CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site Karen Duus 1 , Nicole M. Thielens 2 , Monique Lacroix 2 , Pascale Tacnet 2 , Philippe Frachet 2 , Uffe Holmskov 3 and Gunnar Houen 1 1 Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, Copenhagen, Denmark 2 Laboratoire d’Enzymologie Mole ´ culaire, Institut de Biologie Structurale Jean-Pierre Ebel, Commissariat a ` l’Energie Atomique, CNRS UMR 5075, Universite ´ Joseph Fourier, 41 rue Jules Horowitz, Grenoble Cedex 1, France 3 Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Introduction CD91 (also known as low-density lipoprotein receptor- related protein) is a pattern-recognition receptor that is highly expressed on human macrophages and involved in the recognition and phagocytosis of over 30 differ- ent ligands. It consists of two noncovalently bound polypeptide chains: a 515 kDa alpha-chain with four Keywords CD91; clearance; ficolin, complement; mannan-binding lectin (MBL); scavenging Correspondence K. Duus, Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark Fax: +45 32683149 Tel: +45 32688241 E-mail: kds@ssi.dk (Received 12 August 2010, revised 20 September 2010, accepted 4 October 2010) doi:10.1111/j.1742-4658.2010.07901.x CD91 plays an important role in the scavenging of apoptotic material, pos- sibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found to bind CD91. The MBL–CD91 interaction was time- and concentra- tion-dependent and could be inhibited by known ligands of CD91. MBL- associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP–MBL interaction site. This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic material by CD91. Structured digital abstract l MINT-8040679, MINT-8040706: MBL (uniprotkb:P11226) binds (MI:0407)toCD91 (uni- protkb: Q07954)byenzyme linked immunosorbent assay (MI:0411) l MINT-8040690: L-ficolin (uniprotkb:Q15485) binds (MI:0407)toCD91 (uniprotkb:Q07954) by enzyme linked immunosorbent assay ( MI:0411) l MINT-8040663: C1q B (uniprotkb:P02746), C1q C (uniprotkb:P02747), C1q A (uniprotkb: P02745) and CD91 (uniprotkb:Q07954) physically interact (MI:0915)byenzyme linked immu- nosorbent assay ( MI:0411) l MINT-8040821, MINT-8040869, MINT-8040928: CD91 (uniprotkb:Q07954) binds (MI:0407) to MBL (uniprotkb: P11226)bysurface plasmon resonance (MI:0107) l MINT-8040880: CD91 (uniprotkb:Q07954) binds (MI:0407)toL-ficolin (uniprotkb:Q15485) by surface plasmon resonance ( MI:0107) Abbreviations k a, association rate constant; K D, apparent equilibrium dissociation constant; k d, dissociation rate constant; MASP, MBL-associated serine protease; MBL, mannan-binding lectin; pNPP, para-nitrophenyl phosphate; RAP, receptor-associated protein; SP-A, surfactant protein A; SP-D, surfactant protein D; SPR, surface plasmon resonance; TTN, Tris ⁄ Tween ⁄ NaCl. 4956 FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS ligand-binding clusters and an 85 kDa beta-chain involved in endocytosis and comprising the transmem- brane and intracellular domains. Ligand interaction is mediated by 31 homologus ligand-binding repeats dis- tributed unequally between the four ligand-binding clusters. Each repeat has a bound calcium ion and a pattern of six cysteine residues forming three intramo- lecular disulfide bonds [1–3]. In a number of reports, CD91 and calreticulin have been described as a receptor complex for C1q, mediat- ing removal of apoptotic cells and immune complexes [2,4–6]. Other immune-recognition molecules, includ- ing mannan-binding lectin (MBL) and the lung surfac- tant proteins surfactant protein A (SP-A) and surfactant protein D (SP-D), have also been proposed to enhance phagocytosis through the calreticulin– CD91 complex. Thus, MBL has been shown to recog- nize and stimulate the ingestion of apoptotic cells, and this could be inhibited by antibodies recognizing cal- reticulin or CD91 [5]. Similarly, initiation of phagocy- tosis by the collectin members SP-A and SP-D through the CD91 ⁄ calreticulin pathway could be inhibited by antibodies recognizing CD91 and calreti- culin [2;6]. The more recently identified ficolins have also been proposed to play a role in phagocytosis [7–9], and L-ficolin and H-ficolin have been found to interact with calreticulin [7,10]. CD91 and ⁄ or cal- reticulin may therefore be likely candidates for a ficolin receptor. MBL is the recognition molecule of the MBL–lectin pathway and is a homooligomer composed of 26-kDa polypeptides. The protomers contain a short N-termi- nal cysteine-rich domain that is capable of forming interchain disulfide bonds, a collagen-like region and a C-terminal globular carbohydrate-recognition domain. MBL recognizes patterns of neutral carbo- hydrates, such as mannan, and the binding avidity is correlated to the degree of oligomerization. Upon binding to carbohydrate patterns, MBL activates the complement system, a function that is dependent on its associated serine proteases, the MBL-associated serine proteases (MASPs), which initiate the lectin comple- ment cascade by cleavage of C2 and C4 [11]. The ficolins are recently discovered defence proteins. Three types have been characterized in humans: H-ficolin, L-ficolin and M-ficolin. L-ficolin is a lectin with specificity for N-acetylated and neutral carbohydrates [8] . It is synthesized in the liver and, like the other ficolins, has the potential of initiating the MBL ⁄ lectin complement cascade through the MASPs [12–15]. SP-A and SP-D are lung surfactant proteins. Like MBL and the ficolins they are pattern-recognition molecules but do not share the property of binding the MASPs and therefore do not activate the complement cascade but function as opsonins. The lung surfactant proteins share with MBL and the ficolins a unique domain structure and organization with a collagen tail linked to a carbohydrate-recognition domain. Each polypeptide chain associates into homotrimers and subsequently assembles into oligomers with a sertiform or cruciform structure containing four to eight trimeric subunits [16–18]. Previously, we have reported a direct, specific inter- action between CD91 and C1q. The interaction could be inhibited by the CD91 chaperone, receptor-associ- ated protein (RAP), and partially by the C1q-binding protein calreticulin, where inhibition experiments indi- cated that half of the binding sites on C1q for CD91 were shared with calreticulin [19]. Here, it is shown that CD91 interacts directly with MBL and, to a lesser extent, with L-ficolin and there- fore has the potential to function as an MBL and ⁄ or an L-ficolin receptor mediating the endocytosis of apoptotic material. In contrast to previous reports, CD91 showed direct recognition of both MBL and L-ficolin in the absence of calreticulin. The interaction appeared to be specific because it was time-dependent, concentration-dependent and could be inhibited by protein ligands of both CD91 and the lectins. The interaction site of the CD91 molecule on MBL ⁄ L-fico- lin seemed to be in close proximity to the MASP-asso- ciation site because inhibition was observed with the MASPs and reduced binding was seen for MBL point mutants unable to bind the MASPs. Results CD91 binds MBL and L-ficolin directly We have previously shown that CD91 interacts directly with C1q. This prompted us to investigate the possible role of CD91 as a scavenger receptor for other mole- cules involved in apoptotic cell recognition. For this purpose, we tested the interaction between CD91 and several pattern-recognition molecules, namely MBL, SP-A, SP-D and ficolins, which have previously been shown to be involved in the phagocytosis of apoptotic material [7,10,20–22]. When immobilized onto a polystyrene surface, MBL interacted with soluble biotinylated CD91, to a level comparable to that observed with C1q (Fig. 1). L-fico- lin exhibited a lower, but significant, interaction, whereas SP-A, SP-D, H-ficolin and the control pro- tein, ovalbumin, showed no detectable binding. Fur- ther analyses by surface plasmon resonance (SPR) K. Duus et al. CD91 interacts with mannan-binding lectin FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS 4957 provided no evidence for an interaction between immobilized CD91 and SP-A, SP-D or H-ficolin (data not shown). The interaction between CD91 and MBL was time- dependent, readily detectable after a few minutes and reached a plateau after  400 min (Fig. 2A). Binding was also dependent on the concentration of immobi- lized MBL (Fig. 2B) and soluble CD91 (Fig. 2C). Several ligands of CD91 inhibit the interaction with MBL Different ligands of CD91 were tested for their ability to inhibit the interaction between CD91 and MBL. RAP, a CD91 chaperone known to prevent access of CD91 ligands [23,24], inhibited the interaction between CD91 and MBL in a dose-dependent manner (Fig. 3), whereas no inhibition was observed using the control protein ovalbumin. We have previously char- acterized the interaction between CD91 and C1q [19]. C1q was also tested for its ability to inhibit the CD91–MBL interaction, and addition of this protein decreased CD91 binding, with nearly complete inhibi- tion at a 10-fold C1q:CD91 molar ratio (Fig. 3). Alpha 2-macroglobulin, a known ligand of CD91 and 0 1 2 3 A 0 500 1000 1500 2000 Time (min) CD91 binding (A405) Tris, NaCl, Tween Tris, NaCl, Calcium B C CD91 binding (A405) Immobilized MBL (nM) 0 1 2 3 158 53 16 6 2 0.6 0.2 0.07 0.02 0.01 0 1 2 3 670 224 75 25 8 3 1 0.3 0.1 0.03 Immobilized CD91 (nM) MBL binding (A405) 0 20 40 60 80 100 120 C1q MBL L-ficolin H-ficolin SP-D SP-A ovalbumin CD91 binding (%) Fig. 1. CD91 binds MBL and L-ficolin. Several molecules known to enhance phagocytosis were tested for their ability to interact with CD91. C1q, MBL, L-ficolin, H-ficolin, SP-A, SP-D and ovalbumin (1 lgÆmL )1 ) as a control were immobilized on a polystyrene surface and biotinylated CD91 (1 lgÆmL )1 ) was allowed to bind for 2 h. Bound biotinylated CD91 was quantified using streptavidin-coupled alkaline phosphatase and pNPP. Results are expressed relative to C1q binding. Fig. 2. Interaction between CD91 and MBL is time- and concentra- tion-dependent. (A) MBL was immobilized on the polystyrene sur- face and biotinylated CD91 was added for the indicated intervals of time in 25 m M Tris ⁄ HCl, 0.5% Tween 20, 150 mM NaCl (pH 7.5) or 25 m M Tris ⁄ HCl, 150 mM NaCl, 5 mM CaCl 2 (pH 7.5). The level of bound CD91 was quantified as described in the legend to Fig. 1. (B) MBL was immobilized at the indicated concentrations and CD91 (1 lgÆmL )1 ) was allowed to interact and quantified using streptavidin-coupled alkaline phosphatase and pNPP. (C) CD91 was immobilized at 1 lgÆmL )1 . Biotinylated MBL was added at the indi- cated concentrations, allowed to interact for 1 h, and the amount of bound MBL was quantified using streptavidin-coupled alkaline phosphatase and pNPP. CD91 interacts with mannan-binding lectin K. Duus et al. 4958 FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS MBL, also showed the ability to interfere with the CD91–MBL interaction (Fig. S1). In addition, we tested whether MBL ligands could inhibit the interac- tion. No inhibition was observed using either man- nose or GlcNAc. The proposed MBL receptor calreticulin [5] was also tested but only inhibited the interaction weakly at high calreticulin ⁄ CD91 ratios (Figs 3 and S2). Calcium-dependence of the CD91–MBL interaction CD91 and MBL are both calcium-binding proteins. CD91 holds 31 Ca 2+ ions that are important for main- taining its structure and for ligand recognition [25]. As a C-type lectin, the ligand-binding ability of MBL is also dependent on calcium [26], in contrast to L-ficolin which has been shown to recognize GlcNAc in the absence of calcium ions [27]. We tested the interaction between CD91 and MBL in the presence or absence of 5mm CaCl 2 , and surprisingly, no difference was observed (Fig. 2A). To further investigate this ques- tion, MBL was immobilized and CD91 was allowed to interact at varying Ca 2+ and Mg 2+ concentrations and in the presence of 5 mm EDTA. EDTA markedly decreased the MBL–CD91 interaction but none of the calcium concentrations tested had a significant effect (Fig. 4A). The influence of calcium concentration was also tested by SPR with CD91 immobilized on the sensor chip surface and MBL in solution. In this 0 1 2 3 1010 C1q Ovalbumin Calreticulin RAP Molar excess of indicated inhibitor CD91 binding (A405) Fig. 3. The CD91–MBL interaction can be inhibited by CD91 ligands. MBL was immobilized on a polystyrene surface and allowed to interact for 2 h with biotinylated CD91 alone or in the presence of increasing concentrations of C1q or the control protein ovalbumin, calreticulin or RAP. The level of bound CD91 was quan- tified using streptavidin-coupled alkaline phosphatase and pNPP. 0 1 2 3 A B C 5 mM EDTA 0 0.1 0.5 1 2 5 10 CD91 binding (A405) Mg 2+ Ca 2+ mM Mg 2+ /Ca 2+ 0 40 80 120 0 100 300 Time (s) RU NaCl/Tris + 2 mM EDTA NaCl/Tris NaCl/Tris + 2 m M Ca 2+ 200 0 1 2 3 Binding (A405) b-MBL b-ovalbumin NaCl + Ca 2+ NaCl/Tris NaCl/Tris + mannose Fig. 4. Calcium dependence of the CD91–MBL interaction. (A) MBL was immobilized on a polystyrene surface and biotinylated CD91 was allowed to interact in the presence of 5 m M EDTA or at different concentrations of CaCl 2 or MgCl 2 , as indicated. The level of bound CD91 was quantified using streptavidin-coupled alkaline phosphatase and pNPP. (B) CD91 (14 600 RU) was immobilized on the surface of a sensor chip, and MBL was injected in 10 m M Tris ⁄ HCl, 150 mM NaCl, 0.005% P20 in the presence or absence of 2m M EDTA or 2 mM CaCl 2 . (C) CD91 was immobilized on a poly- styrene surface, and biotinylated MBL or the control protein (bioti- nylated ovalbumin) was allowed to interact alone (NaCl ⁄ Tris) or in the presence of either mannose (NaCl ⁄ Tris + 55 n M mannose) or CaCl 2 (NaCl ⁄ Tris + 5 mM CaCl 2 ). RU, resonance units. K. Duus et al. CD91 interacts with mannan-binding lectin FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS 4959 configuration, the interaction was found to be highly sensitive to calcium. Not only was the interaction abol- ished in the presence of 2 mm EDTA but also in a buf- fer without calcium ions (Fig. 4B). This may indicate that immobilization of MBL on a polystyrene surface restrains MBL in a conformation that, with respect to CD91 binding, is not sensitive to calcium. This hypothesis was confirmed by immobilizing CD91 on a polystyrene surface and measuring the interaction between MBL and L-ficolin, using ELISA, under dif- ferent conditions. The interaction was markedly increased in the presence of Ca 2+ , whereas mannose (55 nm) had no detectable effect (Fig. 4C). In the case of L-ficolin, the interaction with CD91 was abolished in the presence of EDTA, but the presence or absence of Ca 2+ ions in the buffer had no effect on the interac- tion (data not shown). Determination of the kinetic constants To determine the kinetic constants of the interaction between CD91 and MBL, increasing concentrations of recombinant MBL, ranging from 3 to 30 nm, were injected over a sensor chip on which CD91 has been immobilized (Fig. 5A). The sensorgrams were properly fitted using a Langmuir 1 : 1 reaction model, yielding association rate constant (k a ) and dissociation rate constant (k d ) values of 1.6 · 10 5 m )1 Æs )1 and 5.26 · 10 )4 s )1 , respectively, and a resulting apparent equilibrium dissociation constant (K D ) value of 3.3 nm (v 2 = 3.64). The reverse configuration, whereby recombinant MBL was immobilized on a sensor surface and soluble CD91 was injected over the surface, revealed similar binding characteristics (Fig. 5B) and kinetics. The CD91 used contains residual attached RAP, which influences the concentration of active CD91 and there- fore the kinetics of the interaction when CD91 is in solution. The K D value obtained was 9.33 nm with a v 2 = 0.899, and k d and k a values of 5.74 · 10 )4 S )1 and 6.15 · 10 4 MÆs )1 , respectively. In the case of L-ficolin, the kinetics for interaction with CD91 was similarly determined by SPR using plasma-derived L-ficolin concentrations ranging from 120 to 960 nm. This yielded a K D of 2.8 · 10 )7 m, using a 1 : 1 binding model (Fig. 6), which was used for comparison purposes with MBL; it should be noted that the v 2 value was higher (6.52), indicating a poorer fit of the experimental curves to the model than in the case of MBL (Fig. 6). The K D is significantly higher than that determined in the case of MBL, with both higher k a and k d values (k a = 1.98 · 10 4 M )1 Æs )1 and k d = 5.51 · 10 )3 s )1 ). CD91 interacts with MBL at or near the MASP binding site It has previously been shown that calreticulin inter- acts with MBL at the MASP-binding site [28]. 120 240 360 480 0 100 200 Time (s) RU 3 n M 30 n M 6 n M 9 n M 12 n M 18 n M 24 n M 300 A B 150 300 450 0 100 Time (s) RU 1 n M 2 n M 4 n M 8 n M 16 n M 24 n M 150 50 0 Fig. 5. SPR analysis of the MBL–CD91 interaction. (A) The kinetic constants were determined by SPR using CD91 as the immobilized ligand (14 600 RU) and concentrations of soluble MBL ranging from 3to30n M, as indicated. (B) Similar binding data was obtained with immobilized recombinant MBL (13 000 RU) and different concentra- tions (1–24 n M) of soluble CD91. RU, resonance units. 120 240 360 480 150 0 50 100 Time (s) RU 120 nM 240 nM 480 nM 720 nM 960 nM 200 Fig. 6. SPR analysis of the L-ficolin–CD91 interaction. Several con- centrations of plasma-derived L-ficolin, ranging from 120 to 960 n M, were injected over a sensor chip on which CD91 was immobilized (14 600 RU). RU, resonance units. CD91 interacts with mannan-binding lectin K. Duus et al. 4960 FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS To investigate whether CD91 binds in the same area within MBL, we tested the ability of MASP-3 to compete with CD91 for interaction. MBL was pre- incubated with MASP-3 for 5 min before injection over a CD91 surface. As shown in Fig. 7A, MASP-3 clearly inhibited the interaction between MBL and CD91 in a dose-dependent manner. To further inves- tigate whether interaction with CD91 takes place at or near the MASP-binding site, the ability of the MBL mutants K55A and K55Q, which lack the abil- ity to associate with MASPs, to interact with CD91 was investigated. Compared with wild-type MBL, both mutants showed strongly reduced binding (Fig. 7B), providing further support to the hypothesis that, as previously reported for calreticulin [28], CD91 binds at or in the vicinity of the MASP-bind- ing site of MBL. Discussion MBL and L-ficolin are both collectins that mediate activation of the MBL–lectin complement pathway. These molecules are pattern-recognition molecules of the innate immune defence and function as opsonins for the removal of pathogens and apoptotic material [5,8]. We have previously shown that CD91 directly recognizes C1q in vitro independently of calreticulin [19] and here we present data showing that CD91 also directly recognizes MBL and L-ficolin and there- fore has the potential to function as a phagocytotic receptor for these proteins. CD91 interacted directly with MBL and L-ficolin in solid-phase binding assays, both when CD91 was immobilized and when the collectins were immobilized. The interactions were concentration-dependent, time-dependent and satura- ble. The interaction between CD91 and MBL and CD91 and L-ficolin could be inhibited by ligands of both CD91 (RAP, C1q) and MBL (MASP-3) and therefore fulfilled the requirements of a specific inter- action. The binding between MBL and CD91 could be inhibited by EDTA, similarly to the interaction with other ligands of CD91 [29–31]. No requirement for additional calcium ions was observed when MBL was immobilized, whereas additional calcium was obligatory for the CD91–MBL interaction when MBL was in solution. This may suggest that calcium maintains MBL in a conformation necessary for interacting with CD91. Several concentrations of L-ficolin and MBL were injected over a surface onto which CD91 was immobilized to obtain kinetic con- stants of the interactions. K D values of 3.3 and 280 nm were determined for MBL and L-ficolin, respectively, indicating much higher affinity in the case of MBL. CD91 has been suggested to interact with C1q at a site in close proximity to the C1r ⁄ C1s-binding site [19]. It was therefore of interest to investigate whether CD91 also interacts with MBL near the site occupied by the MASPs. MASP-3 induced dose-dependent inhi- bition of the interaction between CD91 and MBL. This result was confirmed by using MBL mutants with a point mutation at residue 55, which rendered them unable to bind the MASPs [32], showing that the MBL mutations K55A and K55Q strongly inhibited interaction with CD91. Ogden and co-workers have previously reported that MBL recognizes apoptotic cells and initiates their phagocytosis through calreticulin and CD91, with calreticulin acting as the MBL-recognition mole- cule and CD91 as the internalizing receptor [5]. Here, we describe direct interaction between CD91 and MBL without the necessity of recognition by calreti- culin. Additionally, we describe L-ficolin as a novel CD91 ligand. As L-ficolin is known as an opsoniza- tion molecule for apoptotic material, CD91 has the potential to function as a receptor that mediates the RU Time (s) 30 110 190 270 0 100 200 300 MBL MBL K55Q MBL K55A 0 120 A B 200100 0 300 40 80 RU Time (s) 6 n M MBL 6 n M MBL + 0.50 n M MASP-3 6 n M MBL + 1 n M MASP-3 0.5 n M MBL + 2 n M MASP-3 Fig. 7. CD91 interacts with MBL at or near the MASP-binding site. (A) Using SPR, 6 n M of MBL was injected over a sensor chip on which CD91 was immobilized (16 000 RU), with or without preincu- bation with increasing concentrations of MASP-3, as indicated. (B) Using SPR, 24 n M of MBL, the MBL K55A mutant or the MBL K55Q mutant were injected over a sensor chip on which CD91 was immobilized (14 600 RU). RU, resonance units. K. Duus et al. CD91 interacts with mannan-binding lectin FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS 4961 phagocytosis of L-ficolin-bound apoptotic cells and debris. Materials and methods Chemicals p-nitrophenyl-phosphate (pNPP), glycerol, Hepes, sodium carbonate, dimethylsulfoxide and N-hydroxy-succinimido- biotin were from Sigma (St Louis, MO, USA). Alkaline phosphatase-conjugated streptavidin was from Dako (Glostrup, Denmark). MaxiSorp microtitre plates were from Nunc (Roskilde, Denmark). CM5 sensorchips, surfac- tant P20, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and ethanolamine were from GE Healthcare (Uppsala, Sweden). Proteins Human C1q, alpha 2-macroglobulin, BSA and ovalbumin were from Sigma. Human placenta-derived CD91 was from BioMac (Chamalie ` res, France). RAP was from Innovative Research (Novi, Michigan, USA). Recombinant MBL was a generous gift of NatImmune (København Ø, Denmark). The MBL K55A and K55Q mutants were produced as described by Teillet et al.[32]. Serum-derived MASP-free L-ficolin and recombinant MASP-3 were purified as described by Teillet et al. [32] with a final purification by high-pressure gel- permeation chromatography on a TSK G-300 SWG column (Tosohaas, King of Prussia, PA, USA) equilibrated in 145 mm NaCl, 1 mm CaCl 2 and 50 mm triethanol- amine ⁄ HCl. Recombinant H-ficolin and L-ficolin was puri- fied as described by Lacroix et al. [33]. Calreticulin was purified as previously described [34,35]. SP-A was purified from proteinosis lavage, as described by Madsen et al. [36], and SP-D was purified from amniotic fluid, as described by Leth-Larsen et al. [37]. The concentrations of MBL, L-ficolin and MASP-3 were estimated using absorption coefficients A1%, 1 cm at 280 nm of 7.8, 17.6 and 13.5, and protomer molecular mass values of 25 300, 33 800 and 87 500 Da, respectively [32]. Molar concentrations of CD91, C1q, calret- iculin, RAP and ovalbumin were estimated using molecular mass values of 595, 459, 39 and 44 kDa, respectively. Protein biotinylation CD91, MBL and ovalbumin were dialysed against 100 mm NaHCO 3 (pH 9.0) at 4 °C, followed by addition of N-hydroxysuccinimidobiotin in dimethylsulfoxide (10 mgÆmL )1 ) to a final ratio of 4 mgÆmg )1 of protein. The solution was incubated for 2 h at room temperature with end-over-end agitation, and then dialysed against NaCl ⁄ P i (10 mm NaH 2 PO 4 ⁄ Na 2 HPO 4 , pH 7.3, 150 mm NaCl) at 4 °C. The biotinylated pr oteins were stored at )20 °C un til use. ELISA Unless otherwise stated, incubations and washings were performed at room temperature on a shaking table using 100 lLÆwell )1 for incubation and 200 lLÆ well )1 for washing and blocking. TTN buffer (25 mm Tris ⁄ 0.5% Tween 20 ⁄ 150 mm NaCl, pH 7.5) was used for blocking, incuba- tion and washing. Proteins were coated at 1 lgÆ mL )1 onto the microtiter plate surface using 50 mm sodium carbonate (pH 9.6) as the coating buffer. After coating overnight at 4 °C, plates were washed three times, for 1 min each wash, followed by blocking for 30 min. Subsequently, incubation with or with- out biotinylated protein diluted to 1 lgÆmL )1 was carried out for 2 h, followed by another three washes. Finally, the plates were incubated for 1 h with alkaline phosphatase- conjugated streptavidin diluted 1:1000. Following another three washes, the bound biotinylated protein was quantified using pNPP (1 mgÆmL )1 )in1m diethanolamine containing 0.5 mm MgCl (pH 9.8). The absorbance was read at 405 nm, with background subtraction at 650 nm, on a VERSAmax microplate reader (Molecular Devices, Sunny- vale, CA, USA). All experiments were repeated at least twice and performed in duplicate. Error bars represent the standard deviation of the mean from two wells in one experiment. SPR experiments Analyses were performed using BIAcore 3000 or Biacore X instruments (GE Healthcare). CD91 was diluted to 100 lgÆmL )1 in 10 mm sodium formate (pH 3), and immobilized on a CM5 sensor chip (GE Healthcare) in 10 mm Hepes, 150 mm NaCl, 3.4 mm EDTA, 0.005% surfactant P20 using the amine-coupling chemistry recom- mended by the manufacturer. Binding of MBL and ficolins to immobilized CD91 were measured at a flow rate of 20 lLÆmin )1 in 10 mm Tris ⁄ HCl, 150 mm NaCl, 2mm CaCl 2 (pH 7.4), containing 0.005% surfactant P20. Regeneration of the surface was achieved by injection of 10 lLof2m NaCl. As a control surface for background subtraction, a quenched (activated ⁄ inactivated) surface was used for plasma-derived proteins, whereas a BSA- coated surface was used for recombinant proteins. Data were analyzed by global fitting to a 1 : 1 Langmuir-bind- ing model of both the association and dissociation phases for at least five concentrations simultaneously, using the BIAevaluation 3.2 software (GE Healthcare). The K D value was calculated from the ratio of the k d and k a values. 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Eur J Biochem 268, 2558–2565. 36 Madsen J, Tornoe I, Nielsen O, Koch C, Steinhilber W & Holmskov U (2003) Expression and localization of lung surfactant protein A in human tissues. Am J Respir Cell Mol Biol 29, 591–597. 37 Leth-Larsen R, Holmskov U & Hojrup P (1999) Struc- tural characterization of human and bovine lung surfac- tant protein D. Biochem J 343 (Pt 3), 645–652. Supporting information The following supplementary material is available: Fig. S1. Alpha 2-macroglobulin and MBL binds over- lapping sites on CD91. Fig. S2. Calreticulin has little inhibitory effect on the CD91-MBL interaction. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. CD91 interacts with mannan-binding lectin K. Duus et al. 4964 FEBS Journal 277 (2010) 4956–4964 ª 2010 The Authors Journal compilation ª 2010 FEBS . CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site Karen Duus 1 , Nicole. (2008) The chaperone and potential mannan- binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine