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).

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