Complement activation by ligand-driven juxtapositionof discrete pattern recognition complexesSøren 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 andTechnology, Aarhus University, 8000 Aarhus C, Denmark; and cDr. John T. Macdonald Department of Human Genetics, Miller School of Medicine, Universityof 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 forreview April 14, 2014)

Defining mechanisms governing translation of molecular bindingevents into immune activation is central to understanding immunefunction. In the lectin pathway of complement, the pattern recog-nition molecules (PRMs) mannan-binding lectin (MBL) and ficolinscomplexed with the MBL-associated serine proteases (MASP)-1 andMASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 wasrecently found to be the exclusive activator of MASP-2 under phys-iological conditions, yet the predominant oligomeric forms of MBLcarry only a single MASP homodimer. This prompted us to inves-tigate whether activation of MASP-2 by MASP-1 occurs throughPRM-driven juxtaposition on ligand surfaces. We demonstratethat intercomplex activation occurs between discrete PRM/MASPcomplexes. PRM ligand binding does not directly escort the tran-sition of MASP from zymogen to active enzyme in the PRM/MASPcomplex; rather, clustering of PRM/MASP complexes directlycauses activation. Our results support a clustering-based mecha-nism of activation, fundamentally different from the conforma-tional model suggested for the classical pathway of complement.

innate immunity | collectin | inflammation | homeostasis

Complement is a central component of humoral immunity (1).Activation of the classical pathway occurs through ligand bind-

ing of complement component C1q, inducing a conformationalchange in the C1qC1r2C1s2 complex and causing complementcomponent C1r to autoactivate and subsequently activate C1s,which in turn cleaves complement components C4 and C2 (2, 3).The lectin pathway proteins are similar, prompting suggestions ofa similar mode of activation (4, 5). However, important differencesdefy this simple analogy. Five different pattern recognition mole-cules (PRMs)—mannan-binding lectin (MBL); H-, L- and M-fico-lin (also known as Ficolin-3, -2, and -1, respectively); and collectin-kidney 1 (CL-K1)—associate withMBL-associated serine proteases(MASP)-1 and -2 to activate complement. In addition, CL-K1 andthe related collectin-liver 1 (CL-L1) form heteromers that also as-sociate with MASPs and activate complement (6). The PRMs arehighly polydisperse oligomers of homotrimeric subunits, whereasC1q is a hexamer of heterotrimeric subunits. In contrast to theC1r2C1s2 tetramer, which associates with C1q to form the C1complex, MASP homodimers independently associate withPRMs, and the predominant oligomers of MBL in serum carryonly a single MASP homodimer (7, 8). Whereas C1r cleaves onlyC1s, MASP-1 cleaves both MASP-2 and C2, and MASP-2, likeC1s, cleaves C4 and C2 (9–11).MASP-1 andMASP-2 must be brought in close proximity during

activation to cooperate. In circulation they associate with distinctoligomeric forms of MBL, indicating their spatial separation athomeostasis (7). Although MASP-2 is able to autoactivate (12),a role of MASP-1 in activating MASP-2 suggested an intercomplexactivation mechanism as opposed to the intracomplex mechanismof C1 (13, 14). This potential mode of activation was never ex-amined experimentally. The recent demonstration that MASP-1 isthe exclusive activator of MASP-2 under physiological conditions(15, 16) prompted us to investigate whether activation of MASP-2

by MASP-1 occurs through juxtaposition of distinct PRM com-plexes on ligand surfaces. Our results support a clustering-basedmechanism of activation for the lectin pathway, fundamentallydifferent from the classical pathway.

ResultsTetrameric MBL Does Not Allow Formation of Cocomplexes of MASPs,but Supports Complement Activation. Serum MBL is a polydispersemixture of oligomers of a trimeric subunit, the most predominant(∼70% of total) forms being trimers and tetramers (9 and 12polypeptide chains) (7) (Fig. S1 A–C). Previous data indicatedno difference in MASP binding between these two forms andsuggested that they bind only a single MASP homodimer (8). IfMASP-1 and MASP-2 were unable to colocalize in tetrameric orsmaller PRM complexes, and this was prerequisite to comple-ment activation, the function of the most abundant MBL formswould be left unaccounted for.Polydisperse recombinant human MBL was fractionated into

(i) mainly trimers, (ii) mainly tetramers, and (iii) higher-orderoligomers (Fig. 1A and Fig. S1 D and E) and then analyzed forcapacity to support formation of cocomplexes of MASP homo-dimers. Only higher-order oligomers had this ability (Fig. 1 B–E).The nature of these complexes was confirmed based on the re-quirement for calcium and sensitivity to high ionic strength (Fig.S1 F–I). We compared the complement-activating potential of thehighest oligomer able to associate with only a single dimer (tet-ramer) and the lowest oligomer able to associate with two dimers(>tetramer), minimizing differences in ligand avidity. Mannan, apolysaccharide from the cell wall of Saccharomyces cerivisiae, is a

Significance

A salient feature of the immune system is its ability to discrimi-nate self from nonself. We define the molecular mechanismgoverning activation of an ancient and central component: thelectin pathway of complement. The basis is the association of twoproteases in distinct complexes with at least five pattern recog-nition molecules. Clustering of these complexes on ligand surfa-ces allows cross-activation of the proteases, which subsequentlyactivate downstream factors to initiate a proteolytic cascade. Thisis conceptually similar to signaling by cellular receptors and couldbe viewed as cellular signaling turned inside out. Different pat-tern recognition complexes “talk to each other” to coordinateimmune activation, which may impart differential activationbased on recognition of simple vs. complex ligand patterns.

Author contributions: S.E.D. and S.T. designed research; S.E.D., T.R.K., L.J., and A.G.H.performed research; M.T. contributed new reagents/analytic tools; R.T.K. and G.R.A. per-formed structural modeling; S.E.D., T.R.K., J.C.J., and S.T. analyzed data; and S.E.D., T.R.K.,R.T.K., J.C.J., G.R.A., and S.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: sdegn@biomed.au.dk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406849111/-/DCSupplemental.

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strong ligand for MBL. The ability of tetrameric and higher-orderoligomeric MBL to reconstitute complement activation in MBL-deficient human serum on mannan was assayed. Both supportedC4 and C3 fragment deposition (Fig. 1 F and G), indicating thatcolocalization ofMASP-1 andMASP-2 in the sameMBL complexis not required for activation. There was a vast difference in thecapacity of tetrameric and >tetrameric MBL fractions to supportcocomplex formation (Fig. 1 B–E), yet minute amounts ofcocomplexes formed by higher-order oligomeric MBL in the tet-rameric fraction (Fig. 1A) could be initiating activation, sub-sequently propagating through intercomplex activation. Thetrimeric fraction contained mainly trimer, with a significantamount of tetramer and dimer, but undetectable pentamer andhigher oligomers (Fig. S1 D and E). Although it had no capacityfor cocomplex formation (Fig. 1 B–E), it supported complementactivation, albeit to a somewhat lesser extent than the tetramericfraction (Fig. 1 F and G). The lower capacity for activation couldbe explained by (i) the significant amount of dimer, which has noactivity, and (ii) the significantly lower maximal binding capacityand higher dissociation rate constants for binding of carbohydrateligands by trimeric MBL compared with tetrameric MBL (8).

Distinct MBL/MASP-1 andMBL/MASP-2 Complexes Cooperate on LigandSurfaces. To directly examine intercomplex activation of MASPsby juxtaposition of distinct complexes of MBL/MASPs on acti-vating surfaces, tetrameric MBL saturated with either MASP-1 orMASP-2 was assayed for C4 deposition capacity on mannan,

individually or combined. Virtually no cocomplexes were formedwith tetrameric MBL (Fig. 1 B–E), and presaturation of MBLwith either MASP-1 or MASP-2 served to isolate each of the twoMASPs on their own pool of tetrameric MBL.When both MASP-1and MASP-2 were present, the tetrameric MBL mediated C4 ac-tivation (Fig. 2 A–D), indicating intercomplex activation. This didnot 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 hu-man pathogen targeted by MBL (17). C4 deposition on S. aureusdepended on cooperation between tetrameric MBL carryingMASP-1 and tetrameric MBL carrying MASP-2 (Fig. 2E). MBLbinding and C4 deposition was inhibited by mannose or EDTA(Fig. S2). Asking whether cooperation of MASP-1 and MASP-2was a consequence of juxtaposition of tetrameric MBL/MASP-1and tetrameric MBL/MASP-2 on the same surface, rather thansimply an effect of both MASP-1 and MASP-2 being present, weincubated S. aureus with both MBL/MASP-1 and MBL/MASP-2or with either separately, followed by admixture of the two dis-crete populations (Fig. 2F). In the former setup, MBL/MASP-1and MBL/MASP-2 are allowed to bind to the same bacteria,whereas in the latter, half the bacteria carry MBL/MASP-1 andhalf carry MBL/MASP-2. The latter combination was insufficientto support complement activation (Fig. 2G). We concluded thatthe driving force for activation was intercomplex activation be-cause the absence of C4 deposition in the scenario with twodiscrete populations of bacteria with MBL/MASP-1 and MBL/MASP-2 demonstrated that no exchange of complexes or dy-namic formation of cocomplexes occurred.

Distinct PRM/MASP Complexes Cooperate on a Mixed Ligand Surface.The observation of inter–MBL-complex cooperation raised thequestion whether distinct PRM/MASP complexes in general co-operate on complex ligand surfaces. We examined cooperation ofH-ficolin/MASP-1 and MBL/MASP-2 on a ligand surface pre-senting both acetylated groups [acetylated BSA (AcBSA)] bindingH-ficolin and carbohydrate groups (mannan) binding MBL. Fourtypes of PRM/MASP complexes were generated: H-ficolin/MASP-1;MBL/MASP-2; mixed complexes of MASP-1 and MASP-2 withH-ficolin and MBL; and, finally, pregenerated H-ficolin/MASP-1combined with pregenerated MBL/MASP-2 (Fig. 3A). The abilityof these four reagents to support C4 deposition was examined onAcBSA, mannan, or a combined AcBSA and mannan coat. At thesame time, the amount of MASP-1, MASP-2, MBL, and H-ficolinbound, as well as MASP-1 activity (using a synthetic peptidesubstrate, methylsulfonyl-D-Phe-Gly-Arg-AMC, FGR-AMC), wasmeasured. H-ficolin and MBL were detected only on surfacescontaining their cognate ligands (Fig. 3 E and F). When MASP-1was incubated with only H-ficolin, or preincubated with H-ficolinbefore mixing with preincubated MBL/MASP-2, MASP-1 wasdetected only on surfaces containing AcBSA (Fig. 3B). Similarly,MASP-2 was detected only on surfaces containing mannan, whenincubated with MBL only or when preincubated with MBL beforemixing with preincubated H-ficolin/MASP-1 (Fig. 3C). Thus,minimal exchange of MASPs occurred between preformed H-ficolin/MASP-1 and preformed MBL/MASP-2 complexes, uponPRM binding to ligand. When MASP-1, MASP-2, H-ficolin, andMBL were mixed, a blend of complexes resulted. Consequently,bothMASP-1 andMASP-2 were bound on all three surfaces in thissample (Fig. 3 B and C), yielding MASP-1 enzymatic activity andC4 deposition on all three surfaces (Fig. 3 D and G ). H-ficolin/MASP-1 and MBL/MASP-2 samples served as controls; i.e.,MASP-1 enzymatic activity was only on AcBSA surfaces (Fig. 3G),and neither sample yielded C4 deposition on any surface (Fig. 3D).When discrete H-ficolin/MASP-1 and MBL/MASP-2 complexeswere present, full C4 activation was achieved only on the mixedligand surface (Fig. 3D), demonstrating direct cooperation betweendiscrete PRM/MASP complexes.

Fig. 1. Only higher-order oligomeric MBL supports cocomplex formation ofMASPs, but both tetrameric and higher-order oligomeric forms of MBL canreconstitute the lectin pathway in MBL-deficient serum. (A) Silver stain ofrecombinant human MBL (lane 1) and purified fractions containing pre-dominantly trimer (lane 2), tetramer (lane 3), and higher-order oligomericforms (lane 4) of the trimeric subunit. Molecular size markers are indicated(lane 5), as are schematic structures of the oligomeric forms. Note that therecombinant human MBL has an oligomer distribution with predominanceof higher oligomers compared with plasma-derived MBL. (B) Analysis ofcocomplex formation between recombinant MASP-1 and MASP-2 affordedby trimeric, tetrameric, or >tetrameric MBL, measured by capture of com-plexes with anti–MASP-1 (5F5) and development with anti–MASP-2 (8B5). (C)As in B, but for MAp44 (2D5) and MASP-1 (rat 3). (D) As in B, but for MASP-2(8B5) and MASP-3 (5F5). (E) As in B, but for MASP-2 (8B5) and MAp44 (5F5).Note that in B–E the trimeric and tetrameric symbols largely overlap. (F) C4fragment deposition from an MBL-deficient serum as a function of re-constitution with increasing amounts of trimeric, tetrameric, or >tetramericMBL. (G) As in F, but measuring C3 deposition. In F and G, the tetrameric and>tetrameric symbols overlap, as do “no MBL” and “no serum.” (B, C, D, andE) Mean and SD of four measurements in two experiments or (F and G)duplicates from one representative experiment of two.

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In a more physiological scenario, we tested the ability of pre-formedMBL/MASP-1 complexes to reconstitute C4 deposition inserum from an individual with a combined MASP1 gene defectand nonproducing MBL genotype and hence deficient in MASP-1,MASP-3, and MAp44 as well as functional MBL (15). Recentstudies indicated that two collectins related to MBL, CL-K1, andCL-L1, or heteromers of these, can activate complement. How-ever, when the serum was reconstituted with preformed MBL/MASP-1 complexes and C4 deposition was assayed, endogenousCL-K1/CL-L1/MASP-2 complexes were insufficient to cooperatewith exogenous MBL/MASP-1 on the mannan surface (Fig. 3H).Thus, functionally, the serum contained only ficolin/MASP-2 com-plexes before reconstitution with the preformed MBL/MASP-1complexes. On a combined mannan and AcBSA coat, the signalwas markedly increased, demonstrating cooperation of exogenousMBL/MASP-1 and endogenous ficolin/MASP-2 complexes. Se-rum reconstituted with MBL complexed with catalytically inactiveMASP-1 (rMASP-1i, Ser646Ala) and MBL/MASP-1 or MBL/MASP-1i without serum yielded no activation (Fig. 3H).

Intercomplex Cooperation Is the Main Driver of Activation. We re-cently found that colocalization of MASP-1 and MASP-2 inhigher-order oligomeric MBL complexes can drive activation ofcomplement when complexes are bound on a ligand or antibodysurface (18). However, in that study we neither demonstratedintracomplex activation directly nor ruled out activation betweenthese complexes. Therefore, we sought to compare the scenario inwhich both intra- and intercomplex activation of MASP-2 byMASP-1 could occur with the scenario in which only intercomplexactivation could occur. Four types of MBL/MASP complexes weregenerated: polydisperse (unfractionated) MBL saturated withMASP-1; withMASP-2; or with a mixture of MASP-1 andMASP-2;and a mixture of presaturated MBL/MASP-1 with presaturatedMBL/MASP-2. The samples were added to mannan-coated wellsand analyzed for C4 activation and the amounts of MASP-1,MASP-2, andMBL bound (Fig. 4A). In parallel, the samples wereanalyzed for formation of MASP-1–MASP-2 cocomplexes eitherby capture with anti–MASP-1 antibody and development withanti-MASP-2 or vice versa. Comparable amounts of MASP-1 and

MASP-2 were bound on mannan for samples containing MASP-1andMASP-2, respectively (Fig. 4E and F). TheMBLwas saturatedwith MASP, and conditions were optimized to bind comparableamounts of each MASP, so twice as much MBL was bound forsamples containing both MASPs as for samples containing eitherMASP (Fig. 4D). Some cocomplexes formed when preformedMBL/MASP-1 complexes were mixed with preformed MBL/MASP-2 complexes, but to lesser extent than when MBL wasmixed with MASP-1 and MASP-2 (Fig. 4 B and C). C4 cleavingactivity was absent for MBL/MASP-1 complexes alone, whereas itwas intermediate for MBL/MASP-2 complexes alone. Of note,MASP-2 was present at unphysiologically high concentration,comparable to that of MASP-1, explaining its partial self-suffi-ciency. Despite the markedly lower level of MASP-1–MASP-2cocomplexes when preformed MBL/MASP-1 complexes weremixed with preformed MBL/MASP-2 complexes, C4 activationwas indistinguishable from that of MBL complexes formed witha mixture of MASP-1 and MASP-2 (Fig. 4G). This suggested thatthe driving force in activation is the colocalization of MASP-1 andMASP-2, whether by cocomplex formation (higher MBL oligom-ers) or juxtaposition on ligand surfaces (both lower and higheroligomers). This was observed despite the use of MBL containingpredominantly higher-than-physiological oligomers, favoring apotential effect of intracomplex activation.

MASP-1 Activates in a Concentration-Dependent Manner. The obser-vation of juxtapositional activation on ligand surfaces, rather thanintracomplex activation, suggested that the lectin pathway acti-vation mechanism differs from that of the classical pathway. Weanalyzed the autoactivating capacity of MASP-1 in the context ofPRM complexes under various conditions. Our results from theexperiments with activation of discrete MBL/MASP or PRM/MASP complexes indicated that the PRM/MASP complex bind-ing and activation events could be separated in time (Fig. S3). Wedirectly examined the importance of the sequence of bindingevents: preformed MBL/MASP complexes binding to mannanversus MASP binding to MBL already bound to mannan. As-suming a conformational activation mechanism driven by glycanbinding, one would expect that only preformed complexes of

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 twocombined 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 C4fragments 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) TetramericMBL 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 titratedonto 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 (bluecurve). Samples were incubated with purified C4 and analyzed by flow cytometry. Mouse IgG1k isotype control (green curve) and S. aureus without additionof MBL/MASP complexes (orange) were included. Representative of two experiments.

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MBL/MASP could activate. If MBL were already bound tomannan, the conformational drive and binding energy released byligand recognition would dissipate before interaction of MASPwith MBL. However, the two scenarios were equivalent (Fig. 5 A–C), indicating that PRM ligand binding does not directly escortthe MASP from zymogen to active conformation.We asked whether activation of MASP-1 could be simply driven

by clustering, akin to the mechanism governing signaling throughmany cellular receptors. Microtiter wells were coated with differ-ent antibodies toward MASP-1 and then incubated with zymogenMASP-1, and activation of the zymogen was assayed by catalyticactivity toward FGR-AMC. MASP-1 activated in a straightfor-ward concentration-dependent manner on the various antibodycapture coats (Fig. 5D). Two anti-MBL capture coats, MBL di-rectly coated, and MBL bound to mannan also demonstrated aconcentration-dependent activation of MASP-1. However, peramount of MASP-1 bound, the MBL-mannan surface proved amore potent activator (Fig. 5E). We confirmed that substrate cleav-age was a function of the catalytic activity of activated MASP-1by assaying in parallel the activity of WT MASP-1 vs. MASP-1i

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 tocross-linking in solution, with the observation that cross-linking ofMASP-1 in solution by mouse monoclonal anti–MASP-1 anda cross-linking anti-mouse Ig antibody, as well as cross-linking ofMBL/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 resultssuggested a fundamentally different mechanism of activation ofthe lectin pathway from that inferred by analogy with the classicalpathway (3–5). Although it is unlikely that directly coated MBL,MBL captured on either of two different antibody coats, as wellas MBL cross-linked in solution and H-ficolin cross-linked insolution (Fig. 5 D and E and Fig. S4 A–D), could all induce aconformational change similar to ligand binding and hence driveactivation; we sought to examine this possibility. A conforma-tional model would predict that even if MASP-1 had an activity inisolation, it would be inactive when in complex with nonligand-bound MBL and would only assume an active conformation fol-lowing a conformational change in the complex driven by MBL

Fig. 3. H-ficolin/MASP-1 and MBL/MASP-2 cooperate on mixed ligand sur-faces, MASP-1 is able to activate MASP-2, and exogenous MBL/MASP-1cooperates with endogenous H-ficolin/MASP-2 in human serum deficient inMBL 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-2was 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 fragmentdeposition (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 wasreconstituted 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 andSD of four measurements in two experiments.

Fig. 4. Intra- and intercomplex vs. intercomplex-only activation scenarios areequivalent. (A) PolydisperseMBLwas saturated with eitherMASP-1 orMASP-2or a mixture of the two. This does not allow cooperation of MASP-1 andMASP-2(either alone with MBL), allows both intra- and intercomplex activation (MBL +MASP-1 +MASP-2), or allows intercomplex activation only [(MBL +MASP-1) +(MBL+MASP-2)]. Note that the potential for intercomplex activation betweenthe two latter conditions is not significantly different when one considers the3D arrangement of activating complexes on a ligand surface (see Fig. 6D). Hencecomparison of these two allows dissection of the contribution of intracomplexactivation. The samples were incubated in anti–MASP-2–coated (B), anti–MASP-1–coated (C), or mannan-coated (D, E, F, and G) wells. Wells were analyzed forcocomplexes of MASP-2–MASP-1 (8B5–5F5) (B), MASP-1–MASP-2 (5F5–8B5) (C),MBL bound on mannan (D), MASP-1 bound on mannan (E), MASP-2 bound onmannan (F), and C4 deposited on mannan (G). Mean and SD of six measure-ments in three experiments. In B, MBL + M1 and MBL + M2 overlap; in C, allcurves except for MBL +M1 overlap at high dilution; inD, MBL +M1 andMBL +M2, andMBL + (M1 +M2) and (MBL+M1) + (MBL +M2), overlap pairwise; in E,all curves except for MBL + M2 overlap; in F, all curves except for MBL + M1overlap; and in G, MBL + (M1 + M2) and (MBL + M1) + (MBL + M2) overlap.

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binding to ligand. In a recent model (5), MASP-1 was tucked awayinside the cone created by the collagen stems of MBL and henceshould not be accessible for antibody binding. To test this, we eitherincubated MASP-1 alone or preformed complexes of excess tet-rameric MBL with MASP-1 in wells coated with anti–MASP-1antibody directed toward the CCP1 domain. Wells were developedin parallel with MASP-1 substrate and anti–MASP-1 antibodyreacting with the C-terminal part of the SP domain. MASP-1 wasreadily available for capture through CCP1, even when in complexwith MBL, and readily activated (Fig. 6 A and B). A slightlystronger activation seen for free MASP-1 (Fig. 6A) was paralleledby a slightly higher degree of capture (Fig. 6B), likely due to ab-sence of sterical hindrance by MBL. However, during incubationon the antibody capture coat, MASP-1 binding by anti–MASP-1could 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 maskedMASP-1 in the MBL/MASP-1 complex. To assess this, we per-formed kinetic measurements of MASP-1 activation in absence ofthe preincubation step on anti–MASP-1. Activation of freeMASP-1was only marginally higher than that of preformed MBL/MASP-1complexes, indicating that MASP-1 in complex with MBL in solu-tion is largely exposed for antibody capture, supporting the notionthat clustering directly drives activation (Fig. 6C). We ruled out anyinterfering preactivation or catalytic activities (Fig. S5).The most parsimonious explanation for the superiority of

ligand-bound MBL in activation of MASP-1 is that mannan pro-vides a roughly planar ligand pattern, which orients MBL/MASPcomplexes and places the catalytic domains at a similar distancefrom the surface, facilitating interaction of neighboringMASPs. Acomplete molecular model of the core LPS layer of Pseudomonasaeruginosa has been simulated. Sugar groups recognizable byMBLare exposed terminally in a near-planar and dense glycan layer(19). This core layer can be modified by less abundant O-antigenglycosylations, lipid rafts, and embedded proteins, introducingnonplanarity, but this may be compensated by the flexibility andpolydispersity of MBL. It therefore seems a reasonable assump-tion that neighboringMBL/MASP complexes are oriented roughlyin a plane. We recently described a molecular model for

tetrameric MBL in a 1:1 complex with a MASP homodimer andthe corresponding MBL/MASP-2–C4 complex (20). In thesemodels, MBL carbohydrate recognition domains are containedwithin a diameter of 240 Å, whereas the two catalytic sites in theprotease domains of a MASP dimer are separated by 290 Å.Therefore, the catalytic sites are well accessible for their very largesubstrates C4, C4bC2, or a MASP protease domain on a neigh-boring zymogen MBL/MASP complex (Fig. 6D). Our modelsuggests that each MBL/MASP complex could be surroundedby several other complexes acting either as MASP activators orin C4/C4bC2 cleavage. Based on their relative abundance (TableS1), almost all C4-cleaving MASP-2 complexes will havea C4bC2-cleaving MASP-1 complex as the closest neighbormaking substrate channeling between complexes efficient.

DiscussionWe demonstrated here that discrete PRM/MASP complexes co-operate on ligand surfaces. Trimers and tetramers of MBL sub-units associate with only one dimer of either MASP-1 or MASP-2,whereas higher oligomers associate with two dimers and maycontain both MASP-1 and MASP-2. We saw no difference in ac-tivation between the situation where MASP-1 and MASP-2 werecolocalized in the same MBL complex vs. the situation where theywere localized in distinct complexes. The majority (∼70%) ofserumMBL is trimeric or tetrameric, implying that intracomplexactivation in higher-order oligomers is not a major driver ofphysiological activation. This agrees with observations that,in blood, MASP-1 and MAp19 are predominantly associatedwith trimeric MBL, whereas MASP-2 and MASP-3 associatepreferentially with tetrameric MBL (7). Thus, intercomplexactivation appears a prerequisite for physiological activation of

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Fig. 5. Activation of MBL/MASP-1 onmannan is independent of sequence ofbinding events, and MASP-1 activates in a concentration-dependent manner.MASP-1 was added to mannan-coated wells preincubated with MBL, orMASP-1 preincubated with MBL was added to mannan-coated wells. At thesame time, MBL bound (A), MASP-1 bound (B), and MASP-1 activity (C) wasmeasured. Mean and SD are from duplicate measurements in one of twoexperiments. Note that the curves overlap. (D) A dilution series of MASP-1was added to wells coated with either of three anti–MASP-1 antibodies (5F5,4H2, and 1E2), and at the same time the level of MASP-1 activity and MASP-1bound (detecting with a fourth anti–MASP-1 antibody) was measured. Rep-resentative experiment of two, with minor variations in setup. Note that thecurves overlap. (E) As in D, but for wells containing mannan-MBL, directlycoated MBL (rMBL), or MBL bound to either of two anti-MBL antibodies(131-01 or 93C). Representative experiment of two, with minor differencesin setup. Note that rMBL, 131–01, and 93C largely overlap.

Fig. 6. Model for intercomplex activation of PRM/MASP. (A) MASP-1 enzy-matic activity as a function of time following capture of MBL/MASP-1 complexesor free MASP-1 in wells coated with anti–MASP-1 CCP1. (B) Measurement ofMASP-1 captured in wells in the setup presented in A. Representative experi-ment from two repeats with minor differences in setup. (C) MASP-1 enzymaticactivity as function of time during capture of MBL/MASP-1 complexes or freeMASP-1 in wells coated with anti–MASP-1 CCP1 (similar to A, but withoutpreincubation on antibody capture coat and washing unboundMASP-1 away).Representative experiment from two repeats with minor differences in setup.Note that at early time points the curves overlap, and for B the curves largelyoverlap throughout. (D) Structure-based model of MBL transactivating com-plexes on a glycan surface. Seven MBL tetramers (green) harboring MASP-1dimers (blue) clustered around one MBL tetramer (gray) binding a MASP-2 di-mer (dark red) placed on an atomic model of the P. aeruginosa core LPS layer.Cylindrical rods at top of theMBLmolecules represent their N-terminal disulfidebridged regions, for which no structural information is available.

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the cascade, yet we cannot exclude that intracomplex activationin higher-order oligomers also plays a role. We recently dem-onstrated that colocalization of MASP-1 and -2 in higher-orderoligomeric MBL complexes can drive activation when com-plexes are bound on a ligand or antibody surface (18). In light ofthe present observations, we conclude that activation by hetero-complexes is a specific instance of the general principle that wedemonstrate here, i.e., juxtaposition- and concentration-dependentactivation. The possibility remains that some physiological ligandsurfaces may allow efficient binding of single PRM/MASP com-plexes with spacing between complexes precluding intercomplexactivation.Our findings contrast with a previous report that isolated zy-

mogen MASP-2 can bind C4, but when zymogen MASP-2 is as-sociated with MBL, C4 binding is diminished (11). Chen andWallis (11) estimated the affinity of zymogen MASP-2 for C4around 7 μM, suggesting that MASP-2 would have to be seques-tered in the MBL complex to prevent spontaneous activation ofcomplement. However, recombinant constitutive zymogen MASP-2in complex withMBL onmannan is unable to activate C4 (15), andwhen MASP-1 is inhibited in serum, preventing activation ofMASP-2, no activation of C4 occurs (16).We propose a mechanistically simple mode of operation that

integrates the available data and fits the primordial origins of thelectin pathway: the PRMs (i) concentrate the MASPs on ligandsurfaces, (ii) orient the MASPs relative to the surface and eachother, and (iii) juxtapose MASP-1 and MASP-2. This serves to(i) tip the balance of MASPs and inhibitors to favor MASP ac-tivation, (ii) allow intercomplex activation of MASP-1, and (iii)facilitate intercomplex activation of MASP-2 by MASP-1. Thissimple mode of activation could represent the primordial originsof the elaborate conformational mechanism proposed for higher-order oligomeric MBL/MASP complexes and the C1 complex.

Materials and MethodsTo assay C4 and C3 fragment deposition based on defined oligomers of MBL,purified tetrameric and >tetrameric MBL serially diluted 1.5-fold was added toMBL-deficient serum, incubated 10 min at room temperature, then added tomannan-coated wells. After incubation for 1 h at 37 °C, wells were developedwith anti-C3c or -C4c. To analyze C4 deposition by tetrameric MBL, MASP-1 orMASP-2 was incubated with tetrameric MBL overnight at 4 °C (∼0.5 μg MASPper 1 μg MBL, i.e., close to a 1:1 stoichiometry). MBL/MASP-1 and MBL/MASP-2complexes (100 ng/mLMASP-saturatedMBL), or a 1:1 mixture of the two, wereserially diluted twofold and then added to mannan-coated wells. Wells wereincubated for 4 h on ice to bindMBL and then received 2 μg/mL purified humanC4 and were incubated for 30 min at 37 °C and then developed for C4 (mAb162–02), MASP-1 (4H2), MASP-2 (8B5), and MBL (131-1). MBL binding and C4deposition on S. aureuswas analyzed by incubating strainWOOD for 2 h at 4 °Cwith 10 ng/mL tetrameric recombinantMBL (rMBL) presaturatedwith zymogen

MASP-2 and decreasing concentrations of tetrameric rMBL presaturated withzymogen MASP-1. C4 was added, and samples were incubated for 30 min at37 °C and thenanalyzed for C4deposition andMBL binding. Formixed samples,5 ng/mL tetrameric rMBL presaturated with zymogen MASP-2 or 5 ng/mL tet-rameric rMBL presaturatedwith zymogenMASP-1 were added to bacteria, andthese were subsequently mixed before incubation with C4. Alternatively, bac-teria were incubated simultaneously with both.

For the mixed ligand assays, purified H-ficolin or rMBL was added torecombinant MASP-1 and MASP-2 supernatants (each 0.5 μg/mL) to a finalconcentration of 1 μg/mL. Similarly, rMBL and purified H-ficolin were added toa mixture of the two supernatants to a final concentration of 0.5 μg/mL eachand then incubated overnight at 4 °C. The preformed H-ficolin/MASP-1,MBL/MASP-2, the (H-ficolin + MBL)+(MASP-1 + MASP-2), or a 1:1 mixture ofH-ficolin/MASP-1 with MBL/MASP-2 were added to microtiter wells coated withmannan, AcBSA (Sigma), or both. Wells were developed with C4 and anti-C4c(162-02); with FGR-AMC substrate (methylsulfonyl-D-Phe-Gly-Arg-AMC; Ameri-can Diagnostica), 0.1 mM, incubated at 37 °C and fluorescence read over time;or with anti-MBL (131-01); anti–H-ficolin (4H5); anti–MASP-1 (4H2); or anti–MASP-2 (8B5). To analyze cooperation of MBL/MASP-1 and ficolin/MASP-2in serum, MBL presaturated with rMASP-1 or rMASP-1(S646A) was added tobuffer or MBL and MASP-1/-3/MAp44–deficient serum (15). Samples wereadded to wells coated with mannan, AcBSA, or both and then developed forC4 deposition. For comparison of intracomplex versus intercomplex scenar-ios, microtiter wells were coated with mannan, 8B5, or 5F5. RecombinantMASP-1 (1 μg/mL), recombinant MASP-2 (1 μg/mL), or a 1:1 mixture of the two,were incubated overnight at 4 °C with 1 μg/mL rMBL (2:1 stoichiometry). Serialdilutions of the MBL/MASP-1, the MBL/MASP-2, or the MBL/(MASP-1+MASP-2)samples were added to wells. Alternatively, MBL/MASP-1 and MBL/MASP-2were mixed 1:1, serially diluted, and added to wells. After incubation for 4 h onice, mannan-coated wells were developed for MASP-1 (4H2), MASP-2 (8B5), oradded C4 and developed for deposition. The 5F5-coated wells were developedwith anti–MASP-2 (8B5) and the 8B5-coated wells with anti–MASP-1 (4H2).

To analyze dependence of activation on sequence of binding events, rMBLwas added at 1 or 0 μg/mL to mannan-coated wells. A twofold dilution series ofMASP-1 was added to wells that received MBL. MBL was added to a twofolddilution series of MASP-1 to a final concentration of 1 μg/mL and was sub-sequently added to wells that had not received MBL. Wells were analyzed forMASP-1 enzymatic activity, MASP-1 (4H2), or MBL (131-01). To examine theconcentration dependence of activation, microtiter wells were coated witha threefold dilution series of anti–MASP-1 antibodies, 5F5, 4H2, or 1E2, andthen 100 ng/mL MASP-1 was added. Parallel wells were developed for MASP-1activity and MASP-1 bound (2B11). Wells were also coated with a twofold di-lution series of mannan or rMBL or two different anti-MBL antibodies (131-01or 93C). To the 131–01- and 93C-coated wells was added 10 μg rMBL/mL. Wellswere developed for MASP-1 activity and MASP-1 bound (4H2). Additionaldetails are in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Professor Uffe Skov Sørensen for theS. aureus. S.E.D. was supported by the Carlsberg and Lundbeck Foundations.S.T. was supported by The Danish Council for Independent Research, MedicalSciences and by the Lundbeck Foundation.

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