Co-Complexes of MASP-1 and MASP-2 Associated with the Soluble Pattern-Recognition Molecules Drive Lectin Pathway Activation in a Manner Inhibitable This information is current as by MAp44 of September 28, 2021. Søren E. Degn, Lisbeth Jensen, Tomasz Olszowski, Jens C. Jensenius and Steffen Thiel J Immunol 2013; 191:1334-1345; Prepublished online 19 June 2013; Downloaded from doi: 10.4049/jimmunol.1300780 http://www.jimmunol.org/content/191/3/1334 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/06/19/jimmunol.130078 Material 0.DC1 References This article cites 43 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/191/3/1334.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Co-Complexes of MASP-1 and MASP-2 Associated with the Soluble Pattern-Recognition Molecules Drive Lectin Pathway Activation in a Manner Inhibitable by MAp44

Søren E. Degn,* Lisbeth Jensen,* Tomasz Olszowski,† Jens C. Jensenius,* and Steffen Thiel*

The lectin pathway of complement is an integral component of innate immunity. It is activated upon binding of mannan-binding lectin (MBL) or ficolins (H-, L-, and M-ficolin) to suitable ligand patterns on microorganisms. MBL and ficolins are polydisperse homo-oligomeric molecules, found in complexes with MBL-associated serine proteases (MASP-1, -2, and -3) and MBL-associated proteins (MAp19 and MAp44). This scenario is far more complex than the well-defined activation complex of the classical pathway,

C1qC1r2C1s2, and the composition of the activating complexes of the lectin pathway is ill defined. We and other investigators Downloaded from recently demonstrated that both MASP-1 and MASP-2 are crucial to lectin pathway activation. MASP-1 transactivates MASP-2 and, although MASP-1 also cleaves C2, MASP-2 cleaves both C4 and C2, allowing formation of the C3 convertase, C4bC2a. Juxtaposition of MASP-1 and MASP-2 during activation must be required for transactivation. We previously presented a possible scenario, which parallels that of the classical pathway, in which MASP-1 and MASP-2 are found together in the same MBL or ficolin complex. In this study, we demonstrate that, although MASPs do not directly form heterodimers, the addition of MBL or

ficolins allows the formation of MASP-1–MASP-2 co-complexes. We find that such co-complexes have a functional role in http://www.jimmunol.org/ activating complement and are present in serum at varying levels, impacting on the degree of complement activation. This raises the novel possibility that MAp44 may inhibit complement, not simply by brute force displacement of MASP-2 from MBL or ficolins, but by disruption of co-complexes, hence impairing transactivation. We present support for this contention. The Journal of Immunology, 2013, 191: 1334–1345.

he complement system is a crucial component of innate and, in turn, activate the two C1s, which can then cleave C4 and immunity that is central to health and disease (1). It consists C2, forming the C3 convertase C4bC2a. T of three pathways of activation, which converge at the level Although the lectin pathway is conceptually similar, the com- by guest on September 28, 2021 of C3 convertase formation, leading to generation of C5 con- position of the activating complexes is far more complex and less vertase and the subsequent formation of the terminal membrane well characterized than that of the classical pathway. Four pattern- attack complex (2). The three pathways of activation are the clas- recognition molecules (PRMs), mannan-binding lectin (MBL), H- sical pathway, the alternative pathway, and the lectin pathway. ficolin, L-ficolin, and M-ficolin, associate with three proteases, The classical and the lectin pathways are conceptually very MBL-associated serine protease (MASP)-1, MASP-2, and MASP- similar. The classical pathway is initiated by a defined complex, C1, 3, as well as two MBL-associated proteins (MAps), MAp19 (also composed of the recognition molecule C1q with a tetramer of two termed sMAP) and MAp44 (also known as MAP-1) (3, 4). MBL serine proteases, C1r2C1s2. C1q itself is a monodisperse homo- and ficolins are highly polydisperse homo-oligomers of homotri- oligomeric molecule composed of six heterotrimeric subunits, each meric subunits. Thus, MBL is found in a number of oligomeric comprising an A-chain, a B-chain, and a C-chain. Upon binding of forms in serum, ranging from dimers to hexamers and even higher- the C1 complex to immune complexes, the two C1r autoactivate order oligomers, the most predominant being trimers (9 polypep- tide chains) and tetramers (12 polypeptide chains) (5). A similar scenario presents for the ficolins, although the degree of oligomeri- *Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark; and zation varies among the three. †Department of Hygiene, Epidemiology and Public Health, Pomeranian Medical University, 70-210 Szczecin, Poland The MASPs and MAps are generated from two genes: MASP-1, MASP-3, and MAp44 are alternative splice products of MASP1, Received for publication March 21, 2013. Accepted for publication May 24, 2013. whereas MASP-2 and MAp19 are alternative splice products of This work was supported by The Danish Council for Independent Research, Medical Sciences. S.E.D. was supported by a postdoctoral fellowship from the Carlsberg MASP2 (3, 4). It is known that MASP-2 is necessary for lectin Foundation. pathway function, by virtue of its ability to cleave both C4 and C2 Address correspondence and reprint requests to Dr. Søren E. Degn, Department of (6). Although MASP-2 was also reported to be sufficient in itself, Biomedicine, Aarhus University, The Bartholin Building, Wilhelm Meyers Alle´ 4, as a result of its ability to autoactivate (7, 8), it was recently found 8000 Aarhus C, Denmark. E-mail address: [email protected] that MASP-1 is crucial in transactivating MASP-2 under physio- The online version of this article contains supplemental material. logical circumstances (9–11). Indeed, MASP-1 was found to au- Abbreviations used in this article: MAp, mannan-binding lectin–associated protein; MASP, mannan-binding lectin–associated serine protease; MBL, mannan-binding toactivate and to cleave MASP-2 much more efficiently than lectin; NHS, normal human serum; PRM, pattern-recognition molecule; rMASP-1i, MASP-2 itself (12). Additionally, MASP-1 cleaves auxiliary C2 recombinant catalytically inactive (active-site serine-to-alanine mutant) mannan- for the C3 convertase formation (10, 13). Based on studies in binding lectin–associated serine protease 1. knockout mice, MASP-1 and MASP-3 were suggested to be re- Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 sponsible for cleavage of pro-fD to active factor D; the latter was www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300780 The Journal of Immunology 1335 also proposed to cleave fB directly (14, 15). However, the exact Recombinant and purified proteins role is still unclear, because we recently found that a patient de- Recombinant MASPs and MAps were produced by Lipofectamine 2000– ficient in both MASP-1 and MASP-3 retained a functional alter- mediated plasmid transfection of HEK293F FreeStyle cells in 293F Ex- native pathway (9). MASP-3 has an important function during pression Medium (Invitrogen), as described in detail (26), using the con- development because its absence causes the so-called 3MC syn- structs presented (3, 9, 19). For coexpressions, plasmids were mixed 1:1 drome (16, 17). A suggested complement-regulatory role for by mass before incubation with Lipofectamine 2000 and subsequent transfection. Recombinant MBL was produced as described (27). L-ficolin MAp19 (18) could not be confirmed by us (19), whereas we found devoid of MASPs was purified from plasma according to a previously that MAp44 competitively inhibited binding of MASP-2 to MBL published method, using polyethylene glycol precipitation, affinity chro- and, hence, attenuated lectin pathway activity (3). This activity of matography, and anion-exchange chromatography (28). H-ficolin from H- MAp44 was subsequently confirmed by other investigators, both ficolin/MASP complexes, purified as described (29), was separated from MASPs by two consecutive rounds of size-exclusion chromatography on in vitro (4) and in vivo (20). a Superose 6 HR 10/30 column in a buffer containing EDTA and high ionic A number of possible scenarios allowing transactivation of strength (10 mM Tris, 10 mM EDTA, 1 M NaCl, 0.01% Tween 20 [pH MASP-2 by MASP-1 present themselves. The first possibility, and 7.4]), followed by concentration and buffer exchange to TBS/Ca2+ (10 mM the most simple, would be for MASP-1 and MASP-2 to interact Tris, 5 mM CaCl2 [pH 7.4]) on Vivaspin 6 10,000 MWCO spin concen- directly. However, the MASPs and MAps reportedly only form trators (Sartorius). homodimers, antiparallel tail-to-tail, by virtue of interactions in Abs their CUB–EGF–CUB regions (21). This is somewhat surprising, The reactivities of Abs used in the following sections are illustrated in considering the high homology of this region between MASP-1 Supplemental Fig. 1. and MASP-2 and its identity among MASP-1, MASP-3, and Downloaded from MAp44 (Supplemental Fig. 1). To our knowledge, no data have Analysis of MASP-2 in complex with other MASPs and MAps been presented to document that MASPs cannot, or do not, form in serum heterodimers. The second possibility would be for MASP-1 and FluoroNunc microtiter wells (Nunc) were coated with anti–MASP-2 MASP-2 to associate in the same MBL or ficolin complex. This B-chain Ab [mAb 8B5 (30); Hycult Biotech] at a concentration of 2 mg/ scenario is partially analogous to that of the classical pathway, ml PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4 [pH 7.4]) overnight at room temperature. The wells were with the important difference that a tetramer composed of two http://www.jimmunol.org/ blocked with TBST (10 mM Tris, 140 mM NaCl, 15 mM NaN3 [pH 7.4], heterodimers of C1r and C1s occur in the C1 complex. We re- containing 0.05% v/v Tween 20) and then washed thrice in TBST. To the cently demonstrated that MASP-1 and MASP-2 can indeed form wells were then added a pool of normal human serum (NHS), diluted 5- 2+ co-complexes with MBL and that co-complexes with MBL and/or fold in serum dilution buffer: TBST/Ca (TBST containing 5 mM CaCl2) containing 100 mg/ml each rat Ig (Lampire), mouse Ig (Lampire), bovine ficolins can be detected in serum (9), despite previous suggestions Ig (Lampire), and heat-aggregated human IgG (Beriglobulin; CSL Beh- that this was not the case (22, 23). The third possible scenario is ring; heat-aggregated by incubating the IgG at 56˚C for 30 min, followed that MASP-1 and MASP-2 could be found in distinct complexes by centrifugation at 10,000 3 g and recovery of the supernatant). Serum with MBL and/or ficolins and that such complexes would be dilution buffer alone, as a control, was added to parallel wells. The samples brought into close proximity upon binding to ligand surfaces, as were incubated in wells overnight at 4˚C, and wells were washed thrice with TBST/Ca2+. This was followed by incubation with biotinylated anti– by guest on September 28, 2021 conceptually suggested previously (24). Of note, these scenarios MASP-1 C-terminal Ab (polyclonal Ab rat 3) (31), anti–MASP-3 C-terminal are not necessarily mutually exclusive. In this study, we exam- Ab (mAb 38.12.3) (32), anti-MAp44 C-terminal Ab (mAb 2D5) (32), or ined the first and second scenarios. anti–MASP-2/MAp19 Ab (mAb 6G12) (30), all at 1 mg/ml TBST/Ca2+. We find that coexpression of MASP-1 and MASP-2 results in Following a 2-h incubation at room temperature, the wells were washed thrice with TBST/Ca2+, and europium-labeled streptavidin, 0.1 mg/ml TBST a detectable level of MASP-1/-2 heterodimer. However, we do not containing 25 mM EDTA, was added. After a 1-h incubation and washing detect significant levels of such heterodimers in serum. Presumably, thrice with TBST/Ca2+, the amount of bound europium-streptavidin was the dimers are very stable, and exclusive expression in distinct tissues measured following the addition of enhancement solution (Perkin-Elmer) by may cause significant formation of only homodimers in vivo. In reading time-resolved fluorescence on a VICTOR3 plate reader. contrast, the addition of MBL or ficolins allows the formation of Analysis of heterodimer formation upon coexpression of MASP-1 and MASP-2 co-complexes, as well as the other combi- MASPs and MAps nations of MASPs and MAps. We demonstrate that these com- plexes have a functional role, because co-complexes of MASP-1 and Microtiter wells were coated with either anti-MASP-1/-3/MAp44 Ab (mAb 5F5) (32) or anti–MASP-2 B-chain Ab (8B5), 2 mg/ml PBS. After over- MASP-2 efficiently activate complement. Such co-complexes of night incubation, the wells were blocked with TBST. Supernatants from MASP-1 and MASP-2 are present in serum, and their abundance recombinant expression of MASP-2 alone or MASP-2 coexpressed with varies from individual to individual, influencing the degree of com- MASP-1, MASP-3, or MAp44 were diluted 5-fold in TBST/Ca2+,withor plement activation. Intriguingly, this raises the novel possibility that without 100 mM mannose and with or without 1 mg recombinant MBL/ml. Inactive versions of the MASPs (recombinant catalytically inactive [active- MAp44 inhibits complement, not simply by complete displacement site serine-to-alanine mutant], in the following referred to as rMASP-1i, of MASP-2 from MBL, but by displacement of either MASP-1 or rMASP-2i and rMASP-3i) were used to exclude interference from catalytic MASP-2, disrupting such co-complexes and, hence, impairing activities. Following overnight incubation at 4˚C, wells were washed thrice 2+ transactivation. Indeed, we present evidence supporting this idea. with TBST/Ca . Wells coated with anti–MASP-2 (8B5) were developed with either biotinylated anti–MASP-2 (8B5) or anti–MASP-1/-3/MAp44 (5F5). Wells coated with anti–MASP-1/-3/MAp44 (5F5) were developed Materials and Methods with either biotinylated anti–MASP-2 (8B5) or anti–MASP-2/MAp19 Blood samples (6G12), followed by europium-labeled streptavidin. Blood was obtained from apparently healthy Danish blood donors after Assay for MAp44 and MASP homodimers and higher-order informed consent and according to the requirements of the Helsinki oligomers formed through MBL binding Declaration. Similarly, blood was obtained from a MASP-2–deficient pa- m tient, homozygous for the D120G mutation, which was described previ- Microtiter wells were coated with anti-MAp44 (2D5), 2 g/ml PBS. The ously in another patient (25). wells were blocked with TBST. Supernatants from recombinant expression of the inactive versions of MASP-1 (16.8 mg/ml), MASP-2 (1.5 mg/ml), Statistical analyses MASP-3 (2.6 mg/ml), and MAp44 (7.5 mg/ml) were mixed in a ratio of 1:1 and then diluted 5-fold in TBST/Ca2+, with or without 1 mg recombinant Statistical analyses were performed using GraphPad Prism 6 software. MBL/ml, and incubated for 2 h at room temperature. The samples were 1336 MASP AND MAp CO-COMPLEXES INFLUENCE COMPLEMENT ACTIVATION then added to the microtiter wells and incubated overnight at 4˚C. The NHS pool was diluted 5-fold and then serially 2-fold in serum dilution wells were washed thrice with TBST/Ca2+ and then biotinylated anti– buffer. The serial dilutions and a serum dilution buffer–only control were MASP-1 (rat 3), anti–MASP-3 (38.12.3), anti–MAp44 (2D5), or anti– incubated in wells overnight at 4˚C. The wells were then washed thrice in MASP-2/MAp19 (6G12), all at 1 mg/ml TBST/Ca2+, was added. Follow- TBST/Ca2+, and biotinylated anti–MASP-3 (38:12-3), anti–MASP-2/MAp19 ing incubation for 2 h at room temperature, the wells were again washed (6G12), or anti–MASP-1 (rat 3), at 1 mg/ml TBST/Ca2+, was added. Fol- thrice and developed with europium-labeled streptavidin, as before. lowing incubation for 2 h at room temperature, the wells were washed and developed as before. Measurement of heterodimers in serum Two-hundred microliters of serum from two individuals were centrifuged briefly at 10,000 3 g and passed through a Superose 6 10/30 HR column Four normal sera and a MASP-2–deficient serum were diluted 10-fold in 2+ (GE) in TBS. Fractions of 250 ml were collected in Tween-20–preblocked either TBST/Ca or in a buffer dissociating MASPs and MAps from MBL microtiter plates. A 2-fold dilution series, from 1/5 to 1/80, for each parent and ficolins, TBST/1 M NaCl/10 mM EDTA (TBST added NaCl to 1 M serum, as well as the fractions diluted 1:1 in serum dilution buffer, were total, and 10 mM EDTA). The samples were incubated in anti–MASP-2 tested as before for MAp44–MASP-3 complexes (anti–MAp44 [2D5] Ab (8B5)-coated microtiter wells, and the wells were washed and subse- capture, biotinylated anti–MASP-3 [38:12-3] development) or MASP-2– quently developed with biotinylated anti–MASP-1/-3/MAp44 (5F5), fol- MASP-1 complexes (anti–MASP-2 [8B5] capture, biotinylated anti–MASP-1 lowed by europium-labeled streptavidin, as above. [rat 3] development). Using previously described assays, fractions were Analysis of heterocomplex formation mediated by MBL and also tested for MBL (fractions diluted 1:5) (34), H-ficolin (1:100) (35), L-ficolin (1:5) (35), MASP-2 (1:2) (30), and/or MAp44 (1:4) (32). L-ficolin Analysis of co-complex levels in sera Supernatants from recombinant expression of MAp44 and inactive versions of the MASPs (rMAp44 [7.5 mg/ml], rMASP-3i [2.6 mg/ml], rMASP-2i Microtiter wells were coated with anti–MASP-2 (8B5) or anti–MAp44 [1.5 mg/ml], and rMASP-1i [16.8 mg/ml]) were mixed 1:1 and then with 2- (2D5), at 2 mg/ml PBS, and blocked with TBST. Sera diluted 10-fold in fold dilution series of recombinant MBL or purified L-ficolin in TBST/Ca2+. serum dilution buffer, as well as serum dilution buffer–only control, were Downloaded from Following a 2-h incubation at room temperature, assays for co-complexes added to the wells. Following incubation overnight at 4˚C, the wells were were performed as described above. washed thrice with TBST/Ca2+ and then developed with biotinylated anti– MASP-1 (rat 3, for anti–MASP-2 [8B5] coat) or biotinylated anti–MASP-3 Analysis of heterocomplex formation mediated by H-ficolin (38.12.3, for anti-MAp44 [2D5] coat), at 1 mg/ml TBST/Ca2+. Matched samples of serum, EDTA plasma, heparin plasma, and citrate plasma were Supernatants from recombinant expression of inactive versions of MASP-2 included for three individuals to compare the different sampling methods. (1.5 mg/ml) and MASP-1 (16.8 mg/ml) were mixed 1:1 and then with 2- 2+ http://www.jimmunol.org/ fold dilution series of purified H-ficolin in TBST/Ca . Following a 2-h MBL concentration–dependent formation of co-complexes incubation at room temperature, assays for MASP-2–MASP-1 co-complexes were performed as described above. The oligomerization pattern of the Microtiter wells were coated with anti–MASP-2 (8B5) or anti-MAp44 (2D5), purified H-ficolin was verified by immunoblotting (as described below) at 2 mg/ml PBS, and blocked with TBST. Supernatants from recombinant and developed with in-house biotinylated polyclonal goat anti-human H- expression of inactive forms of MASP-1 (16.8 mg/ml), MASP-2 (1.5 mg/ ficolin Ab (AF2367; R&D Systems). The amount of endogenous MASP-2 ml), MASP-3 (2.6 mg/ml), and MAp44 (7.5 mg/ml) were mixed in a ratio of remaining in the purified preparation was ,0.2% w/w compared with 1:1 and then diluted 5-fold in TBST/Ca2+ containing a 2-fold dilution series H-ficolin. of recombinant MBL, starting at 1 mg/ml. The samples were incubated for 2 h at room temperature and then added to microtiter wells and incubated Ab capture of complexes and analysis by immunoblotting overnight at 4˚C. The wells were washed thrice with TBST/Ca2+ and de-

veloped with biotinylated anti–MASP-1/-3/MAp44 (5F5) for anti–MASP-2 by guest on September 28, 2021 Twenty-four microtiter wells were coated with anti–MASP-2 Ab (8B5), and m (8B5) coat and biotinylated anti–MASP-1 (rat 3) or biotinylated anti– 24 wells were coated with anti–MASP-1/-3/MAp44 Ab (5F5), at 4 g/ml MASP-3 (38.12.3) for anti-MAp44 (2D5) coat, followed by incubation with carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3 [pH 9.6]) and europium-labeled streptavidin and measurement of bound europium. 3 mg/ml PBS, respectively. The wells were blocked with TBST for 1 h at room temperature and then washed thrice with this buffer. A serum pool Analysis of C4 deposition as a function of MASP-1–MASP-2 2+ was diluted 10-fold in TBST/Ca and added to 12 wells for each Ab coat, co-complexes and, in parallel, in the complex-dissociating buffer TBST/1 M NaCl/10 mM EDTA, and added to the remaining 12 wells for each Ab coat, at 100 Microtiter wells were coated either with anti–MASP-1/-3/MAp44 Ab ml/well. The plate was then incubated overnight at 4˚C. After incubation, (5F5), as described previously, or with mannan (a mannose-rich mem- the microtiter wells were emptied and washed thrice with TBST and then brane fraction isolated from Saccharomyces cerevisiae), at 10 mg/ml coating 200 ml TBS was added. Each set of 12 wells was then serially eluted, first buffer. Wild-type zymogen MASP-2, at a final concentration of 160 ng/ml, removing the 200 ml TBS, adding 100 ml SDS-PAGE sample buffer (62.5 was mixed with a dilution series of wild-type zymogen MASP-1 to give mM Tris, 8 M urea, 10% [v/v] glycerol, 3% [w/v] SDS, 0.001% [w/v] final concentrations ranging from 1.1 mg/ml to 2.1 ng/ml and including 0 ng/ bromophenol blue [pH 6.7]) diluted 1:1 with TBS, incubating for 10 min ml. Recombinant MBL was added to the mixtures to a final concentration and then emptying the next well of TBS and transferring the elution buffer of 50 ng/ml TBST/Ca2+. Following incubation for 2 h at room temperature, to this well, repeating until 12 wells had been serially eluted for each the mixtures were added to the anti–MASP-1/-3/MAp44 Ab (5F5)-coated sample. Forty-five microliters of the eluate was loaded per well on Cri- or mannan-coated wells and incubated overnight at 4˚C. After washing terion XT 4–12% gels (Bio-Rad). The gel was run in XT MOPS running thrice with TBST/Ca2+, anti–MASP-1/-3/MAp44 Ab (5F5)-coated wells buffer before being semidry blotted onto a polyvinylidene difluoride were developed with biotinylated anti–MASP-2 (8B5), followed by europium- membrane (Bio-Rad). The membrane was blocked in 0.1% Tween 20 in labeled streptavidin, as described previously. The mannan-coated wells were TBS and then incubated with primary Ab in primary buffer (TBS, 0.05% also washed thrice with TBST/Ca2+ and then purified human C4 (36), at 2 Tween 20, 1 mM EDTA, 1 mg human serum albumin [CSL Behring]/ml, mg/ml B1 buffer (4 mM barbital, 145 mM NaCl, 2 mM CaCl2, and 1 mM and 100 mg human IgG/ml). The membrane was washed, incubated with MgCl2 [pH 7.4]), was added and incubated for 30 min at 37˚C. After secondary Ab in secondary buffer (TBST, no azide, 1 mM EDTA, and 100 washing, in-house biotinylated anti-C4c Ab (clone 162-2; BioPorto), at 0.5 mg human IgG/ml), and washed again before being developed with Super- mg/ml TBST/Ca2+, was added to the wells, incubated for 2 h, and developed Signal West Dura Extended Duration Substrate (Pierce). Images were with europium-labeled streptavidin, as before. The C4 fragment deposition taken using a charge-coupled device camera (LAS-3000; Fuji) and ana- results obtained in separate experiments were normalized according to the lyzed with the Image Analysis Software supplied with the camera. The total level of C4 fragment deposition of each experiment, to be able to pool primary Abs were biotinylated anti–MASP-2/MAp19 (1.3B7; 1 mg/ml) experiments, because the degree of C4 fragment deposition is highly de- (33), followed by HRP-streptavidin (P0397; DAKO) or affinity-purified pendent on small variations in C4 preparation, concentration, and incu- rabbit anti-human-MAp44 (R74B; 1 mg/ml) (3), followed by HRP-goat bation time. anti-rabbit Ig (P0448; DAKO). C4 deposition as a function of serum level of MASP-1–MASP-2 Titration of co-complexes in serum and analysis by co-complexes size-exclusion chromatography Microtiter wells were coated, as previously described, with anti–MASP-1/- Microtiter wells were coated with anti-MAp44 (2D5) or anti-MASP-2 3/MAp44 (5F5). Serum samples, diluted 1:10 in binding buffer (20 mM (8B5), at 2 mg/ml PBS, and then blocked with TBST, as before. An Tris, 1 M NaCl, 10 mM CaCl2, 1 mg human serum albumin/ml, 0.05% The Journal of Immunology 1337

[v/v] Triton X-100 [pH 7.4], containing heat-aggregated human Ig, mouse Ig, rat Ig, and bovine Ig, all at 100 mg/ml), were added to the wells and incubated overnight at 4˚C. After washing, the wells were incubated, in parallel, with C4 for 2 h and developed with biotinylated anti-C4c, as described, or developed with biotinylated anti–MASP-2 (8B5), at 1 mg/ml TBST/Ca2+. MAp44 inhibition of co-complex formation and C4 deposition Microtiter wells were coated with anti–MASP-2 (8B5), at 2 mg/ml PBS, blocked, and washed with TBST as before. Wild-type zymogen MASP-1 was mixed with wild-type zymogen MASP-2 for final concentrations of 60 ng/ml each and then mixed with a dilution series of recombinant MAp44 to give final concentrations ranging from 2.7 mg/ml to 2.6 ng/ml, and in- cluding 0 ng/ml. Recombinant MBL was added to the mixtures to a final concentration of 50 ng/ml. Following incubation for 2 h at room temper- ature, the mixtures were added to the anti–MASP-2 (8B5)-coated wells. FIGURE 1. MASP-2 is found in complex with other MASPs and MAps The wells were developed as described above for deposition of C4 frag- in serum. Capture of serum MASP-2 in microtiter wells (8B5 coat) de- ments, MASP-1, and MAp44. veloped for MASP-1 (rat 3), MASP-3 (38.12.3), MAp44 (2D5), or MASP- Analysis of MAp44 competition with pre-existing co-complexes 2/MAp19 (6G12). Pairwise comparison of 5-fold diluted serum (stippled bars) versus buffer only (white bars). Note that the serum column for in serum MASP-2/MAp19 development has been truncated for clarity. Also note

Serum was diluted 5- or 2.5-fold in serum dilution buffer and then combined that comparisons cannot be made directly between the different complexes, Downloaded from 1:1 with a 2-fold dilution series of purified recombinant MAp44 in serum because the magnitudes of the responses do not reflect the quantities of the dilution buffer, ranging from 4 mg/ml down to 31 ng/ml, and including components, but rather the sensitivities of the detection systems. Data are a serum dilution buffer–only control. The samples were incubated for 24 h mean with SD based on four measurements in two independent experi- end-over-end at 4˚C, added to microtiter wells coated with anti–MASP-1/- ments. The signals for serum versus buffer alone were compared using an 3/MAp44 Ab (5F5; 2 mg/ml PBS, blocked with TBST), and incubated unpaired t test for each analysis. A cutoff a of 0.01 was used, and p values overnight at 4˚C. Following incubation, the wells were washed, and parallel wells were developed with biotinylated anti–MASP-2 Ab (8B5), followed are indicated above the column pairs. http://www.jimmunol.org/ by europium-streptavidin as before, or incubated with purified C4, at 2 mg/ ml B1 buffer, for 2 h at 37˚C. The latter wells were washed and developed with biotinylated anti-C4c Ab (162-2), followed by europium-labeled dependent co-complexes, with the PRM harboring more than streptavidin, as before. one MASP dimer. These two scenarios are not mutually exclusive.

Results Coexpression of MASP-2 with MASP-1, MASP-3, or MAp44 leads to detectable levels of heterodimers We previously reported preliminary data indicating that MASP-1 and MASP-2 may form MBL-dependent co-complexes in vitro, We sought to address the most simple scenario for colocalization as well as that co-complexes of MASP-1 and MASP-2, with MBL, of MASP-1 and MASP-2 in connection with transactivation: het- ficolins, or both, can be detected in serum (9). erodimer formation. We also included MASP-3 and MAp44 in the by guest on September 28, 2021 analysis, thus including all three splice products from MASP1. MASP-2 can be found in complex with each of the other These three have identical dimerization domains (CUB–EGF– MASPs and MAps in serum CUB), but the possibility remained that the configuration of the We first sought to examine whether MASP-2 might also be found in remainder of the molecule could influence dimerization. We also complex with the other MASPs and MAps. We incubated a serum sought to examine the effect of MBL in this scenario. To control pool diluted 5-fold in buffer, or buffer alone, in wells coated with for unwanted cross-linking due to MBL binding to potential car- a specific anti–MASP-2 Ab (8B5), washed the wells, and then bohydrates on the MASPs, we also included a mannose-containing developed them using either an Ab specific for MASP-1 (rat 3), buffer as control. Mannose, being a ligand for MBL, would compete MASP-3 (mAb 38.12.3), or MAp44 (mAb 2D5) or an Ab reacting out any such interaction. with both MASP-2 and MAp19 (mAb 6G12). Of note, the buffers Supernatants from cells expressing MASP-2 alone, MASP-2 and used throughout for experiments with serum included a large MASP-1, MASP-2 and MASP-3, or MASP-2 and MAp44, as well excess of rat Ig, mouse Ig, bovine Ig, and heat-aggregated human as a buffer-only control, were preincubated alone, with mannose, Ig to prevent any heterophilic Ab, contaminating bovine Ig, or with MBL, or with MBL and mannose. The samples were then rheumatoid factor interference [from any human sera, animal sera, applied to microtiter wells coated with either anti–MASP-2 (8B5) polyclonal Abs, or mAbs used (37)]. An overview of the domain or anti–MASP-1/-3/MAp44 (5F5). The MASP-2 capture wells structure of MASPs and MAps, as well as the specificities of the were developed with either the same anti–MASP-2 (8B5) or with Abs used, is shown in Supplemental Fig. 1. MASP-1, MASP-3, anti–MASP-1/-3/MAp44 (5F5). The MASP-1 capture wells were and MAp44 were all found in complex with MASP-2 (Fig. 1). The developed with two different anti–MASP-2 Abs (8B5 and 6G12, signal seen for MASP-2–MASP-1 complexes confirms our pre- the latter is additionally able to recognize MAp19, which is not vious preliminary findings (9). The strong signal seen when de- present in this set-up). veloping with anti–MASP-2/MAp19 (6G12) is expected from Upon capture of MASP-2 in the absence of MBL, there is complexes of MASP-2, but a contribution of co-complex with a negligible signal when developing for MASP-2 for all of the MAp19 cannot be excluded. Importantly, one cannot directly samples, similar to buffer only (Fig. 2A). Presumably, this is be- compare the magnitudes of the responses seen for the different cause the two epitopes present in dimeric MASP-2 are insufficient combinations of Abs used. Nonetheless, it appears that MASP-2 for capture and development using the same Ab (8B5). However, may be found in complex with each of the other MASPs and when adding MBL, there is a marked (3-fold) increase in the MAps in serum. Two possible scenarios present themselves, as signal, indicating MBL-dependent formation of complexes har- outlined in the introduction: MASP-2 might, to some extent, form boring more than one MASP-2 dimer. The signals are similar in heterodimers with the other MASPs and MAps or MASP-2 and the absence and presence of mannose, both with and without the other MASPs and MAps might form MBL- and/or ficolin- MBL. This indicates that the complex formation is not influenced 1338 MASP AND MAp CO-COMPLEXES INFLUENCE COMPLEMENT ACTIVATION by the binding activity of MBL, and mannose, in itself, has no opposed to 5F5 change, likely reflecting a difference in the ca- effect. Still, the possibility remained that the reason for the in- pacity of the coated mAbs to capture Ag. crease in the observed signal in the anti–MASP-2–anti–MASP-2 We conclude that coexpression of MASP-2 with MASP-1, sandwich assay upon addition of MBL was simply a sterical ef- MASP-3, or MAp44 leads to detectable levels of heterodimers fect. One could imagine that both epitopes in the free MASP-2 and that the addition of MBL causes the formation of larger dimer could easily be bound by the capture Ab, preventing bind- heterocomplexes. ing of the developing Ab, whereas in the presence of MBL, sterical MASP and MAp44 homodimers are stable and do not constraints would prevent the capture Ab from binding both epi- interchange upon admixture topes, freeing one for interaction with the developing Ab. The result of capturing MASP-2 and developing for MASP-1, The possibility remained that MASP and MAp homodimers are not MASP-3, or MAp44 is shown in Fig. 2B. As expected, superna- stable entities but are able to dissociate and reassociate, leading to tant from cells expressing MASP-2 alone does not yield an ap- dynamic formation of heterodimers. To examine this, we incubated preciable signal but is similar to buffer only. On the contrary, MAp44 by itself or with MASP-3, MASP-2, or MASP-1, including following coexpression, MASP-1, MASP-3, and MAp44 all ap- a buffer control, all either in the presence or absence of MBL. We pear to have formed heterodimers with MASP-2, because there is added the samples to wells coated with anti–MAp44-specific Ab a signal independent of the presence of MBL. However, when (2D5), washed, and developed for MASP-3, MASP-2, MASP-1, or adding MBL, there is a significant increase in the signal, indi- MAp44. cating MBL-dependent formation of complexes containing more As can be seen from Fig. 3, in the absence of MBL, no signal than one MASP-1–MASP-2 dimer and/or a combination with was detectable for heterocomplexes in any combination, indicat- Downloaded from MASP-1 and MASP-2 homodimers. Again, the observed complex ing that the MASP and MAp44 homodimers are stable and do formation was independent of the ligand-binding activity of MBL, not interchange. Of note, when capturing MAp44 and developing as evidenced by similar signals when including mannose to com- for MAp44, there was a signal markedly higher than background pete out the carbohydrate recognition–domain binding. In this under all conditions in the absence of MBL, although it was not instance, the above-mentioned sterical considerations cannot be statistically significant. In the presence of MBL, MAp44 was able

invoked to explain the observed effect of MBL. to form complexes with MASP-3, MASP-2, and MASP-1, as well http://www.jimmunol.org/ We see a very similar scenario when reversing the assay set- as with itself (Fig. 3). Markedly lower levels of MBL-dependent up, capturing using anti–MASP-1/-3/MAp44 and developing for MAp44–MAp44 complexes were observed in the presence of each MASP-2 using either of two anti–MASP-2 Abs (8B5 in Fig. 2C, of the MASPs (Fig. 3D), indicating that these compete for co- 6G12 in Fig. 2D). Note, however, that the relative magnitude of complex formation. the different complexes detected when capturing with 8B5 as Thus, homodimers, and presumably the heterodimers generated by coexpression, were stable entities that did not interchange once by guest on September 28, 2021

FIGURE 3. MAp44 and MASP dimers are stable and do not interchange FIGURE 2. Cotransfection of MASP-2 with MASP-1, MASP-3, or upon admixture, and MAp44 can form MBL-dependent heterocomplexes MAp44 leads to detectable levels of heterodimers, whereas the addition of with each of the MASPs. (A) Capture of MAp44 (2D5) developed for MBL causes formation of larger heterocomplexes independent of ligand- MASP-3 (38.12.3), using MAp44 (0.75 mg/ml final) mixed 1:1 with cat- binding activity. (A) Capture of recombinant inactive versions of MASP-2, alytically inactive versions of MASP-3 (0.26 mg/ml final), MASP-2 (0.15 MASP-2 coexpressed with MASP-1, MASP-3, or MAp44, or buffer alone mg/ml final), or MASP-1 (1.68 mg/ml final); MAp44 alone (0.75 mg/ml in microtiter wells (8B5), developed for MASP-2 (8B5). Dilution buffer final); or buffer only in the absence (white bars) or presence (black bars) of without MBL (white bars), with 100 mM mannose (horizontal stripes), MBL (1 mg/ml final). Data are mean with SD of four measurements in two with 1 mg recombinant MBL/ml (black bars), or with 1 mg recombinant experiments. Data were analyzed using two-way ANOVA, followed by the MBL/ml and 100 mM mannose (diagonal stripes). Data are mean and SD Dunnett posttest, comparing each column pairwise with buffer control in of duplicates. (B)Asin(A), but capture of MASP-2 (8B5) and develop- either the absence or presence of MBL. (B)Asin(A), but developing for ment for MASP-1/MASP-3/MAp44 (5F5). (C)Asin(A), but capture of MASP-2 (6G12). (C)Asin(A), but developing for MASP-1 (rat 3). (D)As MASP-1/MASP-3/MAp44 (5F5) and development for MASP-2 (8B5). (D) in (A), but developing for MAp44 (2D5). The p values were corrected for As in (A), but capture of MASP-1/MASP-3/MAp44 (5F5) and develop- multiple comparisons, and a cutoff a of 0.05 (*) was used. ****p , ment for MASP-2/MAp19 (6G12). 0.0001. The Journal of Immunology 1339 formed, under the conditions of the assays presented in this study. MAp44 with MASP-1, and MAp44 with MASP-3. Both MBL Again, we saw the formation of MBL-dependent co-complexes of and L-ficolin possessed the capacity to form such co-complexes MASPs and MAp44. (Fig. 5). This indicates that L-ficolin purified from human plasma also harbors multiple binding sites for MASPs and MAps. The Heterodimers are not present in serum at significant levels prominent prozone effect seen for MBL at 1 mg/ml (Fig. 5A, 5B) Having confirmed that MASP dimers are stable entities and that might be explained as an effect of too much MBL compared with heterodimers can be generated by coexpression of MASPs, we MASP-2 and MAp44, and MASP-2 and MASP-3, respectively, proceeded to examine whether heterodimers might be present in because this would cause the formation of MBL complexes with serum. Complexes of MASPs and MAps with MBL and ficolins are only single MASP dimers. known to be calcium dependent and to dissociate in the presence Because H-ficolin is the predominant humoral complement- of EDTA and high ionic strength (33). In contrast, MASP and activating PRM in serum, we also examined the ability of puri- MAp dimers reportedly are not sensitive to EDTA and high ionic fied H-ficolin to generate co-complexes of MASP-1 with MASP-2 strength (21). Using our assay, capturing with anti–MASP-2 (8B5) (Supplemental Fig. 2). and developing with anti–MASP-1/-3/MAp44 (5F5), we examined four sera for the presence of calcium-dependent co-complexes of Pull-down from serum and analysis by immunoblotting MASP-2 with MASP-1/-3/MAp44 (MBL/ficolin-dependent) ver- confirms the presence of calcium-dependent co-complexes sus calcium-independent co-complexes (this would include, but To further evaluate the existence of the co-complexes detected not necessarily be limited to, heterodimers). We included a back- in the solid-phase assays, we performed pull-down from serum, ground control in the form of a MASP-2–deficient serum. In the followed by immunoblotting. We coated anti–MASP-2 (8B5) or Downloaded from 2+ presence of Ca , we saw high signals in the assay in the four anti–MASP-1/-3/MAp44 (5F5) in microtiter wells and then in- normal sera, which were significantly different from the back- cubated with a serum pool either diluted in buffer containing 5 ground signal of the MASP-2–deficient serum (two-way ANOVA, mM calcium (associating conditions) or in buffer containing 10 followed by pairwise comparison with MASP-2–deficient serum mM EDTA and 1 M NaCl (dissociating conditions). After incu- using the Dunnett posttest) (Fig. 4). This agreed well with our bation, the wells were washed and then serially eluted using SDS-

observation of MBL/ficolin-dependent complex formation. Con- PAGE sample buffer. Resulting samples were run on SDS-PAGE, http://www.jimmunol.org/ 2+ versely, in the absence of Ca , serums 1–3 did not give a signal blotted to polyvinylidene difluoride membrane, and then probed above the MASP-2–deficient serum background (p . 0.05 for with anti-MAp44 (R74B) and anti–MASP-2/MAp19 (1.3B7). 2+ each). Meanwhile, serum 4, in the absence of Ca , gave a signal As can be seen from Fig. 6A, when capturing with anti–MASP-2 that was approximately four times lower than in the presence of (8B5) under associating conditions (calcium-containing buffer, 2+ Ca and only slightly higher than background (p , 0.01). Taken lane 1) and probing the blot for MAp44, we saw a clear band cor- together, this indicates that the formation of MASP heterodimers responding to the size of MAp44 (arrow) (the prominent back- is insignificant compared with co-complex formation through in- ground band ∼150 kDa is presumably IgG, either 8B5 eluted from teraction with MBL and ficolins. the capture coat or heterophilic Ab and/or rheumatoid factor from by guest on September 28, 2021 Co-complexes can be formed with MBL, L-ficolin, and H-ficolin the serum pool binding to the former; cross-reactive with the HRP- labeled secondary Ab). On the contrary, we did not see a band when Thus, it appears that MBL- and/or ficolin-dependent co-complex capturing MASP-2 under dissociating conditions (buffer containing formation was the major driving force behind the association of EDTA and high ionic strength, lane 2). This confirmed the exis- different MASPs and MAps, including MASP-1 and MASP-2, in tence of MBL- and/or ficolin-dependent co-complexes of MASP-2 serum. We provided evidence for this above with regard to MBL. and MAp44 in serum, whereas no heterodimers were detected. We next investigated whether similar co-complexes could be Similarly, in Fig. 6B, we pulled down either MASP-2–containing formed with ficolins. As a proof of principle, we compared the (8B5) or MASP-1/-3/MAp44–containing (5F5) complexes under ability of recombinant MBL and purified L-ficolin to generate co- either associating or dissociating conditions and then developed complexes of MASP-2 with MAp44, MASP-2 with MASP-3, for MASP-2/MAp19 (1.3B7). We detected MAp19 in MASP-2 complexes under associating conditions (lane 1), as well as in MASP-1/-3/MAp44 complexes under both associating (lane 3) and, albeit at a much lower level, dissociating conditions (lane 4). This indicates the existence of MBL- and/or ficolin-dependent MASP-2 and MASP-1/-3/MAp44 co-complexes with MAp19 and the potential existence of low levels of MAp19 hetero- dimerized with MASP-1/-3/MAp44. MASP-2 and a presumed degradation fragment of MASP-2 (∼42 kDa) were also detected upon capture of MASP-2 under associating conditions (lane 1). Although the presumed degradation fragment had a molecular size close to that of MAp44, we could exclude that it was MAp44, FIGURE 4. Measurement of heterocomplexes versus heterodimers in because the Ab is specific for MASP-2/MAp19 and because we serum. Four sera (1–4) and a control MASP-2–deficient serum, all diluted did not detect this band after pull-down of MASP-1/-3/MAp44 10-fold, were analyzed by capture in anti–MASP-2 (8B5)-coated micro- complexes (lanes 3 and 4). The fact that we saw only a very titer wells and developed with anti–MASP-1/-3/MAp44 (5F5) under as- weak band for MASP-2 itself under dissociating conditions when sociating (presence of Ca2+; checkered bars) or dissociating (EDTA and pulling down MASP-2 indicates a rather limited sensitivity of this high salt concentration; white bars) conditions. Data are mean with SD of four measurements in two experiments. Data were analyzed by two-way approach, probably also explaining why we did not detect MASP- ANOVA with Dunnett’s posttest, comparing each column pairwise with 2 after pull-down of MASP-1/-3/MAp44. MASP-2–deficient control under either associating or dissociating con- In summary, the results from pull-down and immunoblotting ditions. The p values were corrected for multiple comparisons, and a cutoff served as a further indication that heterocomplexes are indeed a of 0.05 (*) was used. **p , 0.01, ****p , 0.0001. found in serum. 1340 MASP AND MAp CO-COMPLEXES INFLUENCE COMPLEMENT ACTIVATION

FIGURE 5. Co-complexes can be formed with both MBL and L-ficolin. (A) MASP-2 (0.15 mg/ml final concentration) and MAp44 (0.75 mg/ml final concentration) were incubated with 3-fold dilution series (1, 0.25, and 0.063 mg/ml final concentration) of either recombinant MBL or L-ficolin purified from serum and then assayed for co-complex for- mation. MASP-2–MAp44 (8B5–5F5) co-complexes (black bars, MASP-2 + MAp44) as a function of the concentration of each PRM (x-axis). MASP-2 and PRM (horizontally striped bars), MAp44 and PRM (diagonally striped bars), or PRM in buffer only (white bars) were included as controls. Data are mean with SD of duplicates. (B)Asin(A), but for MASP-2–MASP-3 (8B5–5F5) co-complexes (0.15 and 0.26 mg/ml final concentration, respectively). (C)Asin(A), but for MAp44–MASP-1 (2D5–rat 3)

co-complexes (0.75 and 1.68 mg/ml final concen- Downloaded from tration, respectively). (D)Asin(A), but for MAp44– MASP-3 (2D5-38.12.3) co-complexes (0.75 and 0.26 mg/ml final concentration, respectively). http://www.jimmunol.org/

Co-complexes in serum are titratable and colocalize with their titrated out upon serial dilution of serum. We proceeded to analyze presumed constituents on size-exclusion chromatography the migration of such complexes on size-exclusion chromatog- Further analyzing the co-complexes occurring naturally in serum, raphy. As can be seen in Fig. 7B and 7C, MAp44–MASP-3 co- we first titrated them. As can be seen in Fig. 7A, MAp44–MASP- complexes colocalized with MAp44, H-ficolin, and MBL, whereas 3, MASP-2–MASP-2/MAp19, and MASP-2–MASP-1 complexes the MASP-2–MASP-1 co-complexes colocalized with MASP-2, H-ficolin, and L-ficolin. Of note, in Fig. 7C, co-complexes and their constituent MASP/MAps appeared to preferentially colocal-

ize with H-ficolin, less with MBL, and not at all with the higher- by guest on September 28, 2021 oligomeric MBL. However, this may be a consequence of the much higher relative abundance of H-ficolin and L-ficolin com- pared with MBL and higher-oligomeric forms of MBL, which again means that much more MASP/MAp and co-complexes are associated with H-ficolin and L-ficolin. Nonetheless, our results again supported the nature and stability of these co-complexes.

The levels of different co-complexes vary between sera and are normally distributed Because the levels of MBL and ficolins on the one hand, and MASPs and MAps on the other hand, vary significantly between individuals (31), it seemed plausible that so should the levels of the various co-complexes. We found the levels of MAp44–MASP- 3 and MASP-2–MASP-1 co-complexes in normal human sera to be variable, but they conformed to a normal distribution (both data sets passed Kolmogorov–Smirnov, D’Agostino and Pearson om- nibus, and Shapiro–Wilk normality tests) (Fig. 8A, 8B). As described above, the MASP and MAp dimers are stable, whereas MASP and MAp complexes with MBL and ficolins are sensitive to EDTA and high ionic strength. This raised the pos- FIGURE 6. Pull-down of co-complexes from serum and analysis by sibility that, when analyzing co-complex levels in blood, the sam- immunoblotting. (A) Pull-down from serum with anti-MASP-2 (8B5) pling method used might affect the result. To examine this, under associating (calcium-containing buffer; lane 1) or dissociating (high we obtained matched samples of serum, EDTA plasma, heparin salt and EDTA containing buffer; lane 2) conditions. Samples were run plasma, and citrate plasma from three donors and determined the under nonreducing conditions and blot developed for MAp44 (R74B). levels of MAp44–MASP-3 and MASP-2–MASP-1 co-complexes Autocontrast was used to enhance the clarity of this blot. Molecular size markers are indicated on the side. (B) Pull-down from serum with anti– in each. As is evident from Fig. 8C and 8D, the EDTA plasma MASP-2 (8B5; lanes 1 and 2) and anti–MASP-1/-3/MAp44 (5F5, lanes 3 presents a significantly higher level of both of these co-complexes and 4) under associating (lanes 1 and 3) or dissociating (lanes 2 and 4) compared with the other sample types. conditions. Samples were run under nonreducing conditions and blot de- We proceeded to examine further the observed MBL-dependent veloped for MASP-2/MAp19 (1.3B7). co-complex formation in a simplified recombinant system. The Journal of Immunology 1341 Downloaded from

FIGURE 8. The level of different co-complexes varies between different sera, and sampling method impacts the measurement of co-complexes. (A) Measurement of MAp44–MASP-3 co-complexes (capture 2D5–develop http://www.jimmunol.org/ 38.12.3) in sera from 12 apparently healthy Danish donors (NHS). Each symbol represents a different serum; mean and SD are indicated. (B) Measurement of MASP-2–MASP-1 co-complexes (capture 8B5–develop rat 3) in the 12 sera in (A). Each symbol represents a different serum; mean C FIGURE 7. Titration and size-exclusion chromatographic analysis of and SD are indicated. ( ) Measurement of MAp44–MASP-3 co-complexes heterocomplexes in serum. A dilution series of a serum pool was added to in matched serum, EDTA plasma, heparin plasma, and citrate plasma from Ab-coated wells and developed with Ab; the Ab combinations reflect three apparently healthy Danish donors. Each symbol represents a different various co-complexes. (A) Titration of MASP-2–MASP-2/MAp19 co- sample; mean and SD are indicated. Data were analyzed using repeated- complexes (n, capture 8B5–develop 6G12, left y-axis), MAp44–MASP-3 measures one-way ANOVA (p = 0.0006), followed by pairwise compar- co-complexes (d, capture 2D5–develop 38.12.3, right y-axis), and MASP- isons using the Tukey posttest. Multiplicity-adjusted p values are indicated by guest on September 28, 2021 D 2–MASP-1 co-complexes (:, capture 8B5–develop rat 3, right y-axis). for statistically significant differences. ( ) Measurement of MASP-2– Data are mean with SD of duplicates. The experiment was repeated with MASP-1 co-complexes in matched serum, EDTA plasma, heparin plasma, the same results. (B) Superose 6 gel-permeation chromatography of serum and citrate plasma from the same three apparently healthy Danish donors. with TBS as running buffer. Fractions of 250 ml were collected and ana- Each symbol represents a different sample; mean and SD are indicated. lyzed for MASP-2–MASP-1 co-complexes (s, capture 8B5–develop rat 3, Data were analyzed using repeated-measures one-way ANOVA (p = right y-axis), MASP-2 (;, MASP-2 assay, right y-axis), H-ficolin (⬜,H- 0.0076), followed by pairwise comparisons using the Tukey posttest. ficolin assay, left y-axis), and L-ficolin (:, L-ficolin assay, left y-axis). (C) Multiplicity-adjusted p values are indicated for statistically significant Superose 6 gel-permeation chromatography of another serum. Fractions differences. were analyzed for MAp44–MASP-3 co-complexes (s, capture 2D5–develop 38.12.3, right y-axis), MAp44 (;, MAp44 assay, right y-axis), H-ficolin (⬜, H-ficolin assay, left y-axis), and MBL (:, MBL assay, left y-axis). Keeping the concentrations of MASP-2 and MBL constant, we titrated in the amount of MASP-1, measuring in parallel the level of co-complex formed and the amount of C4 deposition on a mannan- The formation of co-complexes is highly dependent on the coated surface. As can be seen in Fig. 10A, there was a congruence relative levels of the constituents between the two curves. Again, the formation of co-complexes We performed experiments in which we mixed a constant amount was highly dependent on the relative levels of the constituents, of MASP-2 with MASP-1, MAp44, or MASP-3, or MAp44 with peaking close to the 1:1 ratio of MASP-1/MASP-2. The C4- MASP-1 or MASP-3, and then titrated the amount of MBL. As deposition curve on mannan was shifted slightly toward favoring can be seen from Fig. 9A and 9B, the amount of co-complexes an excess of MASP-2. Plotting one versus the other indicated detected depended on the concentration of MBL. a close correlation between the level of C4 deposition and the amount of co-complex formed (Fig. 10B), which was confirmed MASP-1–MASP-2 co-complex level correlates with by Spearman correlation analysis (r = +0.82) and a two-tailed p C4-deposition capacity value , 0.0001. We proceeded to examine whether the degree of complement Based on our observations in the recombinant system, we de- activation correlated with the amount of MASP-1–MASP-2 co- cided to examine whether the level of MASP-1–MASP-2 co- complex. For the previous experiments involving recombinant complexes would influence the degree of lectin pathway activa- proteins, we used active-site serine-to-alanine mutant versions of tion in serum. The levels of MASP-1–MASP-2 co-complexes the MASPs to rule out any interference from their proteolytic were measured in four sera. The sera displayed very low, low, activities. For the present experiment, we switched to wild-type intermediate, or high amounts of co-complexes. As we established MASP-1 and MASP-2, produced in their zymogen states, as we in this study, the complexes may be formed with MBL or H-, L-, described previously (26). or M-ficolin. Noting that the level of C4 deposition on mannan is 1342 MASP AND MAp CO-COMPLEXES INFLUENCE COMPLEMENT ACTIVATION

neously, the direct measurement of MASP-2–MAp44 co-complex formation. As can be seen in Fig. 11A, as the amount of MAp44 increases (x-axis), the level of MASP-2–MAp44 complex also increases, leading to a decrease in the level of MASP-2–MASP-1 complex. The functional read-out of this inhibition of activating co-complex is the observed concomitant drop in C4 deposition. Note that the curves for MASP-2–MASP-1 co-complex and MASP-2–MAp44 co-complex cross each other at the point (in- dicated by the dotted line) where there are equal concentrations of MASP-2, MASP-1, and MAp44 (60 ng/ml of each). However, considering the smaller m.w. of MAp44 compared with MASP-1 and MASP-2, this is not entirely equimolar stoichiometry. We proceeded to analyze the ability of MAp44 to compete out pre-existing co-complexes in serum. To this end, we combined serum with dilution series of purified recombinant MAp44, in- cubated the mixture, and added the samples to wells coated with anti–MASP-1/-3/MAp44 Ab (5F5). The wells were incubated and then developed with either biotinylated anti–MASP-2 Ab (8B5),

as before, or with purified C4 and analyzed for C4 fragment de- Downloaded from FIGURE 9. MBL concentration–dependent formation of co-complexes. position. As can be seen in Fig. 11B, when the amount of MAp44 (A) Catalytically inactive MASP-2 (0.15 mg/ml final) was mixed 1:1 with added increased, the level of C4 deposition decreased, despite catalytically inactive MASP-1 (d; 1.68 mg/ml final), MAp44 (:; 0.75 mg/ a near-constant level of MASP-2 bound in the wells. ml final), or catalytically inactive MASP-3 (n; 0.26 mg/ml final) and in- Thus, we conclude that co-complexes of MASP-1 and MASP-2 cubated with recombinant MBL at varying concentrations before assay- are functionally important in activating the lectin pathway and ing for MASP-2–MASP-1/-3/MAp44 co-complex formation (capture 8B5– that MAp44 may competitively inhibit such co-complexes by http://www.jimmunol.org/ develop 5F5). Data are mean and SD based on four measurements in two virtue of its PRM binding, leading to inhibition of complement independent experiments. (B) Recombinant MAp44 (0.75 mg/ml final concentration) was mixed 1:1 with catalytically inactive MASP-1 (1.68 activation. mg/ml final concentration) or MASP-3 (0.26 mg/ml final concentration), incubated with recombinant MBL at varying concentrations, and assayed Discussion for MAp44–MASP-1 co-complex formation (s, capture 2D5–develop rat Following up on our prior observation that MASP-1 and MASP-2 3) or MAp44–MASP-3 co-complex formation (⬜, capture 2D5–develop may form MBL-dependent co-complexes in vitro and that such co- 38.12.3), respectively. Data are mean and SD based on four measurements complexes can be detected in serum (9), in this article we presented in two independent experiments. a more thorough analysis of co-complex formation of the MASPs and MAp44 and extended this to other soluble PRMs. by guest on September 28, 2021 highly dependent on the concentration of MBL in sera, and that Our results indicate that MASP-2 may be found in complex with we have no good ligand surface for all four PRMs, the ability of each of the other MASPs and MAps in serum (Fig. 1). Two possible the sera to deposit C4 was analyzed upon affinity capture in mi- scenarios present themselves: 1) MASP-2 might, to some extent, crotiter wells coated with anti–MASP-1/-3/MAp44 Ab. We pre- form heterodimers with the other MASPs and MAps, or 2) MASP- cluded any interference from the C1 complex by performing the 2 and the other MASPs and MAps might form MBL- and/or serum incubation in a high salt–concentration MBL-binding ficolin-dependent co-complexes, with the PRM harboring more buffer, followed by addition of exogenous purified human C4 than one MASP dimer. As mentioned before, we previously found for the C4 fragment–deposition step, as previously described (38). evidence of the latter, but the two scenarios are not mutually ex- As can be seen in Fig. 10C, there was a very good correlation clusive. It was noted previously by us and other investigators that between the amount of co-complex measured and the level of MASPs and MAps appear to exclusively form homodimers, which C4 deposition observed in this set-up, indicating that such co- is somewhat of a conundrum (39, 40). This may have been based complexes may play an important functional role in serum. largely on misinterpretations of reports that MASP dimers are stable and do not form hetero-oligomers (i.e., higher-order het- MAp44 inhibits complement activation by virtue of disruption erocomplexes of homodimers) (21, 23). However, such obser- of MASP-1–MASP-2 co-complexes vations could hardly be taken as proof that heterodimers do not Our observations regarding the importance of MASP-1–MASP-2 exist. co-complexes raised the novel possibility that MAp44, which we Addressing scenario 1, we report in this article that cotrans- previously found to inhibit lectin pathway activation, does not do fection of MASP-2 with MASP-1, MASP-3, or MAp44 leads to so simply by the complete displacement of MASP-2 from MBL or detectable levels of heterodimers (Fig. 2); however, they are not ficolins, rather it disrupts co-complexes. This more sophisticated detected in serum to any significant extent (Fig. 4). Importantly, mode of inhibition would separate MASP-1 from MASP-2, hence we find that the observed dimers are stable entities (Fig. 3), in preventing transactivation; presumably, it would be more efficient agreement with previous observations (21–23). Thus, combined because displacement of either MASP would be efficacious. To with our observation that such heterodimers can be formed by test our hypothesis, we titrated MAp44 into a fixed amount of coexpression in vitro, it seems plausible that their nonexistence in MASP-1, MASP-2, or MBL. We then assayed, in parallel, the human serum could be caused by mutually exclusive synthesis of degree of C4 deposition, as well as the amounts of MASP-1 and the MASPs and MAps in different cell types and/or populations: MAp44 bound, all on an anti–MASP-2 (8B5) capture coat. This synthesis in one cell (type and/or population) of MASP-1 might set-up ensures a constant level of MASP-2, while allowing for preclude expression of the other MASPs and MAps and result direct measurement of MASP-2–MASP-1 co-complex formation in the generation of (stable) MASP-1 homodimers, and so forth. by determination of the amount of MASP-1 bound and, simulta- Indeed, MASP-1, MASP-3, and MAp44 arise from the MASP1 The Journal of Immunology 1343

FIGURE 10. MASP-1–MASP-2 co-complex level correlates with C4 deposition capacity. (A) Recombi- nantly expressed catalytically active versions of MASP-1 and MASP-2 were mixed at varying stoichiometries (MASP-1 final concentration ranging from 1.1 mg/ml to 2.1 ng/ml and including 0 ng/ml; MASP-2 final con- centration fixed at 160 ng/ml) with a fixed amount of MBL (50 ng/ml final concentration). The level of C4 fragment deposition on a mannan-coated surface (⬜) and the level of MASP-1–MASP-2 co-complexes formed (s; capture 5F5–develop 8B5) were measured in paral- lel. Data are mean and SD of four measurements of C4 fragment deposition in two experiments, normalized according to the total level of C4 fragment deposition observed in each experiment. (B)PlotofC4deposition on mannan as a function of the level of MASP-1–MASP- 2 co-complex measured. This is another representation of the two data sets represented in (A), with a total of 22 XY pairs from the two experiments. Correlation analysis was performed using a Spearman nonparametric test (two-tailed p , 0.0001, Spearman r = +0.82). (C) Plot Downloaded from of C4 fragment deposition capacity on anti–MASP- 1/-3/MAp44 (5F5) capture coat as a function of the MASP-1/-3/MAp44–MASP-2 co-complex levels mea- sured in four sera at 10-fold dilution. The mean and SD based on four measurements of C4 fragment deposition in two experiments are shown for each serum. Corre- http://www.jimmunol.org/ lation analysis was performed using the Pearson para- metric test (two-tailed p = 0.0254, r2 = +0.95).

gene by mutually exclusive splicing, as do MASP-2 and MAp19 indicate that the EDTA used for preparation of EDTA plasma from the MASP2 gene. Although MASP-3 is broadly expressed, may cause dissociation of some of the pre-existing complexes, MASP-1 is expressed exclusively in the liver, and MAp44 is followed by reassociation when the samples are diluted in the produced mainly in heart and liver (3, 32, 41). MASP-2 and calcium-containing sample buffer, in effect scrambling the com- MAp19 are produced in the liver (19). The exact cell types and plexes (Fig. 8). A further inference from this is that the natural by guest on September 28, 2021 subsets are not known. level of co-complex is lower than that resulting from a scrambling, Addressing scenario 2, we demonstrated that the addition of indicating that certain complexes are preferentially formed. This MBL causes the formation of larger heterocomplexes (Figs. 2, 3). could be the result of restricted coexpression in certain cell types Naturally, such co-complex formation would require the existence or populations (e.g., MASP-2 alone with MBL alone, and so forth, of two or more binding sites for a MASP or MAp dimer on each as discussed above). MBL molecule. Indeed, previous observations in the literature Considering the well-established requirement for both calcium indicate that this could well be the case (23). Importantly, the chelation and high ionic strength to disrupt MBL/MASP and MBL/ MBL-dependent co-complex formation that we observe is inde- MAp complexes, it may seem surprising that EDTA alone has pendent of ligand-binding activity. The MASP binding sites in such an effect on the serum co-complexes measured in this study. MBL and ficolins were suggested to be equivalent (42). We further However, our previous work indicated that the interaction of found in this study that MBL, L-ficolin, and H-ficolin all pos- MASPs and MAps with ficolins is rather sensitive to calcium sessed the capacity to form co-complexes (Fig. 5, Supplemental chelation alone. Thus, we observed previously that, in the presence Fig. 2). This indicates that L-ficolin and H-ficolin purified from of calcium, most of the MASP-1, MASP-2, and MAp19 in serum human plasma also harbor multiple binding sites for MASPs emerged on gel-permeation chromatography as large complexes and MAps. Given the structural and ultrastructural homology of that were not associated with MBL, whereas in the presence of MBL, H-ficolin, L-ficolin, and M-ficolin, we believe that M-ficolin EDTA alone, most of these components formed smaller complexes should have the same property. As mentioned previously, when (33). This was before the characterization of the role of ficolins analyzing the existence of co-complexes in serum, we cannot in the lectin pathway; however, in retrospect, this accounts for readily discriminate between co-complexes formed by these dif- the observation at that time that .95% of the total MASPs ferent PRMs. However, we confirmed the existence of the co- and MAp19 found in serum were not complexed with MBL. The complexes detected in the solid-phase assays by performing molar dominance of ficolins over MBL explains the marked effect pull-down from serum, followed by immunoblotting (Fig. 6). of EDTA alone, because ficolins would be expected to drive the Further supporting the nature and stability of these co-complexes, majority of co-complex formation. we found that the co-complexes in serum colocalize with their For the in vitro generation of such co-complexes of different presumed constituents upon analysis by gel-permeation chroma- MASPs and MAps in a clean system, we find a delicate balance of tography (Fig. 7). the constituents. Too little MBL results in little co-complex for- We found that the levels of various co-complexes of MASPs mation, whereas too much MBL has the same effect (Fig. 9). In and MAps vary from individual to individual (Fig. 8), indicating the former situation, there is too little MBL to generate significant the possibility of a functional impact. Importantly, the sampling levels of co-complexes, and/or each MBL is saturated with either method is critical when analyzing co-complexes, because our data MASP/MAp, whereas in the latter scenario, there is so much MBL 1344 MASP AND MAp CO-COMPLEXES INFLUENCE COMPLEMENT ACTIVATION

sists of MBL dimers bound to MASP-1 or MASP-2 homodimers. According to this model, trimers and tetramers of MBL should be able to form complexes containing up to two MASPs. This sce- nario fits well with the results from this study. However, Teillet et al. (5) reported that there was no difference in MASP binding between the two predominant MBL forms in serum, trimer, and tetramer and that these forms bound only a single MASP dimer. Nonetheless, Phillips et al. (43) more recently modeled MASP, C1r, and C1s interactions with MBL and C1q, arriving at two or four binding sites/dimer, again suggesting that tetrameric MBL could harbor two MASP dimers. A further detailed analysis of this subject awaits the purification of defined oligomers of MBL. Even less is known about the oligomer distribution of the ficolins and their relative capacity to bind one or more MASP dimers. In this study, we demonstrated that both L-ficolin and H-ficolin purified from serum are able to support the formation of co- complexes. To our knowledge, we also provide the first indication of a

functional role of such co-complexes in activation of the lectin Downloaded from pathway. We demonstrate in a clean system in vitro that the level of co-complex of MASP-1 and MASP-2 correlates well with the degree of C4 deposition on a mannan surface (Fig. 10A, 10B). We further find a correlation between C4 deposition and MASP-2 in complex with MASP-1/-3/MAp44 in serum (Fig. 10C). Finally,

FIGURE 11. MAp44 can inhibit lectin pathway activation through we demonstrate that MAp44 may inhibit lectin pathway activa- http://www.jimmunol.org/ disruption of co-complexes. (A) MAp44 was titrated into a fixed amount of tion, not simply by brute force displacement of MASP-2 from MASP-1, MASP-2, and MBL (MAp44 final concentrations ranging from MBL, but also by simple disruption of co-complexes of MASP-2 2.7 mg/ml to 2.6 ng/ml and including 0 ng/ml; MASP-1 or MASP-2 at with MASP-1 (Fig. 11). This elegant mode of inhibition allows for 60 ng/ml final concentration, MBL at 50 ng/ml final concentration). The a more potent effect, because the displacement of either MASP-1 levels of C4 fragment deposition (;), MASP-1 bound (s), and MAp44 or MASP-2 attenuates lectin pathway activation. Of note, in the bound (n), all in parallel on 8B5 coat (MASP-2 capture), are expressed as present system we only considered the effect on C4 deposition, functions of the concentration of MAp44. Note that MASP-2 is held which is directly downstream of MASP-2. Hence, the observed constant, and the level of MASP-1 bound is a direct expression of the effect upon displacement of MASP-1 is due solely to the impor-

amount of MASP-2–MASP-1 co-complex formed and similarly for by guest on September 28, 2021 tance of MASP-1 in transactivating MASP-2. However, MASP-1 MAp44 measured and MASP-2–MAp44 co-complex formed. The dotted line indicates the point at which there are equal concentrations of MASP-2, also was found to cleave a significant amount of the C2 required MASP-1, and MAp44 (60 ng/ml each). Data are mean and SD of dupli- for convertase formation in serum (10, 13). Hence, in a system cates. Experiment was repeated with similar results. (B) MAp44 was ti- examining C3 deposition and further downstream points, one trated into serum, and the levels of C4 deposition (;) and MASP-2 (:) would envision the inhibitory role of MAp44 to be even more were determined on an anti–MASP-1/-3/MAp44 (5F5) capture coat. Data significant. are mean and SD of duplicates. Results are shown for 5-fold serum dilu- In conclusion, for MASP-1 to be able to transactivate MASP-2, tion. Similar results were obtained at 10-fold serum dilution. Experiment the two need to colocalize during activation. Three possible sce- was repeated with similar results. narios were presented in the introduction: heterodimers, co- complexes, or cooperation of distinct complexes. In light of the that each has only a single binding site occupied and, hence, no present findings, the first scenario does not seem significant, co-complex is formed. In between these two extremes is a maxi- whereas the second and third scenarios remain viable and are not mum, which should reflect a balanced stoichiometry of the con- necessarily mutually exclusive options. We addressed the second centrations of the two MASPs/MAps and the available binding option in this study, which conceptually parallels that of the C1 sites on MBL. complex, demonstrating that co-complexes are indeed present and The observed curves fit well with the theoretical considerations functional in human serum and that they may be functional targets underlying co-complex formation of two different binding partners of the endogenous natural inhibitor MAp44. with a third molecule harboring multiple binding sites for these binding partners (Figs. 9, 10). However, this is a highly complex Disclosures and nontrivial scenario, because the MBL used is polydisperse The authors have no financial conflicts of interest. and, hence, contains MBL oligomers with different numbers of binding sites for MASPs and MAps, and we do not know these numbers. Some MBL oligomers may have only one binding site References for MASPs and MAps, whereas others have two or more. Several 1. Degn, S. E., J. C. Jensenius, and S. Thiel. 2011. Disease-causing mutations in genes of the complement system. Am. J. Hum. Genet. 88: 689–705. previous studies made progress toward an understanding of the 2. Ricklin, D., G. Hajishengallis, K. Yang, and J. D. Lambris. 2010. Complement: interaction of MASPs with various oligomers of MBL, but the a key system for immune surveillance and homeostasis. Nat. Immunol. 11: 785– subject remains controversial. Chen and Wallis (23) reported that 797. 3. Degn, S. E., A. G. Hansen, R. Steffensen, C. Jacobsen, J. C. Jensenius, and each MASP dimer contains binding sites for two MBL subunits S. Thiel. 2009. MAp44, a human protein associated with pattern recognition and that both sites had to be occupied by subunits from a single molecules of the complement system and regulating the lectin pathway of complement activation. J. Immunol. 183: 7371–7378. MBL oligomer to form a stable complex. Thus, they concluded 4. Skjoedt, M. O., T. Hummelshoj, Y. Palarasah, C. Honore, C. Koch, K. Skjodt, that the smallest functional unit for complement activation con- and P. Garred. 2010. A novel mannose-binding lectin/ficolin-associated protein The Journal of Immunology 1345

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