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: gh@ssi.dk (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. 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