contain a short N-terminal cyste-
ine-rich domain, capable of forming inter-chain
disulfide bonds, a collagen-like region and a C-termi-
nal globular carbohydrate recognition domain (CRD).
These associate as homotrimeric subunits by formation
of collagen-like triple-helical fibers for subsequent
assembly into higher-order oligomers containing up to
six subunits [7–12]. In the mature MBL oligomer, the
CRD is separated from the collagen-like triple-helical
domain by a short coiled-coil sequence, called the
neck region. MBL recognizes patterns of neutral
Keywords
calreticulin; chaperone; collectin; mannan-
binding lectin; serine protease
Correspondence
G. Houen, Department of Autoimmunology,
Statens Serum Institut, Artillerivej 5,
DK-2300 Copenhagen, Denmark
Fax: +45 32683149
Tel: +45 32683276
E-mail:
(Received 7 August 2007, revised 19

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-

vates the complement system. The complement-activat-
ing function of MBL is dependent on its associated
serine proteases, the mannan-binding lectin-associated
serine proteases (MASPs), of which three forms
(MASP-1, MASP-2 and MASP-3) have been described,
together with a truncated form of MASP-2, named
MBL-associated protein of 19 kDa (MAp19) [11,13–
22]. The MASPs form homodimers, which associate
with oligomeric MBL in a ratio of one MASP dimer
per MBL oligomer [9,23].
Several natural or site-directed MBL mutations
affecting MASP association and/or biological activity
have been described [24–31]. These affect the oligomer-
ization of the protein, indirectly affecting the associa-
tion with the MASPs, or directly affecting the binding
site for the MASPs, which has been localized to the
C-terminal part of the collagen-like region. MASP-1,
MASP-2 and MASP-3 have overlapping, but not iden-
tical binding sites [27,29,31].
C1q, the recognition molecule of the first comple-
ment component (C1) shows structural and functional
homology to MBL in many respects. C1q is a hexamer
of heterotrimers, composed of homologous polypeptide
chains A, B and C. These associate as N-terminal
disulfide-linked A–B and C–C dimers, which subse-
quently oligomerize into two heterotrimeric oligomers,
composed of two A–B dimers and one C–C dimer.
Three sets of two heterotrimers assemble to form the
mature C1q hexamer, which in turn associates with a
tetrameric complex formed of two molecules each of

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].

Calreticulin is an abundant chaperone in the endo-
plasmic reticulum, where it functions as a Ca
2+
stor-
age protein and a key component in the folding and
quality control of glycoproteins and other specific pro-
teins [45,46]. Furthermore, it participates in the peptide
loading of the major histocompatibility complex
class I, for presentation on the surface of antigen-pre-
senting cells [47]. Calreticulin has also been reported to
be present at the surface of various cell types, in com-
plex with cell surface receptors such as the general
scavenger receptor CD91. The calreticulin/CD91 com-
plex was shown to be present on the surface of phago-
cytic cells and to function as a scavenger receptor
complex for apoptotic cells and micro-organisms [48–
52]. Thus, the calreticulin/CD91 complex has been sug-
gested to recognize C1q and collectins bound to apop-
totic target cells, and the interaction between C1q and
calreticulin was shown to require a conformational
change in C1q, such as that occurring upon binding
to aggregated immunoglobulins or to a hydrophobic
polystyrene surface [53]. To characterize the interaction
of calreticulin with MBL, we investigated the binding
of calreticulin to plasma-derived MBL (pMBL) and
recombinant MBL (rMBL) under various conditions.
Results
The interaction of calreticulin with immobilized rMBL
was studied using multi-well format solid-phase assays
and showed the same characteristics as observed for its

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+

had to be conducted in the absence of detergent.
Nevertheless, provided that the wells were preblocked
with Tris-Tween-NaCl (TTN) buffer, the omission of
Tween 20 only resulted in a minor increase in back-
ground signal. Consequently, many control experi-
ments were carried out in both TTN buffer without
the addition of extra Ca
2+
(assuming that enough cal-
cium was naturally present to allow Ca
2+
-dependent
reactions to take place), in TTN buffer with EDTA
added to test whether Ca
2+
was a limiting factor, and
in TN buffer (25 mm Tris, 0.15 m NaCl, pH 7.5) with
Ca
2+
added. Control experiments with non-coated
wells and wells coated with control proteins (ovalbu-
min, lysozyme or BSA) ruled out non-specific inter-
actions between calreticulin and the solid phase
(Fig. 1A). In additional control experiments, BSA was
used instead of Tween 20 as a blocking agent to reveal
similar low non-specific binding of biotin-labelled
calreticulin to non-coated wells, and binding between
calreticulin and rMBL was also demonstrated using
non-biotinylated calreticulin and antibodies recogniz-
ing the C-terminus of calreticulin (results not shown).

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

1
2
3
150100500
150100500
A 405 nm
C1q
rMBL
Time before addition of salt (min)
A
B
Fig. 1. (A) Comparative time-dependent binding of calreticulin to
immobilized rMBL and C1q. For coating, rMBL and C1q were
diluted to a final concentration of 1 lgÆmL
)1
in carbonate buffer, pH
9.6, and the plate was incubated with shaking for 24 h, with
100 lL per well, at 4 °C. Control wells only received coating buffer
(negative control = background). Wells were then washed for
3 · 1 min and blocked for 1 h in TTN buffer (25 m
M Tris, 0.15 M
NaCl, 0.5% Tween 20, pH 7.5). Biotin-labelled calreticulin
(0.33 lgÆmL
)1
) diluted in TTN was added and incubation was con-
tinued at room temperature for the indicated periods followed by
incubation with AP-labelled streptavidin. The results are presented
as the mean ± SD of duplicate absorbance readings at 405 nm. (B)
Time-dependent salt-sensitivity of the interaction of calreticulin with
rMBL and C1q. rMBL and C1q were diluted to a final concentration

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

FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS 517
human plasma [54], and rMBL was produced using a
human embryonic kidney cell expression system [8].
However, when analyzing the MBL-containing frac-
tions for their ability to bind calreticulin, immobilized
rMBL from all fractions showed calreticulin binding,
whereas none of the pMBL fractions showed detect-
able binding (Fig. 3).
Binding to calreticulin was also observed for rMBL
bound to immobilized mannan (Fig. 5). By contrast,
although binding of pMBL to mannan is known to
activate the associated MASPs through a conforma-
tional change, no binding was observed to pMBL
immobilized in mannan-coated wells. Binding to
pMBL was, however, observed after size-exclusion
chromatography at pH 5, conditions reported to cause
dissociation of the MASPs from MBL [55]. Neverthe-
less, we did not obtain complete dissociation of the
bound MASPs (results not shown).
These results indicate that calreticulin is able to bind
directly to immobilized rMBL or to mannan-bound
rMBL through an initial ionic interaction, which, pos-
sibly through a conformational change in calreticulin,
gradually develops into a binding of higher strength,
presumably involving hydrogen bonds and hydropho-
bic interactions. The results also suggest that calreticu-
lin may interact with MBL through the MASP binding
site because no significant binding was observed to
pMBL with associated MASPs, neither after direct
immobilization or after binding to mannan, indepen-

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-

fated polysaccharide known to bind C1q [58] (Fig. 7).
Monoclonal antibodies raised against pMBL and spe-
cific for the CRD of MBL (Hyb 131-1) or its triple-
helical collagen-like region (Hybs 131-10, 131-11), were
also tested for their ability to inhibit the interaction
between calreticulin and rMBL, and did not reveal any
significant effect (results not shown), indicating that
they bind to sites not involved in calreticulin binding,
in agreement with their ability to bind pMBL with
associated MASPs.
Taken together, these results indicate that the
MASPs must dissociate from MBL to allow binding to
calreticulin and that conformational changes may take
place in MBL (e.g. during ligand binding or immobili-
zation). In support of this hypothesis, analysis of the
interaction of immobilized calreticulin with soluble
rMBL showed no binding either in the absence or
presence of soluble mannan (results not shown). Using
surface plasmon resonance analysis, calreticulin bound
immobilized MBL with high on and off rates, indicat-
ing that, in the absence of a conformational change in
MBL, only the initial ionic interaction could occur
(data not shown). Similarly, C1q did not bind to
0
2
4
6
8
10
12

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-

reticulin.
Calreticulin MBL interaction R. Pagh et al.
518 FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS
immobilized calreticulin, unless it was in complex with
IgG as previously described [53].
Discussion
The results obtained in the present study demonstrate
that calreticulin exhibits strong binding to rMBL with
the following characteristics: (a) a fast, saturable, and
salt-sensitive binding phase; (b) a slower binding phase
that is resistant to high salt concentrations, but sensi-
tive to 8 m urea and 10% SDS; (c) the interaction is
inhibited in the presence of MASP-2, MASP-3 and
MAp19, but not by mutant forms of MASP-2 and
MAp19 with defective MBL binding abilities; (d) the
interaction between calreticulin and rMBL may require
conformational changes in MBL, which can be
achieved by immobilization on a polystyrene surface
or through binding to a natural immobilized ligand
such as mannan; (e) binding of calreticulin is inhibited
0
1
2
3
A 405 nm
Coating agent:
1. layer:
2. layer:
3. layer:
Mannan

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-

tion with AP-conjugated streptavidin. Results are presented as the
mean ± SD of duplicate absorbance readings at 405 nm.
A1
A2
B1
B2
*
Fraction number
kDa
250
150
100
75
60
37
25
20
15
kDa
250
100
75
60
37
25
20
15
kDa
250
150

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

binding site on MBL; and (f) calreticulin does not bind
MBL point mutants with defective MASP interaction.
Steinø et al. [53] showed that calreticulin interacts
strongly with immobilized C1q, whereas pMBL (asso-
ciated with the MASPs) only exhibits a low level of
binding to calreticulin after prolonged heating at
57 °C. In the present study, we provide experimental
evidence that calreticulin can interact with MBL in a
way similar to C1q, provided that no MASP is associ-
ated. The finding that inhibition of the MBL-calreticu-
lin interaction was achieved with rMASP-2, rMASP-3
and rMAp19, but not with the D105G variant of
rMASP-2 and the Y59A variant of rMAp19, is consis-
tent with the fact that the variants lack the ability to
associate with rMBL [56,57]. In the same way, the fact
that no binding was observed with pMBL is fully con-
sistent with the latter being associated with MASP-1,
MASP-2, MASP-3 and MAp19 [54]. The most likely
hypothesis, therefore, is that any associated MASP
and MAp19 will sterically prevent binding to calreticu-
lin. However, it cannot be excluded that these may
also bring about constraints preventing conformational
changes necessary for calreticulin binding. Taken
together, the above observations, together with the
observation that MBL point mutants with impaired
ability to associate with the MASPs do not interact
with calreticulin, provide strong experimental support
for the hypothesis that calreticulin binds to the MASP
binding site of MBL.
0

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

control
(Ovalbumin)
rMASP-
3r
MAp1
9r
MAp19
(Y59A)
Positive
control
(MBL alone)
A 405 nm
12
3
150
75
50
25
37
15
0
1
2
0550 100
Excess of MASP-3/MAp19
A 405 nm
MASP-3
MAp19
0
1

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-

tant. (B) Inhibition of calreticulin binding to rMBL by preincubation
with rMASP-2 in the presence of 5 m
M Ca
2+
. Immobilized rMBL
was pre-incubated with rMASP-2 (90 l
M) for 24 h, and then calreti-
culin was added in Tris buffer containing 5 m
M of Ca
2+
. (C) Inhibi-
tion of calreticulin binding to rMBL by purified rMASP-3 (20 l
M),
wild-type rMAp19, and the Y59A MAp19 mutant (80 l
M). To the
right, the purity of the recombinant proteins was verified by SDS/
PAGE, stained with GelCODE blue stain: lane 1, rMAp19; lane 2,
rMASP-3; lane 3, rMASP3 Y59A. (D) Concentration-dependent inhi-
bition of rMASP-3 and rMAp19 inhibition of calreticulin binding to
rMBL. Calreticulin and MASP-3 or Map19 were co-incubated at the
indicated ratio (w : w) over calreticulin on microtitre plates coated
with rMBL. (E) Binding of calreticulin to rMBL and two mutant
rMBL forms (K55A, K55E), each coated at 1 lgÆmL
)1
. To the right,
MBL purity is shown by SDS/PAGE with silver-staining: lane 1,
rMBL K55E; lane 2, rMBL K55A; lane 3, rMBL; lane 4, pMBL.
Calreticulin MBL interaction R. Pagh et al.
520 FEBS Journal 275 (2008) 515–526 ª 2008 The Authors Journal compilation ª 2008 FEBS
The results of the present study also suggest that a

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).

Based on this observation, it may be anticipated that
the C-domain could be involved in binding to CD91,
whereas the N- and P-domains are responsible for
interaction with the collectins and C1q. Alternatively,
calreticulin may bind to CD91 through a site of the
N-domain not involved in the interaction with C1q
and the collectins.
The physiological relevance of the interaction of cal-
reticulin with MBL and C1q cannot be deduced from
the results obtained in the present study. However, it
is generally accepted that the MASPs are activated
upon binding of MBL to its targets and that this initi-
ates activation of the complement cascade, leading to
target lysis and/or opsonization. Inactivation of the
MASPs to control this reaction can be achieved by
binding to serum protease inhibitors, notably C1-inhib-
itor and a
2
-macroglobulin [68]. In this process, the
protease inhibitors themselves change conformation
and, in the case of a
2
-macroglobulin, a binding site for
CD91 is exposed. Upon binding of the protease inhibi-
tors, the MASPs may be released from MBL and the
a
2
-macroglobulin/MASP complex may still possess the
ability to bind to CD91. However, the target-bound
MBL may bind to CD91 as well, provided that it asso-

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

50
100
% of control
Coating agent:
Inhibitor: GLRGLQGPOGKLGPOG Fucoidan
2.layer:
3.layer
rMBL
b-calreticulin
AP-strep.
C1q
b-calreticulin
AP-strep.
rMBL
b-calreticulin
AP-strep.
C1q
b-calreticulin
AP-strep.
Fig. 7. Inhibition ELISA performed with a synthetic peptide and the
algal polysaccharide fucoidan. The inhibitory effects of the synthetic
peptide GLRGLQGPOGKLGPOG-NH
2
(where O = hydroxyproline)
and fucoidan added in a 300-fold (w : w) excess over calreticulin
were assessed. rMBL and C1q were diluted to a final concentration
of 1 lgÆmL
)1
in carbonate buffer, pH 9.6, and wells were incubated
under shaking for 24 h with 100 lL, at 4 °C, followed by washing

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

4
Æ2H
2
O were from
Unikem A/S (Copenhagen, Denmark). Monoclonal MBL
antibodies, pMBL, purified as described previously [69],
and antiserum against the calreticulin C-terminus (peptide
CEDVPGQAKDEL conjugated to ovalbumin [70]) were
from Statens Serum Institut (Copenhagen, Denmark). Tris-
glycine gels were from Invitrogen (Carlsbad, CA, USA).
Pre-stained molecular weight markers for SDS/PAGE were
from Bio-Rad Laboratories (Hercules, CA, USA). Gel-
CODE blue stain reagent was from Pierce (Rockford, IL,
USA). Cell culture supernatants containing recombinant
MASP-2 and the D105G MASP-2 mutant, as well as the
purified K55A and K55E rMBL variants [31], were gener-
ous gifts from S. Thiel (University of Aarhus, Denmark).
Purified MASP-3, MAp19 and the Y59A MAp19 variant
were produced at the Institut de Biologie Structurale Jean-
Pierre Ebel, Grenoble, France, as described previously
[57,71].
Purification of human placenta calreticulin
Human placenta calreticulin was purified using a slight
modification of a well established procedure [72,73]. In
brief, a placenta was homogenized in 20 mm bis-Tris, pH
7.2 and centrifuged, followed by homogenization of the
precipitate in the same buffer with the addition of 1% Tri-
ton X-114. A separation of water and detergent phases of
the last two supernatants was induced by addition of
Triton X-114 to 2% and incubation at 37 °C. Ammonium

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

4
,pH
7.3) at 4 °C. The biotinylated calreticulin was mixed with
an equal volume of glycerol and stored at )20 °C until use.
Chromatography of MBL
Recombinant or plasma-derived human MBL (0.3 mg,
1.5 mgÆmL
)1
) was applied on a column (diameter: 1.6 cm)
packed with 70 mL of Superose 6 and equilibrated with
NaCl/Pi, pH 7.3. The column was connected to an A
¨
kta
Explorer system (Amersham Biosciences/GE Healthcare)
and eluted at a flow rate of 0.5 mLÆmin
)1
using NaCl/Pi as
the buffer. The eluted peaks were collected as fractions of
1 mL for subsequent analysis by 4–12% SDS/PAGE.
Synthetic peptides
Peptides were synthesized as amides by solid-phase peptide
synthesis as described by Atherton and Sheppard [74]. The
identity and purity of the peptides were ascertained by
HPLC and mass spectrometry.
SDS/PAGE
SDS/PAGE was performed according to Laemmli [75] and
Studier [76] using precast gels and following the manufac-
turer’s instructions (Invitrogen). Samples from each fraction
were boiled with an equal volume of sample buffer, and
10 lL was loaded onto wells of 4–12% or 4–20% Tris-gly-

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

coating buffer or an irrelevant protein (ovalbumin or BSA).
After coating overnight at 4 °C, plates were washed three
times for 1 min, followed by blocking for 1 h in TTN. Sub-
sequently, wells were incubated with biotinylated calreticu-
lin diluted 1 : 1000 with or without other proteins/peptides
for 1 h, followed by another three washes. Finally, AP-con-
jugated streptavidin diluted 1 : 1000 was added and the
wells incubated for 1 h. Following another three washes,
bound calreticulin was quantified using pNPP (1 mgÆmL
)1
)
in 1 m diethanolamine, 0.5 mm MgCl
2
, pH 9.8. The absor-
bance was read at 405 nm with background subtraction at
650 nm on a VERSAmax microplate reader, using softmax
pro software (Molecular Devices, Sunnyvale, CA, USA). In
some experiments, calreticulin was used in combination
with specific antibodies instead of biotinylated calreticulin
and streptavidin and, in some cases, BSA was used for
blocking instead of Tween 20. As stated in the text, in some
experiments, Ca
2+
was added to the incubation buffer and,
in these cases, Tween 20 had to be omitted due to precipita-
tion. In other experiments, calreticulin was immobilized
and wells were incubated with biotin-labelled MBL or C1q
in the presence of mannan or IgG, respectively.
All binding experiments were carried out at least twice
with double determination in each experiment. Data are

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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