MASP interactions with plasma-derived MBL

Molecular Immunology 52 (2012) 79–87

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MASP interactions with plasma-derived MBL Inga A. Laursen a,1 , Nicole M. Thielens b,c,d , Michael Christiansen a , Gunnar Houen a,∗ a

Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark CEA, DSV, Institut de Biologie Structurale (IBS), Grenoble F-38027, France c CNRS, UMR 5075, 41 rue Jules Horowitz, Grenoble F-38027, France d Université Joseph Fourier, Grenoble 1, F-38000, France b

a r t i c l e

i n f o

Article history: Received 27 April 2012 Accepted 29 April 2012 Available online 18 May 2012 Keywords: MBL MASP Recombinant protein Plasma Exchange

a b s t r a c t The interaction of mannan-binding lectin (MBL) with its associated serine proteases (MASPs) was investigated using recombinant (r) MBL, plasma-derived (pd) MBL, rMASP-3 and rMAp19. When mixed with MBL-deficient serum, rMBL and pdMBL associated with free MASP-2 to (re)gain complement-activating activity. MASPs already associated with pdMBL did not exchange with rMASP-3 or rMAp19, which bound to non-overlapping sites on MBL. Thus, rMASP-3 and rMAp19 bound to free available sites on rMBL and pdMBL. These results have important implications for the therapeutic use of MBL preparations. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Mannan-binding lectin (MBL) is an important component of the humoral innate immune defence and an acute phase reactant, which exhibits a two- to three-fold increase in response to infections (Dommett et al., 2006). MBL circulates in blood as different oligomeric forms ranging from dimers to hexamers of a basic structural unit assembled from three identical polypeptide chains of ∼25 kDa through formation of an N-terminal collagenlike triple helix (Dommett et al., 2006; Turner, 1996). The basic unit further comprises a neck region and three C-terminal carbohydrate recognition domains (CRDs). The oligomerisation of MBL is essential for the functional activity, allowing a high avidity polyvalent interaction between the CRDs and carbohydrate ligands, and for association with the MBL-associated serine proteases (MASPs), which are found in several variants. The structurally homologous MASP-1 and -2 were identified initially (Matsushita and Fujita, 1992; Thiel et al., 1997). Later, the truncated splice variant of the MASP-2 gene, MBL-associated protein 19 (MAp19) (Stover et al.,

Abbreviations: ECL, enhanced chemiluminescence; MAp, MBL-associated protein; MBL, mannan-binding lectin; MASP, MBL-associated serine protease; PBS, phosphate-buffered saline; r, recombinant; pd, plasma-derived; TBS, Tris-buffered saline. ∗ Corresponding author at: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Ørestads Boulevard 5, DK-2300 Copenhagen, Denmark. Tel.: +45 32683276; fax: +45 32683876. E-mail addresses: [email protected] (N.M. Thielens), [email protected] (G. Houen). 1 Deceased 11.12.2011. 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.04.014

1999; Takahashi et al., 1999), and a splice variant of the MASP-1 gene with a different serine protease domain, MASP-3, were found (Dahl et al., 2001). Recently, another splice variant of the MASP1 gene, MAp44, devoid of the serine protease domain, has been identified (Degn et al., 2009; Skjoedt et al., 2010a). MBL binds to patterns of neutral sugars, e.g. N-acetyl-dglucosamine and d-mannose presented on surfaces of various pathogenic micro-organisms (Dommett et al., 2006; Turner, 1996). Binding of MBL to a target pathogen results in activation of the MASPs with subsequent activation of the lectin complement pathway primarily by the action of MASP-2 (Thiel et al., 1997). This leads to clearance of the pathogen by opsono-phagocytosis and complement-mediated killing. Furthermore, MBL has been shown to modulate the cytokine response to infections and inflammatory conditions and to contribute to the clearance of apoptotic cells (Fraser et al., 2006; Super et al., 1989). Structural and promoter variants of the MBL gene are common and influence the functional activity and expression, which is reflected in a broad range of serum MBL concentrations. Persons carrying the MBL wild-type genotype show levels of 1–5 ␮g/ml serum. Approximately 10–15% of Caucasians carry genotypes associated with concentrations below 100 ng/ml; these persons are defined as being MBL-deficient (Madsen et al., 1995). A larger segment, approximately 35%, of the population carries genotypes associated with decreased MBL concentrations, which may cause MBL insufficiency (Madsen et al., 1995). Individuals with low MBL levels appear to be at risk of severe and recurrent infections, most prominently in childhood before maturation of the adaptive immune system and under immune-compromised conditions

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(Kilpatrick, 2002). Comprehensive research has associated MBL deficiency with several clinical conditions, e.g. autoimmune diseases (Kilpatrick, 2002; Saevarsdottir et al., 2006), septicaemia (Eisen et al., 2006), endocarditis (Tran et al., 2007) and chronic venous leg ulcer (Bitsch et al., 2009a). The association of MBL insufficiency with diseases indicated that selected patient groups might benefit from treatment with a plasma-derived MBL (pdMBL) product. This inspired us to develop and manufacture such a product, “MBL SSI”, from a waste fraction obtained from standard ethanol fractionation (Laursen, 2003; Laursen et al., 2007). The safety of the 1st generation “MBL SSI” was assessed in a phase I study, showing it to be a well tolerated and safe product (Valdimarsson et al., 2004). Furthermore, an open uncontrolled safety and pharmacokinetic MBL-substitution study in 12 paediatric oncology patients with chemotherapy-induced neutropenia has been conducted (Frakking et al., 2009; Brouwer et al., 2009), which confirmed that “MBL SSI” was safe and well tolerated. Over several years, both the 1st and the nanofiltered 2nd generation “MBL SSI” have been given on compassionate grounds to patients with cystic fibrosis (Garred et al., 2002), Staphylococcus aureus septicaemia (Bang et al., 2008), and recently an MBL-deficient leg ulcer patient (Bitsch et al., 2009b). Furthermore, 21 patients suffering from severe infectious diseases have been treated, all with no serious adverse events and showing moderate to good clinical efficacy (Laursen et al., unpublished results). The purity of the 2nd generation pdMBL product was around 50% and it consisted of high-oligomeric MBL, with two dominating forms (MBL-I and -II), associated with MASP-1, -2, -3 and MAp19 (Laursen et al., 2007). The functional activity, assessed by mannanbinding activity and opsonic function, was intact, whereas half of the C4-activating capacity was lost during the production process. For purification of pdMBL, affinity chromatography on immobilised carbohydrate ligands is an important required step to gain sufficient purity and recovery. However, ligand binding induces a conformational change in MBL resulting in auto-activation of the MASPs. Especially during purification of pdMBL for therapeutic use, where no protease inhibitors are added, a significant part of the MASPs in complex with MBL will be activated. This has been shown by a loss of 40–50% of the MASP-2 activity, and the appearance of split products from activated MASPs by immunoblotting and mass spectrometry (MS) analyses (Laursen, 2003; Laursen et al., 2007). After intravenous injection of MBL into volunteers and patients MBL levels have increased accordingly, and no extraordinary/nonbeneficial complement activation has been observed as a result of the presence of activated MASPs (Valdimarsson et al., 2004; Frakking et al., 2009; Garred et al., 2002; Bang et al., 2008). The fate of MBL in complex with activated MASPs in vivo is unknown. The activated MASPs might associate with protease inhibitors, i.e. C1-inhibitor and ␣2-macroglobulin with subsequent elimination (Laursen et al., 2007; Kerr et al., 2008; Ambrus et al., 2003). Another possibility is that it functions similarly to MBL in complex with native MASPs after target binding. It has been suggested that native MASP-2 may be recruited from the circulation of the patients to replace the activated MASP-2 in complex with MBL subsequent to infusion (Brouwer et al., 2009). This would result in fully intact MBL–MASP-2 complexes ready for activation of the lectin pathway upon target surface interaction. However, MASP-2 activity was demonstrated to be suboptimal in chemotherapy-treated oncology patients. Experimentally, it has been shown that recombinant MBL (rMBL) is able to bind MASPs when mixed with serum from MBLdeficient persons or with rMASPs. Similar observations have been made for MASP-stripped pdMBL and demonstrate the ability of MASP-2 to activate C4 (Vorup-Jensen et al., 2000; Teillet et al., 2007). However, whether pdMBL in complex with activated MASPs – eventually with MASPs partly stripped off – is capable of binding

free MASPs to replace activated MASPs and/or occupy remaining free binding sites, if any, has not been investigated. Such knowledge is of importance in the therapeutic use of pdMBL. The aim of the present study has been to investigate the possibility of such MASP replacement or binding in an in vitro model system, using rMAp19 and rMASP-3 as free MASPs, rMBL as a MASP-free control and two pdMBL–MASP preparations further purified from the pdMBL product with activated MASPs associated, one partly MASP-depleted. 2. Material and methods 2.1. Purification of plasma-derived MBL 9 mg of MBL SSI product (350 ␮g/ml) (Laursen et al., 2007) was further purified by immuno-affinity chromatography on a matrix conjugated with rabbit antibodies against ␣2-macroglobulin, IgA and IgM (DAKO Cytomation, Copenhagen, Denmark) and subsequently by an affinity step on mannose-agarose (Sigma, St. Louis, MO, USA) to remove known impurities and albumin added as a stabiliser. The final eluate (in 10 mM Tris, 0.15 M NaCl, pH 7.4 (TBS), and 30 mM mannose) constituted the affinity-purified pdMBL material “pdMBL Affi-1” with MASPs associated. 18 mg of MBL SSI product (350 ␮g/ml) was concentrated 10fold by ultrafiltration, and dialysed against 20 mM Na-acetate, 0.5 M NaCl, 5 mM EDTA, pH 5.0, followed by size exclusion chromatography on a HiLoad Superdex 200 column (GE Healthcare Biosciences, Uppsale, Sweden) to remove bound MASPs. The MBL-containing fractions were collected, pooled, and affinity purified as described above. The final eluate (in TBS, 30 mM mannose) constituted the partly MASP-stripped affinity-purified pdMBL preparation “pdMBL Affi-2”. The preparations were stored at −80 ◦ C until use. 2.2. Characterization of pdMBL Affi-1 and Affi-2 and rMBL Recombinant MBL was kindly provided by NatImmune (Copenhagen, Denmark), and has previously been characterized (Jensen et al., 2005). The concentration was 1.5 mg/ml. The preparation was stored at −80 ◦ C until use. Quantification of pdMBL Affi-1 and Affi-2 was done in a semi-automated time-resolved immuno-fluorescence sandwich assay run on an AutoDelphia platform (PerkinElmer, Shelton, CT, USA) using the monoclonal MBL antibody HYB 131-01 (SSI, Copenhagen, Denmark) with and without biotinylation as capture and detection antibodies respectively, and streptavidin-Eu-chelate essentially as described previously (Bergmann et al., 2003). The concentrations were 730 and 511 ␮g/ml, respectively. Purity and band composition were assessed by SDS PAGE in 4–15% gels (BioRad, Hercules, CA, USA) and immunoblotting using mouse monoclonal MASP-1/3 antibody Hyb 341-1, rabbit MASP1/3 N-terminus antibodies and rabbit anti-MASP-2 N-terminus antibodies (SSI, Copenhagen, Denmark). The secondary antibodies used were HRP-labelled goat immunoglobulins against mouse IgG and rabbit IgG (heavy + light chains) respectively (KPL, Gaithersburg, MD, USA). Bands were developed by TMBplus blotting substrate (Kem-en-Tec, Copenhagen, Denmark). Identification of the Coomassie Brilliant Blue-stained bands has previously been performed by mass spectrometry analysis (Laursen et al., 2008). 2.3. MASP-binding studies in microtitre plates Recombinant MASP-3 (1.25 mg/ml and 1.0 mg/ml) and MAp19 (1.1 mg/ml) were produced in baculovirus-insect cells and characterized as described previously (Zundel et al., 2004; Thielens et al., 2001).

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rMAp19 and rMASP-3 interactions with the three preparations were investigated using MBL immobilised by antibodies in wells of microtitre plates (NUNC, Roskilde, Denmark) or in fluid phase with subsequent binding of the MBL–MASP complexes to MBL antibodycoated microtitre wells. The MBL–MASP complexes were eluted with non-reducing sample buffer and analysed by SDS-PAGE and immunoblotting with enhanced chemiluminescence (ECL) detection. In some experiments with MASP-3 binding, immunoblotting was supplemented by a MASP-1/3 ELISA.

2.4. MASP-1/3 ELISA A microtitre plate was coated with 8 ␮g/ml of MBL antibody (Hyb 131-1) in 10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 (PBS) overnight. The plate was then blocked for 1/2 h in dilution buffer (PBS and 0.35 M NaCl, 0.05% Tween 20, 30 mM mannose, and pH 7.2) or binding buffer (TBS, 2 mM CaCl2 , 1 mM MgCl2 , and 0.02% Tween 20). MBL preparations were prediluted to 1 ␮g/ml for further 3/4-fold serial dilutions to 0.06 ␮g/ml, 100 ␮l/well. After 1½ h incubation and subsequent washing (PBS, 0.35 M NaCl, 0.5% Tween 20, and pH 7.2), biotinylated anti-MASP-1/3 (Hyb 341-1 and 0.8 mg/ml) diluted 1/1200 was added. After 1 h incubation and washing, HRP–Streptavidin (2.5 mg/ml, Invitrogen, NY, USA) diluted 1/5000 was added. After washing, colour was developed by TMB X-tra (Kem-En-Tec, Copenhagen, Denmark). Colour development was stopped by 0.18 M H2 SO4 and A450 was read.

2.5. Fluid-phase interaction between MBL and MASP-3 rMASP-3 was preincubated 1/2 h at 37 ◦ C in binding buffer (TBS, 2 mM CaCl2 , 1 mM MgCl2 , and 0.02% Tween 20) or inhibition buffer (TBS, 5 mM EDTA, and 0.02% Tween 20) before addition of rMBL, pdMBL-Affi-1 or pdMBL-Affi-2, and further incubation for 2 h at 37 ◦ C (or 1 h at 37 ◦ C and overnight at 4 ◦ C). The final rMASP-3 and MBL concentrations were 7.5 and 15 ␮g/ml, respectively. Subsequently, samples were serially diluted 3/4-fold, starting from 0.75 ␮g/ml MBL, and applied in a row of the ELISA plate, in binding or inhibition buffer, and the MASP-3 ELISA was performed as described above. For catching and elution of MBL–MASP-3 complexes, two rows received 100 ␮l of 1 ␮g/ml MBL per well for each of the different mixtures. After incubation overnight at 4 ◦ C, the wells were washed 5 times with their respective binding or inhibition buffer, and emptied upside down for 1/2 h. 100 ␮l of non-reducing sample buffer was added in the first well of each row, incubated for 10 min and transferred to the second well, and so on. The combined eluted protein material from wells 1–12 was pooled with that from the second row with the same mixture and analysed by immunoblotting and ECL. The same experiments were also performed in the presence of 10 mM mannose in the binding buffer to prevent unwanted interaction between the oligomannose-type N-linked carbohydrates of rMASP-3 and the lectin domain of MBL (Teillet et al., 2007, 2008).

2.6. Fluid-phase interaction between MBL and MAp19 Mixtures of MBL and rMAp19 were prepared in binding or inhibition buffer as described above, except that the final rMAp19 concentration was 1.5 ␮g/ml. After incubation for 1 h at 37 ◦ C and overnight at 4 ◦ C, the mixtures were diluted to 0.75 ␮g/ml MBL in binding or inhibition buffer. Catching and elution of MAp19–MBL complexes was performed as described above for the MASP-3–MBL complexes.

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2.7. Interaction of MASP-3 with immobilised MBL A microtitre plate was coated with 8 ␮g/ml of MBL antibody (Hyb 131-1) overnight and. blocked for 1/2 h in dilution buffer (PBS, 0.35 M NaCl, 0.05% Tween 20, 30 mM mannose, and pH 7.2). The three MBL preparations were prediluted to 0.6 ␮g/ml for further 2/3-fold dilutions to 0.05 ␮g/ml in inhibition or binding buffer (100 ␮l/well). After 1½ h incubation and subsequent washing, 100 ␮l of 2.5 ␮g/ml rMASP-3 in inhibition or binding buffer (preincubated 1/2 h at 37 ◦ C) was added to the wells. After 1.5 h incubation at RT followed by 1.5 h at 37 ◦ C and washing, biotinylated anti-MASP-1/3 (Hyb 341-1) diluted 1/1200 was added. After 1 h incubation and subsequent washing, HRP–Streptavidin diluted 1/5000 was added. After washing, colour was developed with TMB X-tra (Kem-en-tec), stopped by 0.18 M H2 SO4 and A450 was read. For catching and elution of MBL–MASP-3 complexes, 0.5 ␮g/ml of the three MBL preparations were added to four rows of the antibody-coated microtitre plate. After incubation and washing, rMASP-3 was added as described above. After 3 h incubation, the plate was washed and the protein material was eluted from 2 rows with non-reducing sample buffer and analysed by immunoblotting and ECL, as described in Section 2.5. 2.8. MASP-binding analysis by immunoblotting and ECL The material eluted in sample buffer was analysed by SDS PAGE in 4–15% gels and immunoblotting using mouse monoclonal MASP1/3 antibody (Hyb 341-1) and HRP-labelled goat anti-mouse IgG as secondary antibodies. When detecting MAp19, rat monoclonal MAp19 antibody 6G12 (Hycult, Plymouth, PA, USA) was used as the primary antibody, and HRP-conjugated goat-anti rat IgG (KPL, Gaithersburg, MD, USA) as the secondary antibody. Precision Plus Protein unstained Standards with Strep-tag and StrepTactin–HRP Conjugate (BioRad, Hercules, CA, USA) were used as MW-markers. Bands were developed by ECL with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, US). Photos were taken by a CCD camera (BioSpectrum) and bands were scanned and quantified by densitometry using the VisionWork software (UVP, Upland, CA, USA). 2.9. Activation of the lectin complement pathway Activation of the lectin pathway was measured by spiking MBLdeficient serum with the MBL preparations (Laursen et al., 2007). 3. Results 3.1. Characterization of MBL preparations The three MBL preparations were characterized by SDS-PAGE and immunoblotting. From Fig. 1A–C it clearly appears that recombinant MBL, as expected, has no associated MASPs. Furthermore, it consists of higher oligomeric forms than pdMBL, which is dominated by the two known major forms, MBL-I and -II, consisting of 3 × 3 = 9 and 3 × 4 = 12 monomers, respectively (Teillet et al., 2005). It also appears that pdMBL Affi-2 is partly MASP-depleted compared to pdMBL Affi-1 (Fig. 1A). Furthermore, both MASP-1/3 and MASP-2 in complex with pdMBL appear mainly to be proteolytically activated as assessed by the presence of split products upon reduction (Fig. 1B and C). When assayed for activation of the complement lectin pathway upon spiking into an MBL-deficient serum, the three preparations showed 45% (rMBL), 40% (pdMBL Affi-2) and 16% (pdMBL Affi-1) of the activity of a control serum. This indicated that rMBL associated with free MASP-2 from the serum to gain complement activating activity. The MASP-depleted pdMBL Affi-2 regained activity

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Fig. 1. SDS PAGE and immunoblotting analyses of the three MBL preparations. (A) 2 ␮g of rMBL (lanes 1 and 4), pdMBL Affi-1 (lanes 2 and 5) and pdMBL Affi-2 (lanes 3 and 6), unreduced (lanes 1–3) and reduced (lanes 4–6) were analysed by SDS-PAGE and Coomassie Brilliant Blue staining. The Mw marker is All blue (BioRad). Bands a–e have been identified as deriving from MASP-2 (a and d) and MASP-1/3 (b, c, and e) (Laursen et al., 2008). (B) 0.75 ␮g of rMBL (lanes 1 and 4), pdMBL Affi-2 (lanes 2 and 5) and pdMBL Affi-1 (lanes 3 and 6) were submitted to SDS-PAGE analysis under nonreducing and reducing conditions (lanes 1–3 and 4–6, respectively). The western blot was probed using rabbit antibodies to MASP-2 N-terminus (1/500 dilution) and HRP-conjugated secondary antibodies (1/8000 dilution). (C) The same samples as in (B) were probed with mouse anti-MASP-1/3 diluted 1/8000 (unreduced samples) and rabbit anti-MASP-1/3 N-terminus diluted 1/500 (reduced samples).

to almost the same level as rMBL, whereas pdMBL Affi-1 showed the lowest relative activity, presumably due to already associated MASPs with no or much lower complement-activating activity. This suggested that free MASP-2 bound to available free sites on MBL but did not exchange with already bound MASPs. This hypothesis was further investigated using rMAp19 and rMASP-3 as available models for the association of MASPs with the three MBL preparations.

Fig. 2. MAp19 interaction with the three MBL preparations. (A) Immunoblotting with ECL analysis of MBL antibody-captured complexes after fluid phase incubation of rMAp19 with rMBL (lanes 1 and 2), pdMBL Affi-2 (lanes 3 and 4) or pdMBL Affi-1 (lanes 5 and 6) in the presence of Ca2+ or EDTA. Lane 7: rMAp19 control. (B) Immunoblotting and ECL analysis of MBL antibody-captured complexes after incubation of rMAp19 with immobilised rMBL (lanes 2 and 3), pdMBL Affi-2 (lanes 4 and 5) or pdMBL Affi-1 (lanes 6 and 7) in the presence of Ca2+ or EDTA. Lane: 1: rMAp19 control. Lane 8: pdMBL Affi-1 control. (C) Data from densitometric scanning of bands deriving from rMAp19 associated with MBL in fluid phase or immobilised. rMAp19 binding to rMBL is set to 100%. Bands were immunostained by a rat MASP-2/MAp19 antibody.

3.2. MAp19 binding to MBL Fluid-phase incubation of rMAp19 with pdMBL Affi-1, Affi-2 and rMBL was performed at a ratio of 1.5 ␮g/ml rMAp19 to 15 ␮g/ml MBL in the presence of CaCl2 or EDTA. After incubation, MBL complexes were captured by a monoclonal MBL antibody coated in ELISA plate wells followed by elution with non-reducing SDSPAGE sample buffer. Analysis of the eluted MBL–MASP complexes by immunoblotting (Fig. 2A) revealed that rMAp19 bound to the

three MBL preparations in a Ca2+ -dependent manner increasing from Affi-1 to rMBL, with the bands mainly representing MAp19 monomers. Interaction of rMAp19 with the three MBL preparations immobilised in microtitre wells was also investigated in the presence of CaCl2 or EDTA. Immunoblot analysis of the eluted MBL–MASP complexes (Fig. 2B) showed an association of MAp19 (appearing both as monomer and dimer forms) to immobilised MBL in a

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MBL (µg/mL) Fig. 3. ELISA analysis of MASP-1/3 interactions with the three MBL preparations. (A) The MBL preparations rMBL, pdMBL Affi-1 and pdMBL Affi-2 were titrated in ELISA wells coated with monoclonal MBL antibody. Endogenous MASP-1/3 in complex with MBL was detected by biotinylated MASP-1/3 antibody, and visualized by colour development after addition of HRP–Streptavidin. (B) The three MBL preparations were incubated with rMASP-3 in the presence of 30 mM mannose with CaCl2 or EDTA added. pdMBL Affi-1 without rMASP-3 addition was used as control. Subsequent titration of MASP-1/3 bound to MBL was performed in ELISA wells coated with MBL antibody as above.

Ca2+ -dependent manner increasing from pdMBL Affi-1 to rMBL, as also seen for fluid phase binding. A weak binding was detected to immobilised MBL in the presence of EDTA (Fig. 2B, lane 2). However, in both experiments incubation of non-stripped and partly MASP-stripped pdMBL with EDTA not only inhibited association of rMAp19, but EDTA contributed to further stripping of pdMBL (Fig. 2B, lanes 6 and 8). Data from densitometric scanning of the bands from the eluted MAp19 after association with MBL both in fluid phase (Fig. 2A) and immobilised (Fig. 2B) is presented in Fig. 2C. It is clearly seen that binding increased from the “native” pdMBL Affi-1, over the MASP-depleted pdMBL Affi-2 to the completely MASP-free rMBL. No significant difference in the degree of binding to the different MBL preparations was observed for fluid phase or immobilised conditions when setting the binding to rMBL as 100%. 3.3. MASP-3 binding to MBL We set up an ELISA to detect MASP-1/3 in complex with MBL and titrated the three MBL preparations (Fig. 3A). This confirmed the relative MASP-1/3 content seen by immunoblot analysis in Fig. 1C. Furthermore, fluid phase incubation of rMASP-3 with pdMBL Affi1, Affi-2 and rMBL was performed (ratio: 5 ␮g/ml rMASP-3 to 15 ␮g/ml MBL) in the presence of CaCl2 or EDTA. Subsequent titration of the MBL–MASP-3 mixtures in the MASP-1/3 ELISA (Fig. 3B) showed that EDTA prevented MASP-3 binding, and actually eluted bound MASPs, as also observed from Fig. 2. Moreover, binding of rMASP-3 to both pdMBL Affi-2 and Affi-1 reached a plateau, and the titration curve from pdMBL Affi-1 control (without rMASP-3 added) was similar, indicating that fluid phase binding of rMASP-3 to pdMBL is limited by the already associated MASPs. The titration curve after fluid phase binding of rMASP-3 to rMBL did not reach

Fig. 4. Immunoblotting with ECL analysis of MBL–MASP-1/3 complexes after incubation of rMASP-3 with fluid phase or immobilised MBL. (A) rMBL (lanes 1 and 2) and pdMBL Affi-2 (lanes 3 and 4) after fluid phase incubation with rMASP-3, analysed under non-reducing conditions in the presence of CaCl2 or EDTA. The blot has been incubated with mouse anti-MASP-1/3 (1/7000 dilution), and HRP-conjugated secondary antibody (1/10,000 dilution). (B) Analysis of rMASP-3 binding to immobilised rMBL (lanes 1 and 2), pdMBL Affi-2 (lanes 3 and 4) and pdMBL Affi-1 (lanes 5–7) after incubation in the presence of Ca2+ or EDTA. Lane 6 corresponds to 2/3 load of lane 5. Lane 7: pdMBL Affi-1 + EDTA.

the plateau observed for total MASP-1/3 binding to pdMBL in this experiment. The catched and eluted proteins from the mixtures of rMBL or pdMBL Affi-2 and rMASP-3 were analysed by immunoblotting/ECL. As shown in Fig. 4A, rMBL bound rMASP-3 to a higher degree than the partly MASP-stripped pdMBL Affi-2, with a band density ratio of 1–0.86. However, the initial MASP-1/3 content of the latter contributed to the higher OD levels in the ELISA, thus making immunoblotting/ECL analysis a better tool to determine rMASP-3 binding to non-stripped pdMBL. Binding of rMASP-3 to pdMBL Affi-1, pdMBL Affi-2, and rMBL immobilised in microtitre wells was also tested in the presence of CaCl2 or EDTA. The MBL–MASP complexes eluted from the microtitre plate were analysed by SDS PAGE and immunoblotting. It appears from the band intensities in Fig. 4B that rMASP-3 interacted with the immobilised MBL preparations in a similar manner to MAp19. This means that rMBL, partly MASP-stripped pdMBL, and pdMBL bound rMASP-3 and rMAp19 in a similar way, with

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Fig. 5. MAp19 and MASP-3 binding to immobilised MBL, alone and in competition. 1–6: data from densitometric scanning of bands immunodetected by the rat anti-MASP2/MAp19 antibody, deriving from loading of MAp19 incubated with immobilised MBL, alone (1–3, data from Fig. 2B) and together with MASP-3 (4–6, data from Fig. 6A). 7–12: scanning data of bands immunodetected by the mouse anti-MASP-1/3 antibody, deriving from loading of rMASP-3 associated with immobilised MBL, alone (7–9, data from Fig. 4B) and together with rMAp19 (10–12, data from Fig. 6B). MAp19 and MASP-3 binding to rMBL were set to 100%.

decreasing amounts from rMBL over MASP-depleted pdMBL to native pdMBL. It should be mentioned that rMASP-3 binding was fully maintained in the presence of 10 mM mannose (Fig. S1), showing that the observed interactions did not involve N-linked glycans of rMASP-3 and the carbohydrate recognition domain (CRD) of MBL. Here too, the lanes loaded with the three MBL preparations after incubation with rMASP-3 in the presence of EDTA (Fig. 4B, lanes 1, 3 and 7) revealed no bands, thus indicating no rMASP-3 binding in the absence of Ca2 . The bands detected by anti-MASP-1/3 in Fig. 4B were quantified by densitometry (Fig. 5). Comparison with binding of MAp19 under the same conditions (Fig. 2B and C, as reported in Fig. 5) indicated a relatively higher binding of MASP-3 to rMBL compared to the pdMBLs, which might reflect more occupied sites with MASP-1/3 than with MASP-2/MAp19. 3.4. Competition between MAp19 and MASP-3 for MBL binding The interactions of rMAp19 and rMASP-3 with the three immobilised MBL preparations were also investigated in competitive experiments with rMAp19 and rMASP-3 added together or sequentially in order to allow for one of the two to bind first. Immunoblot analysis showed that rMAp19 yielded similar relative binding to the three MBL preparations, whenever added in competition with rMASP-3 (Fig. 6A) or alone (Fig. 5). The same result was obtained upon analysis of rMASP-3 binding to the MBL preparations in competition with rMAp19 (Figs. 6B and 7). Again, MASP-3 binding was not influenced by mannose, ruling out interactions with the CRDs (Fig. S2). Finally when rMAp19 and rMASP-3 were added sequentially, identical results were obtained, independent of the order of addition (Fig. 7A and B). 4. Discussion The present study confirms previous results on the oligomer status of rMBL and pdMBL, with rMBL forming higher oligomers than pdMBL, which mainly consists of two forms, MBL-I and -II, with 3 and 4 trimeric subunits each (Teillet et al., 2005). These two forms are both associated with a variety of MASPs, and the MASP status of pdMBL was consistent with previous reports with MASP-1/MASP-3 and MASP-2/MAp19 as the dominating and minor

Fig. 6. Immunoblotting and ECL analysis of rMAp19 and rMASP-3 competitive binding to immobilised MBL. (A) Detection of rMAp19 bound to rMBL (lanes 2 and 3), pdMBL Affi-2 (lanes 4 and 5) or pdMBL Affi-1 (lanes 6 and 7) in the presence or absence of Ca2+ , as indicated; lane 1: MAp19 control. (B) Detection of MASP-3 bound to rMBL (lanes 2 and 3), pdMBL Affi-2 (lanes 4 and 5) or pdMBL Affi-1 (lanes 6 and 7) in the presence or absence of Ca2+ , as indicated; lane 1: MASP-3 control.

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Fig. 7. Immunoblotting and ECL analysis of binding of sequentially added MAp19 and MASP-3 to immobilised MBL. (A) Addition of rMAP19 followed by rMASP-3 and vice versa every second lane, analysed with anti-MAp19. Lanes: 1: MAp19 control; 2 and 3: rMBL; 4 and 5: pdMBL Affi-2; 6 and 7: pdMBL Affi-1. (B) Addition of MAP19 followed by MASP-3 and vice versa every second lane, analysed with anti-MASP-3. Lanes: 1: MASP-3 control; 2 and 3: rMBL; 4 and 5: pdMBL Affi-2; 6 and 7: pdMBL Affi-1.

components, respectively (Laursen et al., 2007, 2008). Apparently, the absence of MASPs on rMBL allows for more extensive oligomerisation, suggesting a regulating role for the MASPs in the assembly of MBL–MASP complexes. The complement-activating ability of pdMBL has mainly been attributed to MASP-2, the protease responsible for efficient C4 and C2 cleavage (Thiel et al., 1997; Vorup-Jensen et al., 2000; Matsushita et al., 2000). Although previous studies have suggested that there is no free MASP-2 in serum (Moller-Kristensen et al., 2003), the available assays for measuring activation of the lectin complement pathway, based on reconstitution of MBL- or ficolins-deficient serum with recombinant MBL or ficolins, show that serum MASP-2 is available for association with free binding sites on MBL and ficolins (Petersen et al., 2001; Lacroix et al., 2009; Munthe-Fog et al., 2009). In agreement with this, the complement-activating ability of rMBL and partly MASP-depleted pdMBL were found to be almost equal when reconstituted in MBL-deficient serum, whereas the complement-activating ability of affinity-purified pdMBL was only 16% of that of a control serum, presumably mainly due to inactivation of associated MASP-2 by interaction with alpha-2-macroglobulin or C1-inhibitor (Laursen et al., 2007). These results indicate that available MASP-2 in

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serum associates with free binding sites on pdMBL but does not exchange with already bound MASPs, whether active or not. This was also found when pdMBL was incubated with rMASP-3 and rMAp19, either alone or in competition. In all cases, it was found that added rMASP-3 and rMAp19 bound to free sites on pdMBL in a non-competitive manner, but did not exchange with already bound MASPs. This means that the binding of MASPs to MBL is essentially irreversible and that the number of binding sites for MASPs on MBL is limited. The affinities of the full-length MASPs and of MAp44 for MBL have been found to be in the nM range (Thielens et al., 2001; Zundel et al., 2004; Degn et al., 2009; Phillips et al., 2009) whereas that of MAp19 was about 16 times lower (Thielens et al., 2001). The difference between MAp19 and the longer fragments is explained by the fact that the MBL–MASP interaction involves primarily the N-terminal CUB1–EGF modules of the protease, but is stabilised by the following CUB2 module that is absent MAp19. Recent mutagenesis and crystallographic studies have shown that the MASPs–MBL interactions involve major electrostatic interactions between acidic Ca2+ ligands of the protease CUB domains and a conserved lysine residue of the collagen-like region of MBL (Gregory et al., 2004; Wallis et al., 2004; Teillet et al., 2007, 2008; Gingras et al., 2011). The reason for the variety of MASPs associated with serum MBL is not clear and the number of binding sites is not known in detail. However, MBL and its associated proteases take part in different biological processes including complement activation and coagulation (Krarup et al., 2007; Takahashi et al., 2011; Degn et al., 2011) and the association with different MASPs/MAps allows for regulation and minimization of cross-interference of these processes. Although the relative contribution of the various MASPs is not known in complete detail, a picture emerges of two dominating MBL–MASP/MAp complexes, one responsible for coagulation (MBL3×3 /MASP-1/MAp19) and the other one responsible for complement activation (MBL3×4 /MASP-2/MAp44). The role of MAp44 in regulation of complement activation has been clearly demonstrated (Degn et al., 2009; Skjoedt et al., 2010a) whereas the role of MAp19 remains unclear. MASP-3 has been identified previously in association with MBL3×4 and proposed to regulate activation of the lectin pathway by competition with MASP-2 (Dahl et al., 2001), but the fact that the non-protease MAp44 protein fulfils this function suggests another yet unidentified additional role for MASP-3 (Degn et al., 2009; Skjoedt et al., 2010b). The absence of MASP-3 in MBL3×3 complexes was confirmed recently (Tateishi et al., 2011) whereas another report demonstrated a major association of MASP-3 with H-ficolin and a role in the control of H-ficolin-mediated complement activation (Skjoedt et al., 2010b). In all cases, it appears somehow puzzling that MASP-1, MASP-3 and MAp44, which share the same CUB1–EGF–CUB2 interaction region, associate with different MBL or ficolin complexes. In vitro studies using recombinant MASPs and serum-derived, MASPs-free, trimeric and tetrameric MBL did not provide evidence for a difference in the affinity of MASP-3 (or of MAp19) for both types of MBL (Teillet et al., 2005). Our observation that MASP-3 and MAp19 bind to pdMBL molecules in a non-competitive manner suggests that both molecules could bind to each of the oligomeric MBL forms. The present results indicate that the reason for a lag of opsonic function compared to MBL concentration in MBL-deficient patients treated with pdMBL (Brouwer et al., 2009) must be sought in the lower C4-deposition activity of the pdMBL used for the trial. Finally, the results also show that optimal treatment of patients with rMBL is far from realization, since the production of complexes with the same composition and specificity as pdMBL is far from trivial.

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5. Conclusions The aim of this study has been to investigate whether pdMBL in complex with activated MASPs – eventually partly stripped of the MASPs – is capable of binding free MASPs to replace activated MASPs and/or solely to occupy remaining free binding sites, in an in vitro model system using recombinant MASP-3. Our results have shown that rMBL, pdMBL, and partly MASP-stripped pdMBL all possess the ability to bind rMASP-3 in a Ca2+ -dependent way both in fluid and solid-phase. The degree of binding appears to reflect the free versus the occupied MASP-binding sites of the individual MBL molecules, and proteolytically activated/fragmented MASPs associated with pdMBL do not appear to be replaced by MASP-3 even in excess. Acknowledgements Inga Alice Laursen wrote the major part of this manuscript before she passed away 11.12.2011. It was her wish that this paper should be published posthumously and she asked GH to be responsible for this. We thank her family for permission to publish the work. Michael Pfeiffer is thanked for excellent technical work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molimm.2012.04.014. References Ambrus, G., Gal, P., Kojima, M., Szilágyi, K., Balczer, J., Antal, J., Gráf, L., Laich, A., Moffatt, B.E., Schwaeble, W., Sim, R.B., Závodszky, P., 2003. Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments. Journal of Immunology 170, 1374–1382. Bang, P., Laursen, I., Thornberg, K., Schierbeck, J., Nielsen, B., Valdimarsson, H., Koch, C., Christiansen, M., 2008. The pharmacokinetic profile of plasma-derived mannan-binding lectin in healthy adult volunteers and patients with Staphylococcus aureus septicaemia. Scandinavian Journal of Infectious Diseases 40, 44–48. Bergmann, J., Christiansen, M., Laursen, I., Bang, P., Hansen, N.E., Ellegaard, J., Koch, C., Andersen, V., 2003. Low levels of mannose-binding lectin do not affect occurrence of severe infections or duration of fever in acute myeloid leukaemia during remission induction therapy. European Journal of Haematology 70, 91–97. Bitsch, M., Laursen, I., Engel, A.-M., Christiansen, M., Larsen, S.O., Iversen, L., Holstein, P.E., Karlsmark, T., 2009a. Epidemiology of chronic wound patients and relation to serum levels of mannan-binding lectin. Acta Dermato-Venereologica 89, 607–611. Bitsch, M., Christiansen, M., Laursen, I., Engel, A.-M., Holstein, P.E., Jørgensen, B., Karlsmark, T., 2009b. Is there a connection between MBL and chronic ulcers? Acta Dermato-Venereologica 89, 307–308. Brouwer, N., Frakking, F.N., van de Wetering, M.D., van Houdt, M., Hart, M., Budde, I.K., Strengers, P.F., Laursen, I., Houen, G., Roos, D., Jensenius, J.C., Caron, H.N., Dolman, K.M., Kuijpers, T.W., 2009. Mannose-binding lectin (MBL) substitution: recovery of opsonic function in vivo lags behind MBL serum levels. Journal of Immunology 183, 3496–3504. Dahl, M.R., Thiel, S., Matsushita, M., Fujita, T., Willis, A.C., Christensen, T., Vorup-Jensen, T., Jensenius, J.C., 2001. MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15, 127–135. Degn, S.E., Hansen, A.G., Steffensen, R., Jacobsen, C., Jensenius, J.C., Thiel, S., 2009. MAp44, a human protein associated with pattern recognition molecules of the complement system and regulating the lectin pathway of complement activation. Journal of Immunology 183, 7371–7378. Degn, S.E., Jensenius, J.C., Bjerre, M., 2011. The lectin pathway and its implications in coagulation, infections and auto-immunity. Current Opinion in Organ Transplantation [Epub ahead of print]. Dommett, R.M., Klein, N., Turner, M.W., 2006. Mannose-binding lectin in innate immunity: past, present and future. Tissue Antigens 68, 193–209. Eisen, D.P., Dean, M.M., Thomas, P., Marshall, P., Gerns, N., Heatley, S., Quinn, J., Minchinton, R.M., Lipman, J., 2006. Low mannose-binding lectin function is associated with sepsis in adult patients. FEMS Immunology and Medical Microbiology 48, 274–282.

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