Complement Activation by Ligand-Driven Juxtaposition of Discrete Pattern Recognition Complexes

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

Søren E. Degna,1, Troels R. Kjaera, Rune T. Kidmoseb, Lisbeth Jensena, Annette G. Hansena, Mustafa Tekinc, Jens C. Jenseniusa, Gregers R. Andersenb, and Steffen Thiela

aDepartment of Biomedicine, Health, Aarhus University, 8000 Aarhus C, Denmark; bDepartment of Molecular Biology and Genetics, Science and Technology, Aarhus University, 8000 Aarhus C, Denmark; and cDr. John T. Macdonald Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL 33136

Edited by Douglas T. Fearon, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, and approved August 12, 2014 (received for review April 14, 2014)

Defining mechanisms governing translation of molecular binding by MASP-1 occurs through juxtaposition of distinct PRM com- events into immune activation is central to understanding immune plexes on ligand surfaces. Our results support a clustering-based function. In the lectin pathway of complement, the pattern recog- mechanism of activation for the lectin pathway, fundamentally nition molecules (PRMs) mannan-binding lectin (MBL) and ficolins different from the classical pathway. complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was Results recently found to be the exclusive activator of MASP-2 under phys- Tetrameric MBL Does Not Allow Formation of Cocomplexes of MASPs, iological conditions, yet the predominant oligomeric forms of MBL but Supports Complement Activation. Serum MBL is a polydisperse carry only a single MASP homodimer. This prompted us to inves- mixture of oligomers of a trimeric subunit, the most predominant tigate whether activation of MASP-2 by MASP-1 occurs through (∼70% of total) forms being trimers and tetramers (9 and 12 PRM-driven juxtaposition on ligand surfaces. We demonstrate polypeptide chains) (7) (Fig. S1 A–C). Previous data indicated that intercomplex activation occurs between discrete PRM/MASP no difference in MASP binding between these two forms and complexes. PRM ligand binding does not directly escort the tran- suggested that they bind only a single MASP homodimer (8). If MASP-1 and MASP-2 were unable to colocalize in tetrameric or

sition of MASP from zymogen to active enzyme in the PRM/MASP INFLAMMATION complex; rather, clustering of PRM/MASP complexes directly smaller PRM complexes, and this was prerequisite to comple- IMMUNOLOGY AND causes activation. Our results support a clustering-based mecha- ment activation, the function of the most abundant MBL forms nism of activation, fundamentally different from the conforma- would be left unaccounted for. tional model suggested for the classical pathway of complement. Polydisperse recombinant human MBL was fractionated into (i) mainly trimers, (ii) mainly tetramers, and (iii) higher-order oligomers (Fig. 1A and Fig. S1 D and E) and then analyzed for innate immunity | collectin | inflammation | homeostasis capacity to support formation of cocomplexes of MASP homodimers. Only higher-order oligomers had this ability (Fig. 1 B–E). omplement is a central component of humoral immunity (1). The nature of these complexes was confirmed based on the re- CActivation of the classical pathway occurs through ligand bind- quirement for calcium and sensitivity to high ionic strength (Fig. ing of complement component C1q, inducing a conformational S1 F–I). We compared the complement-activating potential of the change in the C1qC1r2C1s2 complex and causing complement highest oligomer able to associate with only a single dimer (tet- component C1r to autoactivate and subsequently activate C1s, ramer) and the lowest oligomer able to associate with two dimers which in turn cleaves complement components C4 and C2 (2, 3). (>tetramer), minimizing differences in ligand avidity. Mannan, a The lectin pathway proteins are similar, prompting suggestions of polysaccharide from the cell wall of Saccharomyces cerivisiae,isa a similar mode of activation (4, 5). However, important differences defy this simple analogy. Five different pattern recognition mole- Significance cules (PRMs)—mannan-binding lectin (MBL); H-, L- and M-ficolin (also known as Ficolin-3, -2, and -1, respectively); and collectin- A salient feature of the immune system is its ability to discrimi- kidney 1 (CL-K1)—associate with MBL-associated serine proteases nate self from nonself. We define the molecular mechanism (MASP)-1 and -2 to activate complement. In addition, CL-K1 and governing activation of an ancient and central component: the the related collectin-liver 1 (CL-L1) form heteromers that also as- lectin pathway of complement. The basis is the association of two sociate with MASPs and activate complement (6). The PRMs are proteases in distinct complexes with at least five pattern recog- highly polydisperse oligomers of homotrimeric subunits, whereas nition molecules. Clustering of these complexes on ligand surfa- C1q is a hexamer of heterotrimeric subunits. In contrast to the ces allows cross-activation of the proteases, which subsequently C1r2C1s2 tetramer, which associates with C1q to form the C1 activate downstream factors to initiate a proteolytic cascade. This complex, MASP homodimers independently associate with is conceptually similar to signaling by cellular receptors and could PRMs, and the predominant oligomers of MBL in serum carry be viewed as cellular signaling turned inside out. Different pat- only a single MASP homodimer (7, 8). Whereas C1r cleaves only tern recognition complexes “talk to each other” to coordinate C1s, MASP-1 cleaves both MASP-2 and C2, and MASP-2, like immune activation, which may impart differential activation C1s, cleaves C4 and C2 (9–11). based on recognition of simple vs. complex ligand patterns. MASP-1 and MASP-2 must be brought in close proximity during activation to cooperate. In circulation they associate with distinct Author contributions: S.E.D. and S.T. designed research; S.E.D., T.R.K., L.J., and A.G.H. oligomeric forms of MBL, indicating their spatial separation at performed research; M.T. contributed new reagents/analytic tools; R.T.K. and G.R.A. per- homeostasis (7). Although MASP-2 is able to autoactivate (12), formed structural modeling; S.E.D., T.R.K., J.C.J., and S.T. analyzed data; and S.E.D., T.R.K., a role of MASP-1 in activating MASP-2 suggested an intercomplex R.T.K., J.C.J., G.R.A., and S.T. wrote the paper. activation mechanism as opposed to the intracomplex mechanism The authors declare no conflict of interest. of C1 (13, 14). This potential mode of activation was never ex- This article is a PNAS Direct Submission. amined experimentally. The recent demonstration that MASP-1 is 1To whom correspondence should be addressed. Email: [email protected]. the exclusive activator of MASP-2 under physiological conditions This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (15, 16) prompted us to investigate whether activation of MASP-2 1073/pnas.1406849111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406849111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 individually or combined. Virtually no cocomplexes were formed with tetrameric MBL (Fig. 1 B–E), and presaturation of MBL with either MASP-1 or MASP-2 served to isolate each of the two MASPs on their own pool of tetrameric MBL. When both MASP-1 and MASP-2 were present, the tetrameric MBL mediated C4 activation (Fig. 2 A–D), indicating intercomplex activation. This did not preclude dynamic formation of cocomplexes on a minor con- taminant of higher-order oligomeric MBL upon ligand binding, which could trigger activation. To approach the physiological scenario, we analyzed activation on the surface of Staphylococcus aureus, a clinically relevant human pathogen targeted by MBL (17). C4 deposition on S. aureus depended on cooperation between tetrameric MBL carrying MASP-1 and tetrameric MBL carrying MASP-2 (Fig. 2E). MBL binding and C4 deposition was inhibited by mannose or EDTA (Fig. S2). Asking whether cooperation of MASP-1 and MASP-2 was a consequence of juxtaposition of tetrameric MBL/MASP-1 and tetrameric MBL/MASP-2 on the same surface, rather than simply an effect of both MASP-1 and MASP-2 being present, we incubated S. aureus with both MBL/MASP-1 and MBL/MASP-2 or with either separately, followed by admixture of the two discrete populations (Fig. 2F). In the former setup, MBL/MASP-1 Fig. 1. Only higher-order oligomeric MBL supports cocomplex formation of and MBL/MASP-2 are allowed to bind to the same bacteria, MASPs, but both tetrameric and higher-order oligomeric forms of MBL can whereas in the latter, half the bacteria carry MBL/MASP-1 and reconstitute the lectin pathway in MBL-deficient serum. (A) Silver stain of half carry MBL/MASP-2. The latter combination was insufficient recombinant human MBL (lane 1) and purified fractions containing pre- to support complement activation (Fig. 2G). We concluded that dominantly trimer (lane 2), tetramer (lane 3), and higher-order oligomeric forms (lane 4) of the trimeric subunit. Molecular size markers are indicated the driving force for activation was intercomplex activation be- (lane 5), as are schematic structures of the oligomeric forms. Note that the cause the absence of C4 deposition in the scenario with two recombinant human MBL has an oligomer distribution with predominance discrete populations of bacteria with MBL/MASP-1 and MBL/ of higher oligomers compared with plasma-derived MBL. (B) Analysis of MASP-2 demonstrated that no exchange of complexes or dy- cocomplex formation between recombinant MASP-1 and MASP-2 afforded namic formation of cocomplexes occurred. by trimeric, tetrameric, or >tetrameric MBL, measured by capture of complexes with anti–MASP-1 (5F5) and development with anti–MASP-2 (8B5). (C) Distinct PRM/MASP Complexes Cooperate on a Mixed Ligand Surface. As in B, but for MAp44 (2D5) and MASP-1 (rat 3). (D)AsinB, but for MASP-2 The observation of inter–MBL-complex cooperation raised the (8B5) and MASP-3 (5F5). (E)AsinB, but for MASP-2 (8B5) and MAp44 (5F5). question whether distinct PRM/MASP complexes in general co- Note that in B–E the trimeric and tetrameric symbols largely overlap. (F)C4 operate on complex ligand surfaces. We examined cooperation of fragment deposition from an MBL-deficient serum as a function of re- H-ficolin/MASP-1 and MBL/MASP-2 on a ligand surface pre- > constitution with increasing amounts of trimeric, tetrameric, or tetrameric senting both acetylated groups [acetylated BSA (AcBSA)] binding MBL. (G)AsinF, but measuring C3 deposition. In F and G, the tetrameric and > “ ” “ ” H-ficolin and carbohydrate groups (mannan) binding MBL. Four tetrameric symbols overlap, as do no MBL and no serum. (B, C, D, and types of PRM/MASP complexes were generated: H-ficolin/MASP-1; E) Mean and SD of four measurements in two experiments or (F and G) duplicates from one representative experiment of two. MBL/MASP-2; mixed complexes of MASP-1 and MASP-2 with H-ficolin and MBL; and, finally, pregenerated H-ficolin/MASP-1 combined with pregenerated MBL/MASP-2 (Fig. 3A). The ability strong ligand for MBL. The ability of tetrameric and higher-order of these four reagents to support C4 deposition was examined on oligomeric MBL to reconstitute complement activation in MBL- AcBSA, mannan, or a combined AcBSA and mannan coat. At the deficient human serum on mannan was assayed. Both supported same time, the amount of MASP-1, MASP-2, MBL, and H-ficolin C4 and C3 fragment deposition (Fig. 1 F and G), indicating that bound, as well as MASP-1 activity (using a synthetic peptide colocalization of MASP-1 and MASP-2 in the same MBL complex substrate, methylsulfonyl-D-Phe-Gly-Arg-AMC, FGR-AMC), was is not required for activation. There was a vast difference in the measured. H-ficolin and MBL were detected only on surfaces capacity of tetrameric and >tetrameric MBL fractions to support containing their cognate ligands (Fig. 3 E and F). When MASP-1 cocomplex formation (Fig. 1 B–E), yet minute amounts of was incubated with only H-ficolin, or preincubated with H-ficolin cocomplexes formed by higher-order oligomeric MBL in the tet- before mixing with preincubated MBL/MASP-2, MASP-1 was rameric fraction (Fig. 1A) could be initiating activation, sub- detected only on surfaces containing AcBSA (Fig. 3B). Similarly, sequently propagating through intercomplex activation. The MASP-2 was detected only on surfaces containing mannan, when trimeric fraction contained mainly trimer, with a significant incubated with MBL only or when preincubated with MBL before amount of tetramer and dimer, but undetectable pentamer and mixing with preincubated H-ficolin/MASP-1 (Fig. 3C). Thus, higher oligomers (Fig. S1 D and E). Although it had no capacity minimal exchange of MASPs occurred between preformed H- for cocomplex formation (Fig. 1 B–E), it supported complement ficolin/MASP-1 and preformed MBL/MASP-2 complexes, upon activation, albeit to a somewhat lesser extent than the tetrameric PRM binding to ligand. When MASP-1, MASP-2, H-ficolin, and fraction (Fig. 1 F and G). The lower capacity for activation could MBL were mixed, a blend of complexes resulted. Consequently, be explained by (i) the significant amount of dimer, which has no both MASP-1 and MASP-2 were bound on all three surfaces in this activity, and (ii) the significantly lower maximal binding capacity sample (Fig. 3 B and C), yielding MASP-1 enzymatic activity and and higher dissociation rate constants for binding of carbohydrate C4 deposition on all three surfaces (Fig. 3 D and G ). H-ficolin/ ligands by trimeric MBL compared with tetrameric MBL (8). MASP-1 and MBL/MASP-2 samples served as controls; i.e., MASP-1 enzymatic activity was only on AcBSA surfaces (Fig. 3G), Distinct MBL/MASP-1 and MBL/MASP-2 Complexes Cooperate on Ligand and neither sample yielded C4 deposition on any surface (Fig. 3D). Surfaces. To directly examine intercomplex activation of MASPs When discrete H-ficolin/MASP-1 and MBL/MASP-2 complexes by juxtaposition of distinct complexes of MBL/MASPs on acti- were present, full C4 activation was achieved only on the mixed vating surfaces, tetrameric MBL saturated with either MASP-1 or ligand surface (Fig. 3D), demonstrating direct cooperation between MASP-2 was assayed for C4 deposition capacity on mannan, discrete PRM/MASP complexes.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406849111 Degn et al. Downloaded by guest on September 30, 2021 Fig. 2. Tetrameric MBL permits cooperation of MASP-1 and MASP-2 on a ligand surface. Complexes of tetrameric MBL with MASP-1, MASP-2, or the two combined were incubated on a mannan surface. In parallel was measured the level of MBL bound (A), MASP-1 bound (B), MASP-2 bound (C), and C4 fragments deposited after addition of purified C4 (D). Mean and SD are from four measurements in two experiments. In A and D, MBL/MASP-1 and MBL/ MASP-2 overlap; in B, MBL/MASP-1 and MBL/MASP-1+MBL/MASP-2 overlap; and in C, MBL/MASP-2 and MBL/MASP-1+MBL/MASP-2 overlap. (E) Tetrameric MBL was saturated with MASP-1 or MASP-2. A fixed amount of MBL saturated with MASP-2 was added to S. aureus. MBL saturated with MASP-1 was titrated onto the bacteria. The bacteria were incubated with purified C4 and analyzed for C4 deposition by flow cytometry. Representative of three experiments. (F and G) MASP-1–saturated MBL and MASP-2–saturated MBL were incubated with S. aureus together (red curve) or separately followed by admixture (blue

curve). Samples were incubated with purified C4 and analyzed by flow cytometry. Mouse IgG1k isotype control (green curve) and S. aureus without addition INFLAMMATION of MBL/MASP complexes (orange) were included. Representative of two experiments. IMMUNOLOGY AND

In a more physiological scenario, we tested the ability of pre- MASP-2 were bound on mannan for samples containing MASP-1 formed MBL/MASP-1 complexes to reconstitute C4 deposition in and MASP-2, respectively (Fig. 4 E and F). The MBL was saturated serum from an individual with a combined MASP1 gene defect with MASP, and conditions were optimized to bind comparable and nonproducing MBL genotype and hence deficient in MASP-1, amounts of each MASP, so twice as much MBL was bound for MASP-3, and MAp44 as well as functional MBL (15). Recent samples containing both MASPs as for samples containing either studies indicated that two collectins related to MBL, CL-K1, and MASP (Fig. 4D). Some cocomplexes formed when preformed CL-L1, or heteromers of these, can activate complement. How- MBL/MASP-1 complexes were mixed with preformed MBL/ ever, when the serum was reconstituted with preformed MBL/ MASP-2 complexes, but to lesser extent than when MBL was MASP-1 complexes and C4 deposition was assayed, endogenous mixed with MASP-1 and MASP-2 (Fig. 4 B and C). C4 cleaving CL-K1/CL-L1/MASP-2 complexes were insufficient to cooperate activity was absent for MBL/MASP-1 complexes alone, whereas it with exogenous MBL/MASP-1 on the mannan surface (Fig. 3H). was intermediate for MBL/MASP-2 complexes alone. Of note, Thus, functionally, the serum contained only ficolin/MASP-2 com- MASP-2 was present at unphysiologically high concentration, plexes before reconstitution with the preformed MBL/MASP-1 comparable to that of MASP-1, explaining its partial self-suffi- complexes. On a combined mannan and AcBSA coat, the signal ciency. Despite the markedly lower level of MASP-1–MASP-2 was markedly increased, demonstrating cooperation of exogenous cocomplexes when preformed MBL/MASP-1 complexes were MBL/MASP-1 and endogenous ficolin/MASP-2 complexes. Se- mixed with preformed MBL/MASP-2 complexes, C4 activation rum reconstituted with MBL complexed with catalytically inactive was indistinguishable from that of MBL complexes formed with MASP-1 (rMASP-1i, Ser646Ala) and MBL/MASP-1 or MBL/ a mixture of MASP-1 and MASP-2 (Fig. 4G). This suggested that MASP-1i without serum yielded no activation (Fig. 3H). the driving force in activation is the colocalization of MASP-1 and MASP-2, whether by cocomplex formation (higher MBL oligom- Intercomplex Cooperation Is the Main Driver of Activation. We re- ers) or juxtaposition on ligand surfaces (both lower and higher cently found that colocalization of MASP-1 and MASP-2 in oligomers). This was observed despite the use of MBL containing higher-order oligomeric MBL complexes can drive activation of predominantly higher-than-physiological oligomers, favoring a complement when complexes are bound on a ligand or antibody potential effect of intracomplex activation. surface (18). However, in that study we neither demonstrated intracomplex activation directly nor ruled out activation between MASP-1 Activates in a Concentration-Dependent Manner. The obser- these complexes. Therefore, we sought to compare the scenario in vation of juxtapositional activation on ligand surfaces, rather than which both intra- and intercomplex activation of MASP-2 by intracomplex activation, suggested that the lectin pathway acti- MASP-1 could occur with the scenario in which only intercomplex vation mechanism differs from that of the classical pathway. We activation could occur. Four types of MBL/MASP complexes were analyzed the autoactivating capacity of MASP-1 in the context of generated: polydisperse (unfractionated) MBL saturated with PRM complexes under various conditions. Our results from the MASP-1; with MASP-2; or with a mixture of MASP-1 and MASP-2; experiments with activation of discrete MBL/MASP or PRM/ and a mixture of presaturated MBL/MASP-1 with presaturated MASP complexes indicated that the PRM/MASP complex bind- MBL/MASP-2. The samples were added to mannan-coated wells ing and activation events could be separated in time (Fig. S3). We and analyzed for C4 activation and the amounts of MASP-1, directly examined the importance of the sequence of binding MASP-2, and MBL bound (Fig. 4A). In parallel, the samples were events: preformed MBL/MASP complexes binding to mannan analyzed for formation of MASP-1–MASP-2 cocomplexes either versus MASP binding to MBL already bound to mannan. As- by capture with anti–MASP-1 antibody and development with suming a conformational activation mechanism driven by glycan anti-MASP-2 or vice versa. Comparable amounts of MASP-1 and binding, one would expect that only preformed complexes of

Degn et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 and a constitutive zymogen form of MASP-1 (MASP-1 Arg448Gln), and the activation on anti-MBL coats was dependent on MBL (Fig. S4A). The finding was generalized from surface clustering to cross-linking in solution, with the observation that cross-linking of MASP-1 in solution by mouse monoclonal anti–MASP-1 and a cross-linking anti-mouse Ig antibody, as well as cross-linking of MBL/MASP-1 or H-ficolin/MASP-1 by anti-MBL and anti–H- ficolin, respectively, could also drive activation (Fig. S4 B–D).

Structural Model for Activation of the Lectin Pathway. Our results suggested a fundamentally different mechanism of activation of the lectin pathway from that inferred by analogy with the classical pathway (3–5). Although it is unlikely that directly coated MBL, MBL captured on either of two different antibody coats, as well as MBL cross-linked in solution and H-ficolin cross-linked in solution (Fig. 5 D and E and Fig. S4 A–D), could all induce a conformational change similar to ligand binding and hence drive activation; we sought to examine this possibility. A conformational model would predict that even if MASP-1 had an activity in isolation, it would be inactive when in complex with nonligand- bound MBL and would only assume an active conformation following a conformational change in the complex driven by MBL

Fig. 3. H-ficolin/MASP-1 and MBL/MASP-2 cooperate on mixed ligand surfaces, MASP-1 is able to activate MASP-2, and exogenous MBL/MASP-1 cooperates with endogenous H-ficolin/MASP-2 in human serum deficient in MBL and MASP-1. (A) MASP-1 was preincubated with H-ficolin (M1 + H-fic); MASP-2 was preincubated with MBL (M2 + MBL); or MASP-1 and MASP-2 was mixed with MBL and H-ficolin (M1 + M2 + H-fic + MBL). The H-fic + M1, MBL + M2, or these two mixed together [(M1 + H-fic) + (M2 + MBL)] and the (M1 + M2 + H-fic + MBL) sample were added to AcBSA, mannan, or AcBSA + mannan-coated wells. MASP-1 bound (B), MASP-2 bound (C), C4 fragment deposition (D), MBL bound (E), H-ficolin bound (F), and MASP-1 activity (G) were measured. Mean and SD of six measurements in three experiments. (H) Human serum deficient in MASP-1/-3/MAp44 and functional MBL was reconstituted with preformed MBL/MASP-1 or catalytically inactive MBL/ MASP-1(Ser646Ala) complexes, incubated in wells coated with AcBSA, mannan, or AcBSA + mannan, and C4 deposition was measured. Mean and SD of four measurements in two experiments.

MBL/MASP could activate. If MBL were already bound to mannan, the conformational drive and binding energy released by ligand recognition would dissipate before interaction of MASP with MBL. However, the two scenarios were equivalent (Fig. 5 A– Fig. 4. Intra- and intercomplex vs. intercomplex-only activation scenarios are equivalent. (A) Polydisperse MBL was saturated with either MASP-1 or MASP-2 C), indicating that PRM ligand binding does not directly escort or a mixture of the two. This does not allow cooperation of MASP-1 and MASP-2 the MASP from zymogen to active conformation. (either alone with MBL), allows both intra- and intercomplex activation (MBL + We asked whether activation of MASP-1 could be simply driven MASP-1 + MASP-2), or allows intercomplex activation only [(MBL + MASP-1) + by clustering, akin to the mechanism governing signaling through (MBL + MASP-2)]. Note that the potential for intercomplex activation between many cellular receptors. Microtiter wells were coated with differ- the two latter conditions is not significantly different when one considers the ent antibodies toward MASP-1 and then incubated with zymogen 3D arrangement of activating complexes on a ligand surface (see Fig. 6D). Hence MASP-1, and activation of the zymogen was assayed by catalytic comparison of these two allows dissection of the contribution of intracomplex activity toward FGR-AMC. MASP-1 activated in a straightfor- activation. The samples were incubated in anti–MASP-2–coated (B), anti–MASP- ward concentration-dependent manner on the various antibody 1–coated (C), or mannan-coated (D, E, F,andG) wells. Wells were analyzed for – – – – capture coats (Fig. 5D). Two anti-MBL capture coats, MBL di- cocomplexes of MASP-2 MASP-1 (8B5 5F5) (B), MASP-1 MASP-2 (5F5 8B5) (C), rectly coated, and MBL bound to mannan also demonstrated a MBL bound on mannan (D), MASP-1 bound on mannan (E), MASP-2 bound on mannan (F), and C4 deposited on mannan (G). Mean and SD of six measure- concentration-dependent activation of MASP-1. However, per ments in three experiments. In B,MBL+ M1 and MBL + M2 overlap; in C,all amount of MASP-1 bound, the MBL-mannan surface proved a curves except for MBL + M1 overlap at high dilution; in D,MBL+ M1 and MBL + more potent activator (Fig. 5E). We confirmed that substrate cleav- M2, and MBL + (M1 + M2) and (MBL + M1) + (MBL + M2), overlap pairwise; in E, age was a function of the catalytic activity of activated MASP-1 all curves except for MBL + M2 overlap; in F, all curves except for MBL + M1 by assaying in parallel the activity of WT MASP-1 vs. MASP-1i overlap; and in G,MBL+ (M1 + M2) and (MBL + M1) + (MBL + M2) overlap.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1406849111 Degn et al. Downloaded by guest on September 30, 2021 MBL bound MASP-1 activity vs. MASP-1 bound tetrameric MBL in a 1:1 complex with a MASP homodimer and A 3 C MASP-1 activity E ) – ) -6 2.0 Mannan-MBL the corresponding MBL/MASP-2 C4 complex (20). In these -3 2.5 ) rMBL 2 -3 1.5 2.0 131-01 models, MBL carbohydrate recognition domains are contained 93C MBL 1 1.0 1.5 within a diameter of 240 Å, whereas the two catalytic sites in the 0.5 1.0 (counts/s * 10 protease domains of a MASP dimer are separated by 290 Å. 0 0.5 0 10 100 0 (counts/s/min * 10 0 10 100 Therefore, the catalytic sites are well accessible for their very large (counts/s/min * 10 0 MASP-1 added (ng/ml) 4 5 6 Initial rate enzymatic activity MASP-1 added (ng/ml) 10 10 10

Initial rate enzymatic activity substrates C4, C4bC2, or a MASP protease domain on a neigh- MASP-1 bound MASP-1-1 bound (counts/s)) B 2.0 D MASP-1 activity vs. MASP-1 bound boring zymogen MBL/MASP complex (Fig. 6D). Our model ) -6 suggests that each MBL/MASP complex could be surrounded 1.5 1.0 )

-2 5F5 1.0 0.8 4H2 byseveralothercomplexesactingeitherasMASPactivatorsor 1E2 0.5 MASP-1 0.5 0.6 in C4/C4bC2 cleavage. Based on their relative abundance (Table

(counts/s * 10 (counts/s 0.4 0 0 S1), almost all C4-cleaving MASP-2 complexes will have 0 10 100 0.2 105 106 a C4bC2-cleaving MASP-1 complex as the closest neighbor MASP-1 added (ng/ml)

(counts/s/min * 10 0 MASP-1 bound (counts/s) 103 104 105 106 107 making substrate channeling between complexes efficient. Mannan-MBL - MASP-1 wt Initial rate enzymatic activity Mannan - MBL/MASP-1 wt MASP-1 bound (counts/s) Discussion Fig. 5. Activation of MBL/MASP-1 on mannan is independent of sequence of We demonstrated here that discrete PRM/MASP complexes co- binding events, and MASP-1 activates in a concentration-dependent manner. operate on ligand surfaces. Trimers and tetramers of MBL sub- MASP-1 was added to mannan-coated wells preincubated with MBL, or MASP-1 preincubated with MBL was added to mannan-coated wells. At the units associate with only one dimer of either MASP-1 or MASP-2, same time, MBL bound (A), MASP-1 bound (B), and MASP-1 activity (C) was whereas higher oligomers associate with two dimers and may measured. Mean and SD are from duplicate measurements in one of two contain both MASP-1 and MASP-2. We saw no difference in ac- experiments. Note that the curves overlap. (D) A dilution series of MASP-1 tivation between the situation where MASP-1 and MASP-2 were was added to wells coated with either of three anti–MASP-1 antibodies (5F5, colocalized in the same MBL complex vs. the situation where they 4H2, and 1E2), and at the same time the level of MASP-1 activity and MASP-1 were localized in distinct complexes. The majority (∼70%) of bound (detecting with a fourth anti–MASP-1 antibody) was measured. Rep- serum MBL is trimeric or tetrameric, implying that intracomplex resentative experiment of two, with minor variations in setup. Note that the activation in higher-order oligomers is not a major driver of curves overlap. (E)AsinD, but for wells containing mannan-MBL, directly physiological activation. This agrees with observations that, coated MBL (rMBL), or MBL bound to either of two anti-MBL antibodies in blood, MASP-1 and MAp19 are predominantly associated

(131-01 or 93C). Representative experiment of two, with minor differences with trimeric MBL, whereas MASP-2 and MASP-3 associate INFLAMMATION – IMMUNOLOGY AND in setup. Note that rMBL, 131 01, and 93C largely overlap. preferentially with tetrameric MBL (7). Thus, intercomplex activation appears a prerequisite for physiological activation of binding to ligand. In a recent model (5), MASP-1 was tucked away inside the cone created by the collagen stems of MBL and hence should not be accessible for antibody binding. To test this, we either incubated MASP-1 alone or preformed complexes of excess tetrameric MBL with MASP-1 in wells coated with anti–MASP-1 antibody directed toward the CCP1 domain. Wells were developed in parallel with MASP-1 substrate and anti–MASP-1 antibody reacting with the C-terminal part of the SP domain. MASP-1 was readily available for capture through CCP1, even when in complex with MBL, and readily activated (Fig. 6 A and B). A slightly stronger activation seen for free MASP-1 (Fig. 6A) was paralleled by a slightly higher degree of capture (Fig. 6B), likely due to absence of sterical hindrance by MBL. However, during incubation on the antibody capture coat, MASP-1 binding by anti–MASP-1 could drive a shift in equilibrium between free MASP-1 and MBL/ MASP-1 or a shift in equilibrium between spontaneously dynami- cally exposed MASP-1 in the MBL/MASP-1 complex and masked MASP-1 in the MBL/MASP-1 complex. To assess this, we performed kinetic measurements of MASP-1 activation in absence of the preincubation step on anti–MASP-1. Activation of free MASP-1 was only marginally higher than that of preformed MBL/MASP-1 complexes, indicating that MASP-1 in complex with MBL in solution is largely exposed for antibody capture, supporting the notion that clustering directly drives activation (Fig. 6C). We ruled out any interfering preactivation or catalytic activities (Fig. S5). Fig. 6. Model for intercomplex activation of PRM/MASP. (A) MASP-1 enzy- The most parsimonious explanation for the superiority of matic activity as a function of time following capture of MBL/MASP-1 complexes ligand-bound MBL in activation of MASP-1 is that mannan pro- or free MASP-1 in wells coated with anti–MASP-1 CCP1. (B)Measurementof vides a roughly planar ligand pattern, which orients MBL/MASP MASP-1 captured in wells in the setup presented in A. Representative experi- complexes and places the catalytic domains at a similar distance ment from two repeats with minor differences in setup. (C) MASP-1 enzymatic activity as function of time during capture of MBL/MASP-1 complexes or free from the surface, facilitating interaction of neighboring MASPs. A – complete molecular model of the core LPS layer of Pseudomonas MASP-1 in wells coated with anti MASP-1 CCP1 (similar to A, but without aeruginosa preincubation on antibody capture coat and washing unbound MASP-1 away). has been simulated. Sugar groups recognizable by MBL Representative experiment from two repeats with minor differences in setup. are exposed terminally in a near-planar and dense glycan layer Note that at early time points the curves overlap, and for B the curves largely (19). This core layer can be modified by less abundant O-antigen overlap throughout. (D) Structure-based model of MBL transactivating com- glycosylations, lipid rafts, and embedded proteins, introducing plexes on a glycan surface. Seven MBL tetramers (green) harboring MASP-1 nonplanarity, but this may be compensated by the flexibility and dimers (blue) clustered around one MBL tetramer (gray) binding a MASP-2 di- polydispersity of MBL. It therefore seems a reasonable assump- mer (dark red) placed on an atomic model of the P. aeruginosa core LPS layer. tion that neighboring MBL/MASP complexes are oriented roughly Cylindrical rods at top of the MBL molecules represent their N-terminal disulfide in a plane. We recently described a molecular model for bridged regions, for which no structural information is available.

Degn et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 the cascade, yet we cannot exclude that intracomplex activation MASP-2 and decreasing concentrations of tetrameric rMBL presaturated with in higher-order oligomers also plays a role. We recently dem- zymogen MASP-1. C4 was added, and samples were incubated for 30 min at onstrated that colocalization of MASP-1 and -2 in higher-order 37 °C and then analyzed for C4 deposition and MBL binding. For mixed samples, oligomeric MBL complexes can drive activation when com- 5 ng/mL tetrameric rMBL presaturated with zymogen MASP-2 or 5 ng/mL tet- plexes are bound on a ligand or antibody surface (18). In light of rameric rMBL presaturated with zymogen MASP-1 were added to bacteria, and the present observations, we conclude that activation by hetero- these were subsequently mixed before incubation with C4. Alternatively, bac- complexes is a specific instance of the general principle that we teria were incubated simultaneously with both. demonstrate here, i.e., juxtaposition- and concentration-dependent For the mixed ligand assays, purified H-ficolin or rMBL was added to recombinant MASP-1 and MASP-2 supernatants (each 0.5 μg/mL) to a final activation. The possibility remains that some physiological ligand μ surfaces may allow efficient binding of single PRM/MASP com- concentration of 1 g/mL. Similarly, rMBL and purified H-ficolin were added to a mixture of the two supernatants to a final concentration of 0.5 μg/mL each plexes with spacing between complexes precluding intercomplex and then incubated overnight at 4 °C. The preformed H-ficolin/MASP-1, activation. MBL/MASP-2, the (H-ficolin + MBL)+(MASP-1 + MASP-2), or a 1:1 mixture of Our findings contrast with a previous report that isolated zy- H-ficolin/MASP-1 with MBL/MASP-2 were added to microtiter wells coated with mogen MASP-2 can bind C4, but when zymogen MASP-2 is as- mannan, AcBSA (Sigma), or both. Wells were developed with C4 and anti-C4c sociated with MBL, C4 binding is diminished (11). Chen and (162-02); with FGR-AMC substrate (methylsulfonyl-D-Phe-Gly-Arg-AMC; Ameri- Wallis (11) estimated the affinity of zymogen MASP-2 for C4 can Diagnostica), 0.1 mM, incubated at 37 °C and fluorescence read over time; around 7 μM, suggesting that MASP-2 would have to be seques- or with anti-MBL (131-01); anti–H-ficolin (4H5); anti–MASP-1 (4H2); or anti– tered in the MBL complex to prevent spontaneous activation of MASP-2 (8B5). To analyze cooperation of MBL/MASP-1 and ficolin/MASP-2 complement. However, recombinant constitutive zymogen MASP-2 in serum, MBL presaturated with rMASP-1 or rMASP-1(S646A) was added to in complex with MBL on mannan is unable to activate C4 (15), and buffer or MBL and MASP-1/-3/MAp44–deficient serum (15). Samples were when MASP-1 is inhibited in serum, preventing activation of added to wells coated with mannan, AcBSA, or both and then developed for MASP-2, no activation of C4 occurs (16). C4 deposition. For comparison of intracomplex versus intercomplex scenar- We propose a mechanistically simple mode of operation that ios, microtiter wells were coated with mannan, 8B5, or 5F5. Recombinant integrates the available data and fits the primordial origins of the MASP-1 (1 μg/mL), recombinant MASP-2 (1 μg/mL), or a 1:1 mixture of the two, lectin pathway: the PRMs (i) concentrate the MASPs on ligand were incubated overnight at 4 °Cwith1μg/mL rMBL (2:1 stoichiometry). Serial surfaces, (ii) orient the MASPs relative to the surface and each dilutions of the MBL/MASP-1, the MBL/MASP-2, or the MBL/(MASP-1+MASP-2) other, and (iii) juxtapose MASP-1 and MASP-2. This serves to samples were added to wells. Alternatively, MBL/MASP-1 and MBL/MASP-2 (i) tip the balance of MASPs and inhibitors to favor MASP ac- were mixed 1:1, serially diluted, and added to wells. After incubation for 4 h on tivation, (ii) allow intercomplex activation of MASP-1, and (iii) ice, mannan-coated wells were developed for MASP-1 (4H2), MASP-2 (8B5), or added C4 and developed for deposition. The 5F5-coated wells were developed facilitate intercomplex activation of MASP-2 by MASP-1. This with anti–MASP-2 (8B5) and the 8B5-coated wells with anti–MASP-1 (4H2). simple mode of activation could represent the primordial origins of the elaborate conformational mechanism proposed for higher- To analyze dependence of activation on sequence of binding events, rMBL wasaddedat1or0μg/mL to mannan-coated wells. A twofold dilution series of order oligomeric MBL/MASP complexes and the C1 complex. MASP-1 was added to wells that received MBL. MBL was added to a twofold dilution series of MASP-1 to a final concentration of 1 μg/mL and was sub- Materials and Methods sequently added to wells that had not received MBL. Wells were analyzed for To assay C4 and C3 fragment deposition based on defined oligomers of MBL, MASP-1 enzymatic activity, MASP-1 (4H2), or MBL (131-01). To examine the purified tetrameric and >tetrameric MBL serially diluted 1.5-fold was added to concentration dependence of activation, microtiter wells were coated with MBL-deficient serum, incubated 10 min at room temperature, then added to a threefold dilution series of anti–MASP-1 antibodies, 5F5, 4H2, or 1E2, and mannan-coated wells. After incubation for 1 h at 37 °C, wells were developed then 100 ng/mL MASP-1 was added. Parallel wells were developed for MASP-1 with anti-C3c or -C4c. To analyze C4 deposition by tetrameric MBL, MASP-1 or activity and MASP-1 bound (2B11). Wells were also coated with a twofold di- MASP-2 was incubated with tetrameric MBL overnight at 4 °C(∼0.5 μgMASP lution series of mannan or rMBL or two different anti-MBL antibodies (131-01 per 1 μg MBL, i.e., close to a 1:1 stoichiometry). MBL/MASP-1 and MBL/MASP-2 or 93C). To the 131–01- and 93C-coated wells was added 10 μg rMBL/mL. Wells complexes (100 ng/mL MASP-saturated MBL), or a 1:1 mixture of the two, were were developed for MASP-1 activity and MASP-1 bound (4H2). Additional serially diluted twofold and then added to mannan-coated wells. Wells were details are in SI Materials and Methods. incubated for 4 h on ice to bind MBL and then received 2 μg/mL purified human C4 and were incubated for 30 min at 37 °C and then developed for C4 (mAb ACKNOWLEDGMENTS. We thank Professor Uffe Skov Sørensen for the – 162 02), MASP-1 (4H2), MASP-2 (8B5), and MBL (131-1). MBL binding and C4 S. aureus. S.E.D. was supported by the Carlsberg and Lundbeck Foundations. deposition on S. aureus wasanalyzedbyincubatingstrainWOODfor2hat4°C S.T. was supported by The Danish Council for Independent Research, Medical with 10 ng/mL tetrameric recombinant MBL (rMBL) presaturated with zymogen Sciences and by the Lundbeck Foundation.

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