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Structural and Functional Aspects of Complement Activation by Mannose-binding Protein
Immunobiol. (2002) 205, pp. 433 – 445 © 2002 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol
The Lectin-Pathway of Complement Activation: MBL, other Collectins and Ficolins Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK
Structural and Functional Aspects of Complement Activation by Mannose-binding Protein RUSSELL WALLIS
Abstract Serum mannose-binding protein (MBP) is the first component of the lectin pathway of the complement cascade. It binds to sugars on the surface of pathogenic microorganisms and triggers complement fixation by activating an associated serine protease, designated MBP-associated serine protease-2 (MASP-2). Recent studies have provided insight into the interactions between MBP and MASP-2 that trigger complement activation. MBP/MASP complexes share many features with the C1 complex of the classical pathway. The relatively simple MBP/MASP complexes serve as useful models for understanding activation of the classical pathway of the complement cascade.
Introduction Serum mannose-binding protein (MBP, also known as mannose-binding lectin) binds to sugars present in high-density arrays on the surfaces of bacteria, fungi and parasites and activates complement in an antibody-independent manner (1–3). Complement fixation is triggered by activation of an associated serine protease, designated MBP-associated serine proteases-2 (MASP-2), which in turn cleaves and activates downstream complement components (4, 5). Microorganisms are cleared following opsonization and phagocytosis by host leukocytes or are lysed following activation of the lytic pathway of complement. MBP also acts directly as an opsonin (6), although the identities of the receptor(s) on phagocytic cells have not been established. Serum MBP is a member of the collectin family of animal lectins (7). Polypeptides comprise an N-terminal cysteine-rich domain, a collagenous domain, a neck consisting of an a-helical region and a C-terminal Ca2+-dependent carbohydrate-recognition domain (CRD). Three identical polypeptides self-associate to form subunits that in turn interact to generate larger oligomers, resembling bouquet-like structures. Serum MBPs
Abbreviations: MBP = mannose-binding protein; MASP = MBP-associated serine protease; CRD = carbohydrate-recognition domain; MAP19 = MBP-associated protein; EGF = epidermal growth factor; CUB = domain found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1. 0171-2985/02/205/04-05-433 $ 15.00/0
434 · R. WALLIS isolated from different organisms consist of different mixtures of oligomers. For example, human MBPs consist of oligomers containing up to eight subunits (8, 9), of which dimers, trimers and tetramers are the predominant forms, whereas rat MBP forms monomers to tetramers of subunits (10). Dimers, trimers and tetramers of rat MBP all activate complement, although trimers and tetramers have higher specific activities than dimers (11). Two MBP genes have been identified in mammals, encoding proteins designated MBP-A and MBP-C (12). In rats, and many other mammals, the predominant serum protein is MBP-A. Rat MBP-C is found mainly in the liver and consists of single trimeric subunits that do not form larger oligomers (13–15). It has a low complement-fixing activity compared to serum MBP (11). In humans and chimpanzees, only MBP-C is produced (16, 17). Human MBP-C is a serum protein and has similar properties to rat MBP-A. The MBP-A gene is present as a pseudogene that is expressed but is not translated (18). A common immunodeficiency caused by mutations to the human MBP gene highlights the important role of MBP in the immune system (2). MBP-associated immunodeficiency is characterised by increased susceptibility to infections caused by a wide range of pathogenic microorganisms. Subjects are particularly vulnerable in the first few years of life before the adaptive immune system is fully developed. The disorder also manifests itself when adaptive immunity is compromised, for example during HIV infection or following chemotherapy (19, 20). Serum MBP circulates as complexes with three different MASPs (MASPs-1, -2 and -3) and a truncated form of MASP-2, designated MAP19 or sMAP (4, 21–24). MASP-1 and MASP-3 are alternatively spiced products of the same gene, while MAP19 is formed by alternative splicing of the MASP-2 gene product. MASPs are homologues of components C1r and C1s of the classical pathway and have the same domain organisation, consisting of two CUB domains at the N-terminus, separated by an EGF-like domain and followed by two complement control protein modules and a C-terminal serine protease domain. MASPs are Ca2+-independent homodimers formed through interactions involving the N-terminal CUB domain (25, 26). They bind to MBP in a Ca2+-dependent manner through the N-terminal CUB and EGF modules. MAP19 comprises the N-terminal two domains of MASP-2. It binds to MBP but with lower affinity than full-size MASP-2. MASPs are synthesized as zymogens that become activated when MBP binds to foreign cells. Each MASP polypeptide is cleaved near the N-terminal end of the serine protease domain to generate an active protease that remains covalently bound to the N-terminal fragment through a single disulfide bond. Only MASP-2 is thought to trigger complement fixation. When MBP binds to a target cell, MASP-2 auto-activates and cleaves complement components C2 and C4 to generate fragments that form the C3 convertase (5). The biological roles of MASP-1, MASP-3 and MAP19 are not known. Characterisation of the interactions between MBP and MASP-2 has highlighted a number of features in common with the C1 complex of the classical pathway. The underlying mechanism of activation is likely to be similar in each system. The aim of this review is to describe the structural organisation of MBP/MASP complexes and to compare and evaluate and models for complement fixation by the lectin and classical pathways.
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Results Structural organisation of MBP oligomers Oligomeric structure of MBP
Heterogeneity in the oligomeric structure of serum MBP probably arises because of the way in which MBP polypeptides associate during biosynthesis. MBP subunits assemble in a C- to N-terminal direction, so that trimerization of polypeptides and formation of the collagen helix is initiated by interactions between the CRDs and the a-helices that form the neck region (15). After folding of the collagen triple helix, polypeptides are linked by disulfide bonds formed between cysteine residues within the short, N-terminal domains. Disulfide bonding is heterogeneous and asymmetrical. Most subunits in rat serum MBP consist of polypeptides that are linked by disulfide bonds between cysteine residues at positions 13 and 18 arranged in a Cys13:Cys13, Cys13:Cys18 and Cys18:Cys18 pattern (11, 15). Oligomerization of MBP subunits is mediated through interactions involving the N-terminal cysteine-rich domain and the first part of the collagenous domain (11). Disulfide bonds formed between corresponding cysteine residues near the N-terminus of each MBP polypeptide (Cys6 in rat serum MBP) link separate subunits together to form the larger oligomers. Cysteine residues that do not form these interchain disulfide bonds probably form disulfide bonds with free cysteine or glutathione. Attachment of such additional material to polypeptide chains almost certainly blocks further self-association of subunits. Competition between formation of the disulfide bonds that link subunits together and capping of cysteine residues through attachment of small sulfhydryl groups would account for the heterogeneity in the population of MBP oligomers. Once secreted, oligomers do not interact with each other. Rod-like stalks flanked by regions of flexibility in MBP oligomers
Hydrodynamic analysis has shown that rat and human MBPs are highly asymmetrical, reflecting the fact that the collagenous domain of each subunit forms an extended, rodlike structure (9, 11, 15). Images of human MBP produced with rotary shadowing electron microscopy reveal globular heads connected to central stalks of length 13.2 ± 2.0 nm that are broadly consistent with estimated length based on the structure of synthetic collagen peptides (8, 27). 4-Hydroxyproline and glucosylgalactosyl-5-hydroxylysine residues have been identified within the consensus sequences Pro-Gly-Xaa and Lys-Gly-Xaa in MBPs (8, 11, 15, 27). As in vertebrate collagens, hydroxyproline residues are believed to stabilise the collagen triple helix (28). The role of glycosylated hydroxylysine residues is not known. However, substitution of two residues in the first part of the collagenous domain caused structural changes that decreased the complement-fixing activity of rat MBP (29), implying that these residues might be important for folding and oligomerisation of MBP subunits. There are two sites of potential flexibility in MBPs: a region within the collageous domain, referred to as the hinge and a region at the junction between the collagenous domain and the neck, called the swivel (Fig. 1). An interruption to the Gly-X-Y repeats of the collagen consensus sequence causes flexibility at the hinge (12, 16). Measurements based on images observed by rotary shadowing electron microscopy are consistent with the hinge being the point at which individual subunits diverge from each other in the
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Figure 1. Structural organisation of MBP and MASP-2. An MBP trimer is shown. Dimers, trimers and tetramers of subunits have similar organisations. A schematic representation of MASP-2 dimer is depicted. The disulfide bond that links the serine protease to the N-terminal portion is shown. MASP-1 and MASP-3 have the same domain organisations as MASP-2. CCP: complement-control protein module.
bouquet-like structures (8). The swivel is believed to be flexible because polypeptides in the collagenous domain are staggered while the a-helices that form the neck are in register (15). Thus, each of the three polypeptides must adopt a different configuration at the junction between the two domains. Although the structure of this region in MBP is not known in detail, the corresponding junction in class A macrophage scavenger receptors is highly flexible (30). The swivel might be important for orienting the CRDs when MBP binds to a ligand (31). Recognition of exogenous sugar ligands by MBP
Recognition of a foreign cell by MBP is the event that triggers complement activation by the lectin pathway. MBP binds to a wide variety of sugar structures but still discriminates between exogenous and endogenous ligands. Binding selectivity is achieved as a result of the sugar specificity of the CRDs combined with the requirement for multiple interactions in order to form a stable complex (7). Monosaccharide recognition by CRDs of MBP
The CRDs of MBP bind to monosaccharides such as mannose, fucose and N-acetylglucosamine (32). These structures occur only rarely at the terminal positions of oligosaccharides on mammalian glycoproteins and glycolipids but they are present in high-density arrays on many bacterial, fungal and parasitic cells. Crystal structures of CRDs of rat MBPs in complex with mannose-containing sugars reveal that the sugar-binding site is localised around one of two Ca2+ sites of the CRD (33, 34). Equatorial hydroxyl groups at the 3- and 4-OH positions of the sugar residue serve as coordination ligands for the Ca2+. Additional coordination ligands are provided by asparagine and glutamic acid residues in the CRD that also form hydrogen bonds with the 3- and 4-OH groups of the
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mannose residue. Relatively few additional interactions provide binding energy. The limited nature of the contacts between sugar and protein enables MBP to recognize a broad range of target ligands. Thus, sugars such as fucose and N-acetylglucosamine, which have equatorial OH-groups equivalent to those of the 3- and 4-OH groups of mannose are also ligands (32). However, galactose and related sugars that have an axial 4-OH group have a different stereochemistry and consequently are not recognized by MBP. High-avidity binding to sugar structures on foreign cells
Biophysical analysis has revealed that individual CRDs of MBP bind to monosaccharide ligands with affinities typically in the mM range (35). Interactions between multiple CRDs and a target ligand provide the binding energy that is necessary for MBP to trigger complement activation. Crystal structures of fragments comprising the CRDs and neck region of rat and human MBPs reveal that the three CRDs of each subunit are maintained in a fixed geometry through hydrophobic contacts between the CRD of one polypeptide and the upper part of the a-helix that forms the neck region in the adjacent polypeptide (36, 37) (Fig. 2). The sugar binding sites are too far apart (53 Å and 45 Å in rat and human proteins) for multivalent interactions with a single high-mannose oligosaccharide. Because sugars terminating in mannose are relatively rare on the surfaces of mammalian cells, MBPs cannot make the multiple contacts necessary for activation. However, foreign cells such as bacteria, fungi and parasites that are covered in mannoselike sugars will be targets for binding and hence complement activation by MBP.
Figure 2. Structure of the C-terminal portion of a trimeric subunit of rat serum MBP in complex with carbohydrate ligands. The picture was created using the coordinates from the crystal structure of a trimeric fragment of rat serum MBP and the coordinates of the structure of an isolated CRD in complex with a mannose-containing oligosaccharide (33, 37).
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Figure 3. Lateral interactions between the CRDs of adjacent trimers in the crystal structure of a trimeric fragment of rat serum MBP in complex with a mannose-containing oligosaccharide. Adjacent trimers are packed laterally in sheets in the crystal lattice and are crosslinked by sugars (shown in black). The sugar structures presented by adjacent trimers resemble ligands projecting from a cell surface. Each trimer is crosslinked to three neighbours in the lattice (38).
In a recent crystal structure of a C-terminal fragment of rat serum MBP in complex with a high-mannose oligosaccharide, each CRD makes lateral contacts with a CRD in an adjacent subunit within the crystal lattice (38) (Fig. 3). The MBP fragments are arranged in sheets, in which the sugar-binding sites of subunits project in the same orientation, as would be expected when an MBP molecule binds to the surface of a microorganism. Lateral interactions between the CRDs of different subunits would be predicted induce a fixed conformation at the hinge and swivel regions when MBP binds to a target cell. These changes probably trigger auto-activation of MASP-2, thus initiating complement fixation (see below). MASP binding and complement activation by MBP Interactions between MBP and MASPs
Naturally occurring mutations within the first part of the collagenous domain of MBP cause structural changes that disrupt interactions with MASPs (26, 39), suggesting that MASPs bind near to the hinge of MBP subunits. This conclusion is consistent with analysis of chimeras of rat serum and liver MBPs, in which high-level complement-fixing activity of serum MBP was found to be associated with the first part of the collagenous domain and the cysteine-rich domain (11).
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Figure 4. Comparison of the proposed structural organisation of the MBP/MASP complex with models of the C1 complex of the classical pathway. The proposed structure of a complex between an MBP trimer and a MASP-2 dimer is based on the known biophysical properties of the interacting proteins, in which the N-terminal three domains of each MASP protomer bind to a separate MBP subunit (40). Two alternative models of the C1 complex are shown. C1r is shaded in dark grey and C1s is shaded in light grey. The larger spheres correspond to the catalytic domains of the serine proteases and the smaller spheres represent the N-terminal domains.
The N-terminal CUB and EGF domains of MASPs mediate binding to MBP (26, 40). Both CUB modules probably interact directly with MBP. Hydrodynamic analysis indicates that the CUB-1-EGF-CUB-2 domains adopt an extended conformation. The most likely arrangement for the complexes is one in which each protomer of a MASP dimer extends along the N-terminal portion of the collagenous domain of an MBP subunit (Fig. 4). It is of interest to consider whether the MASP-binding site extends beyond the hinge of MBP subunits. The first part of the collagen-like region of rat MBP comprises five Gly-X-Y repeats (12). Based on the crystal structure of a collagen-like peptide, the average unit height for a single repeat is 8.4 Å, so the overall length of the collagenous domain up to the hinge would be 42 Å (27). From crystal structures, CUB domains are globular with dimensions of 42 Å × 27 Å × 23 Å, while EGF domains have a more extended ellipsoidal conformation with dimensions of 32 Å × 15 Å × 15 Å (41, 42). Because EGF-like domains have a loop-through topology, the CUB modules would be linked to opposite ends of the long axis of the EGF-like domain. The possibility that the MASP binding site extends beyond the hinge region of MBP cannot be ruled out. However, based on the dimensions of the domains, it would be possible for the N-terminal MASP modules to adopt an extended conformation and still be configured in such a way that both CUB domains contact only the first part of the collagenous domain of MBP.
440 · R. WALLIS Stoichiometry of MBP/MASP complexes for complement activation
The stoichiometry of MBP/MASP interactions has been determined using hydrodynamic analysis of reconstituted complexes assembled from recombinant components (40). MBP dimers form complexes with single MASP dimers, while trimers and tetramers of MBP subunits bind to up to two MASP dimers. Each protomer of a MASP dimer probably interacts with a separate MBP subunit. Because MBP dimers are able to fix complement in the absence of trimers and tetramers of subunits, complexes consisting of MASP2 dimers bound to MBP dimers must be sufficient to activate complement. This one-step activation mechanism contrasts with activation of the classical pathway, in which binding of C1q to immune complexes triggers auto-activation of C1r that in turn cleaves and activates C1s (43). The mechanism by which MBP interacts with MASPs allows a description of why dimers, trimers and tetramers of MBP activate complement but single subunits have relatively low activity: dimers of MBP subunits, as well as larger oligomers, form stable complexes with at least one MASP-2 dimer and therefore fix complement effectively. In contrast, single MBP subunits bind only weakly to MASPs and have very low complementfixing activities. Biophysical analysis indicates that binding of a second MASP dimer to an MBP trimer or tetramer is not cooperative (40). Thus, it is likely that activation of each MASP dimer occurs independently when more than one MASP dimer is bound to the same MBP oligomer. Rat MASP-1 and MASP-2 bind to different MBP oligomers with different binding affinities (40). MASP-2 binds to dimers, trimers and tetramers with comparable affinities, while MASP-1 binds preferentially to trimers and tetramers of subunits. Because the CUB-1, EGF-like and CUB-2 modules that interact with MBP are identical in MASP1 and MASP-3, it is likely that MASP-3 has similar MBP-binding properties to MASP1. Thus, MBP dimers probably circulate in complex with MASP-2 whereas trimers and tetramers bind to all three MASPs. MASPs-1, MASP-2 and MASP-3 compete for binding sites on MBP oligomers (40). MBP trimers and tetramers and the larger oligomers of human MBP could potentially form complexes with two different MASPs simultaneously. While MASP-2 auto-activates (26, 44), the activation mechanisms of MASP-1 and MASP-3 are not known. MASP-2 might activate MASP-1 or MASP-3 in a fashion analogous to the way in which C1r activates C1s. However, the zymogen form of MASP-2 does not interact with MASP-1 and it is more likely that MASP-1, MASP-3 or both zymogens also auto-activate. MAP19 binds to MBP with lower affinity than the full-size MASP because it lacks the second CUB domain that is directly involved in the interaction (25, 40). Because it also lacks the protease domain, it is unable to activate complement directly. MAP19 could potentially form a complex with an MBP trimer, tetramer or larger MBP oligomer together with an intact MASP. Thus, it might form part of an MBP/MASP complex that activates complement. Alternatively, it could compete for MASP-binding sites on MBP, thereby regulating the activation process.
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Discussion Comparison of MBP/MASP complexes with the C1 complex of the classical complement pathway
It is of interest to compare the structural organisation of the MBP/MASP complexes with the arrangement of the C1 complex of the classical complement pathway. C1q is a hexamer of trimeric subunits assembled from three different polypeptides, each of which contains a collagenous domain at the N-terminal end and a globular C-terminal domain (43). C1q forms bouquet-like structures that resemble MBP oligomers in images obtained by rotary-shadowing electron microscopy (45). C1r and C1s form a heterotetramer in which the catalytic domains of C1r interact with each other to form a homodimeric core (46). A single C1s polypeptide interacts with each protomer of the C1r dimer, in a Ca2+-dependent manner, through interactions involving the N-terminal CUB and EGFlike domains of each component (47). The tetramer interacts with C1q in a Ca2+-dependent manner through the N-terminal domains of C1s (48). The configuration of the complex between C1r2C1s2 and C1q is not known, although a number of different models have been proposed based on biochemical and biophysical properties of the complex (49). For example, it has been suggested that the tetramer forms a compact figure-of-eight shaped conformation, in which the catalytic domains form a central core that is located inside the cone formed from the six collagenous stalks of C1q and the N-terminal domains of C1r and C1s loop around the rod-like stalks (Fig. 4). However, neutron-scattering data are more consistent with a model in which each protomer of C1r2C1s2 interacts with a separate subunit of C1q (50). The overall structure of C1r2C1s2 resembles a ‘W’ in which each stalk represents a separate protomer and the two central stalks comprise the C1r dimeric core (Fig. 4). Because there are six subunits in C1q and only four protomers in the C1r2C1s2 complex, C1r2C1s2 binds on one side of C1q, in a manner very similar to that proposed here for MASP dimers binding to MBP subunits. Human MBP can bind and activate C1r2C1s2 complexes in vitro, suggesting that the interaction between C1r2C1s2 and C1q is similar to the interaction between MBP and MASP-2 (8). Interestingly, in these studies, MBP tetramers were the smallest subunits able to activate C1r2C1s2, whereas MBP dimers are sufficient to activate MASPs. The difference probably arises because MASPs are dimers and consequently bind with high affinity to a pair of MBP subunits, whereas C1r2C1s2 is a tetramer and requires a minimum of four subunits to form a stable complex. Mechanism of activation by the lectin pathway of complement
The molecular mechanism of complement activation by MBP is poorly understood. The sugar-binding sites on the CRDs are a considerable distance away from the MASP-binding site, so it is likely that binding to the surface of a microorganism causes a conformational change in MBP that initiates MASP activation. It is theoretically possible that ligand binding causes a structural change in each subunit that is transmitted from the CRDs in a C- to N-terminal direction through the collagenous domain to the MASP. However, sugar binding does not induce a structural change in the CRD, so it is hard to envisage how information could be transmitted along the polypeptide (33, 51). It seems more likely that multivalent ligand interactions involving CRDs of different subunits initiate a global conformational change in MBP, resulting in a displacement in
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Figure 5. Proposed mechanism of complement activation by MBP. In the proposed model, lateral interactions between CRDs are stabilised by CRD/carbohydrate interactions when MBP binds to the surface of a microorganism. Binding imparts a fixed geometry as a result of a locking the conformation at the hinge and swivel regions of MBP. These changes in turn induce a conformational change in MASP-2 that leads to auto-activation.
the relative positions of subunits (Fig. 5). This hypothesis would explain why a dimer of MBP subunits is the smallest oligomer that activates complement efficiently. The model would also explain why MASPs are dimers, because each protomer interacts with a separate MBP subunit. The arrangement of subunits seen in the crystal structure shown in Figure 3 might represent the type of configuration that occurs when MBP binds to a target cell. Binding of CRDs to sugar-rich surfaces would induce lateral interactions between CRDs of separate subunits. In order to accommodate these interactions, the conformations of the swivel and hinge would become fixed with a relatively acute angle between the collagen stalks (Fig. 5). Because MASPs bind immediately adjacent to the hinge regions of MBP, they would be able to detect the conformation of the stalks, leading to activation of the protease domain. A similar molecular mechanism might cause activation of the classical pathway when C1q binds to immune complexes. Concluding remarks Detailed analysis of the interactions of MBP with its oligosaccharide ligands has afforded insight into a way in which the lectin pathway selectively targets pathogenic microorganisms. Recent characterisation of MBP/MASP complexes has provided a basis for understanding the downstream steps that lead to pathogen neutralisation. Structural and functional analysis will likely provide a more complete understanding of the mechanism of complement activation in the near future. Increasing evidence underlines the impor-
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tant role of the lectin branch of the complement cascade in the immune system. Understanding the molecular details of complement fixation will provide a useful starting point for the development of therapeutic agents aimed at modulating immune function. Acknowledgements
I am very grateful to KURT DRICKAMER for critical reading of the manuscript, for ideas with regard to figures and nomenclature and for helpful discussion. I also thank Hadar Feinberg for providing the image for Figure 3. Funding is provided by Wellcome Trust grant 041845.
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444 · R. WALLIS 17. MOGUES, T., T. OTA, A. I. TAUBER, and K. N. SASTRY. 1996. Characterization of two mannosebinding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications. Glycobiology 6: 543. 18. GUO, N., T. MOGUES, S. WEREMOWICZ, C. C. MORTON, and K. N. SASTRY. 1998. The human ortholog of rhesus mannose-binding protein-A gene is an expressed pseudogene that localizes to Chromosome 10. Mamm. Genome 9: 246. 19. NETH, O., I. HANN, M. W. TURNER, and N. J. KLEIN. 2001. Deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study. Lancet 358: 614. 20. GARRED, P., H. O. MADSEN, U. BALSLEV, B. HOFMANN, C. PEDERSEN, J. GERSTOFT, and A. SVEJGAARD. 1997. Susceptibility to HIV infection and progression of AIDS in relation to varient alleles of mannose-binding lectin. Lancet 349: 236. 21. DAHL, M. D., S. THIEL, M. MATSUSHITA, T. FUJITA, A. C. WILLIS, T. CHRISTENSEN, T. VORUPJENSEN, and J. C. JENSENIUS. 2001. MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15: 127. 22. MATSUSHITA, M., and T. FUJITA. 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J. Exp. Med. 176: 1497. 23. TAKAHASHI, M., Y. ENDO, T. FUJITA, and M. MATSUSHITA. 1999. A truncated form of mannosebinding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway. Int. Immunol. 11: 859. 24. STOVER, C. M., S. THIEL, N. J. LYNCH, T. VORUP-JENSEN, J. C. JENSENIUS, and W. J. SCHWAEBLE. 1999. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene. J. Immunol. 162: 3481. 25. THIELENS, N. E., S. CSEH, S. THIEL, T. VORUP-JENSEN, V. ROSSI, J. C. JENSENIUS, and G. J. ARLAUD. 2001. Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL. J. Immunol. 166: 5068. 26. WALLIS, R., and R. B. DODD. 2000. Interaction of mannose-binding protein with associated-serine proteases: effects of naturally occuring mutations. J. Biol. Chem. 275: 30962. 27. BELLA, J., M. EATON, B. BRODSKY, and H. M. BERMAN. 1994. Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution. Science 266: 75. 28. KADLER, K. 1994. Extracellular matrix. : fibril-forming collagens. Protein-profile 2: 491. 29. HEISE, C. T., J. R. NICHOLLS, C. E. LEAMY, and R. WALLIS. 2000. Impaired Secretion of Rat Mannose-Binding Protein Resulting from Mutations in the Collagen-Like Domain. J. Immunol. 165: 1403. 30. RESNICK, D., J. E. CHATTERTON, K. SCHWARTZ, H. SLAYTER, and M. KRIEGER. 1996. Structures of class A macrophage scavenger receptors. J. Biol. Chem. 271: 26924. 31. WALLIS, R. 2000. C-Type lectins and collectins, in oligosaccharides in chemistry and biology: A comprehensive handbook (ERNST, B., HART, G. and SINAY, P. eds.). Wiley-VCH, Weinheim, 597 pp. 32. LEE, R. T., Y. ICHIKAWA, M. FAY, K. DRICKAMER, M.-C. SHAO, and Y. C. LEE. 1991. Ligandbinding characteristics of rat serum-type mannose-binding protein (MBP-A): homology of binding site architecture with mammalian and chicken hepatic lectins. J. Biol. Chem. 266: 4810. 33. WEIS, W. I., K. DRICKAMER, and W. A. HENDRICKSON. 1992. Structure of a C-type mannosebinding protein complexed with an oligosaccharide. Nature 360: 127. 34. NG, K. K.-S., K. DRICKAMER, and W. I. WEIS. 1996. Structural analysis of monosaccharide recognition by rat liver mannose-binding protein. J. Biol. Chem. 271: 663. 35. IOBST, S. T., M. R. WORMALD, W. I. WEIS, R. A. DWEK, and K. DRICKAMER. 1994. Binding of sugar ligands to Ca2+-dependent animal lectins: I. Analysis of mannose binding by site-directed mutagenesis and NMR. J. Biol. Chem. 269: 15505. 36. SHERIFF, S., C. Y. Y. CHANG, and R. A. B. EZEKOWITZ. 1994. Human mannose-binding protein carbohydrate-recognition domain trimerizes through a triple a-helical coiled coil. Nature Struct. Biol. 1: 789. 37. WEIS, W. I., and K. DRICKAMER. 1994. Trimeric structure of a C-type mannose-binding protein. Structure 2: 1227.
Structure and function of mannose-binding protein · 445 38. NG, K.-S., A. R. KOLATAR, S. PARK-SNYDER, H. FEINBERG, D. A. CLARK, K. DRICKAMER, and W. I. WEIS. 2002. Orientation of bound ligands in mannose-binding proteins: implications for multivalent ligand recognition. J. Biol. Chem. 277: 16088. 39. WALLIS, R., and J. Y. T. CHENG. 1999. Molecular defects in variant forms of mannose-binding protein associated with immunodeficiency. J. Immunol. 163: 4953. 40. CHEN, C.-B., and R. WALLIS. 2001. Stoichiometry of Complexes between Mannose-binding Protein and Its Associated Serine Proteases. defining functional units for complement activation. J. Biol. Chem. 276: 25894. 41. RAO, Z., P. A. HANDFORD, M. MAYHEW, V. KNOTT, G. G. BROWNLEE, and D. STUART. 1995. The structure of a Ca2+-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82: 131. 42. VARELA, P. F., A. ROMERO, L. SANZ, M. J.,ROMAO, E. TOPFER-PETERSEN, and J. J. CALVETE. 1997. The 2.4 A resolution crystal structure of boar seminal plasma PSP-I/PSP-II: a zona pellucida-binding glycoprotein heterodimer of the spermadhesin family built by a CUB domain architecture. J. Mol. Biol. 274: 635. 43. REID, K. B. M. 1983. Proteins involved in the activation and control of the two pathways of human complement. Biochem. Soc. Trans. 11: 1. 44. VORUP-JENSEN, T., S. V. PETERSEN, A. G. HANSEN, K. POULSEN, W. SCHWAEBLE, R. B. SIM, K. B. M. REID, S. J. DAVIS, S. THIEL, and J. C. JENSENIUS. 2000. Distinct Pathways of MannanBinding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2. J. Immunol. 165: 2093. 45. BRODSKY-DOYLE, B., K. R. LEONARD, and K. B. M. REID. 1976. Circular-dichroism and electronmicroscopy studies of human subcomponent C1q before and after limited proteolysis by pepsin. Biochem. J. 159: 279. 46. LACROIX, M., V. ROSSI, C. GABORIAUD, S. CHEVALLIER, M. JAQUINOD, N. M. THIELENS, J. GAGNON, and G. J. ARLAUD. 1997. Structure and assembly of the catalytic region of human complement protease C1r: a three-dimensional model based on chemical cross-linking and homology modeling. Biochemistry 36: 6270. 47. THIELENS, N. M., K. ENRIE, M. LACROIX, M. JAQUINOD, J.-F. HERNANDEZ, A. F. ESSER, and G. J. ARLAUD. 1999. The N-terminal CUB-Epidermal Growth Factor Module Pair of Human Complement Protease C1r Binds Ca2+ with High Affinity and Mediates Ca2+-dependent Interaction with C1s. J. Biol. Chem. 274: 9149. 48. BUSBY, T. F., and K. C. INGHAM. 1990. NH2-terminal calcium-binding domain of human complement C1s- mediates the interaction of C1r- with C1q. Biochemistry 29: 4613. 49. ARLAUD, G. J., N. M. THIELENS, and C. ILLY. 1990. Arrangement of the C1 complex of complement. Biochem. Soc. Trans. 18: 1148–1151. 50. PERKINS, S. J. 1985. Molecular modelling of human complement subcomponent C1q and its complex with C1r2C1s2 derived from neutron-scattering curves and hydrodynamic data. Biochem. J. 228: 13. 51. WEIS, W. I., R. KAHN, R. FOURME, K. DRICKAMER, and W. A. HENDRICKSON. 1991. Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science 254: 1608. Dr. RUSSEL WALLIS, Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. Tel.: 44 1865 275762; Fax: +44 1865 275339; E-mail: [email protected]
The Lectin-Pathway of Complement Activation: MBL, other Collectins and Ficolins Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK
Structural and Functional Aspects of Complement Activation by Mannose-binding Protein RUSSELL WALLIS
Abstract Serum mannose-binding protein (MBP) is the first component of the lectin pathway of the complement cascade. It binds to sugars on the surface of pathogenic microorganisms and triggers complement fixation by activating an associated serine protease, designated MBP-associated serine protease-2 (MASP-2). Recent studies have provided insight into the interactions between MBP and MASP-2 that trigger complement activation. MBP/MASP complexes share many features with the C1 complex of the classical pathway. The relatively simple MBP/MASP complexes serve as useful models for understanding activation of the classical pathway of the complement cascade.
Introduction Serum mannose-binding protein (MBP, also known as mannose-binding lectin) binds to sugars present in high-density arrays on the surfaces of bacteria, fungi and parasites and activates complement in an antibody-independent manner (1–3). Complement fixation is triggered by activation of an associated serine protease, designated MBP-associated serine proteases-2 (MASP-2), which in turn cleaves and activates downstream complement components (4, 5). Microorganisms are cleared following opsonization and phagocytosis by host leukocytes or are lysed following activation of the lytic pathway of complement. MBP also acts directly as an opsonin (6), although the identities of the receptor(s) on phagocytic cells have not been established. Serum MBP is a member of the collectin family of animal lectins (7). Polypeptides comprise an N-terminal cysteine-rich domain, a collagenous domain, a neck consisting of an a-helical region and a C-terminal Ca2+-dependent carbohydrate-recognition domain (CRD). Three identical polypeptides self-associate to form subunits that in turn interact to generate larger oligomers, resembling bouquet-like structures. Serum MBPs
Abbreviations: MBP = mannose-binding protein; MASP = MBP-associated serine protease; CRD = carbohydrate-recognition domain; MAP19 = MBP-associated protein; EGF = epidermal growth factor; CUB = domain found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1. 0171-2985/02/205/04-05-433 $ 15.00/0
434 · R. WALLIS isolated from different organisms consist of different mixtures of oligomers. For example, human MBPs consist of oligomers containing up to eight subunits (8, 9), of which dimers, trimers and tetramers are the predominant forms, whereas rat MBP forms monomers to tetramers of subunits (10). Dimers, trimers and tetramers of rat MBP all activate complement, although trimers and tetramers have higher specific activities than dimers (11). Two MBP genes have been identified in mammals, encoding proteins designated MBP-A and MBP-C (12). In rats, and many other mammals, the predominant serum protein is MBP-A. Rat MBP-C is found mainly in the liver and consists of single trimeric subunits that do not form larger oligomers (13–15). It has a low complement-fixing activity compared to serum MBP (11). In humans and chimpanzees, only MBP-C is produced (16, 17). Human MBP-C is a serum protein and has similar properties to rat MBP-A. The MBP-A gene is present as a pseudogene that is expressed but is not translated (18). A common immunodeficiency caused by mutations to the human MBP gene highlights the important role of MBP in the immune system (2). MBP-associated immunodeficiency is characterised by increased susceptibility to infections caused by a wide range of pathogenic microorganisms. Subjects are particularly vulnerable in the first few years of life before the adaptive immune system is fully developed. The disorder also manifests itself when adaptive immunity is compromised, for example during HIV infection or following chemotherapy (19, 20). Serum MBP circulates as complexes with three different MASPs (MASPs-1, -2 and -3) and a truncated form of MASP-2, designated MAP19 or sMAP (4, 21–24). MASP-1 and MASP-3 are alternatively spiced products of the same gene, while MAP19 is formed by alternative splicing of the MASP-2 gene product. MASPs are homologues of components C1r and C1s of the classical pathway and have the same domain organisation, consisting of two CUB domains at the N-terminus, separated by an EGF-like domain and followed by two complement control protein modules and a C-terminal serine protease domain. MASPs are Ca2+-independent homodimers formed through interactions involving the N-terminal CUB domain (25, 26). They bind to MBP in a Ca2+-dependent manner through the N-terminal CUB and EGF modules. MAP19 comprises the N-terminal two domains of MASP-2. It binds to MBP but with lower affinity than full-size MASP-2. MASPs are synthesized as zymogens that become activated when MBP binds to foreign cells. Each MASP polypeptide is cleaved near the N-terminal end of the serine protease domain to generate an active protease that remains covalently bound to the N-terminal fragment through a single disulfide bond. Only MASP-2 is thought to trigger complement fixation. When MBP binds to a target cell, MASP-2 auto-activates and cleaves complement components C2 and C4 to generate fragments that form the C3 convertase (5). The biological roles of MASP-1, MASP-3 and MAP19 are not known. Characterisation of the interactions between MBP and MASP-2 has highlighted a number of features in common with the C1 complex of the classical pathway. The underlying mechanism of activation is likely to be similar in each system. The aim of this review is to describe the structural organisation of MBP/MASP complexes and to compare and evaluate and models for complement fixation by the lectin and classical pathways.
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Results Structural organisation of MBP oligomers Oligomeric structure of MBP
Heterogeneity in the oligomeric structure of serum MBP probably arises because of the way in which MBP polypeptides associate during biosynthesis. MBP subunits assemble in a C- to N-terminal direction, so that trimerization of polypeptides and formation of the collagen helix is initiated by interactions between the CRDs and the a-helices that form the neck region (15). After folding of the collagen triple helix, polypeptides are linked by disulfide bonds formed between cysteine residues within the short, N-terminal domains. Disulfide bonding is heterogeneous and asymmetrical. Most subunits in rat serum MBP consist of polypeptides that are linked by disulfide bonds between cysteine residues at positions 13 and 18 arranged in a Cys13:Cys13, Cys13:Cys18 and Cys18:Cys18 pattern (11, 15). Oligomerization of MBP subunits is mediated through interactions involving the N-terminal cysteine-rich domain and the first part of the collagenous domain (11). Disulfide bonds formed between corresponding cysteine residues near the N-terminus of each MBP polypeptide (Cys6 in rat serum MBP) link separate subunits together to form the larger oligomers. Cysteine residues that do not form these interchain disulfide bonds probably form disulfide bonds with free cysteine or glutathione. Attachment of such additional material to polypeptide chains almost certainly blocks further self-association of subunits. Competition between formation of the disulfide bonds that link subunits together and capping of cysteine residues through attachment of small sulfhydryl groups would account for the heterogeneity in the population of MBP oligomers. Once secreted, oligomers do not interact with each other. Rod-like stalks flanked by regions of flexibility in MBP oligomers
Hydrodynamic analysis has shown that rat and human MBPs are highly asymmetrical, reflecting the fact that the collagenous domain of each subunit forms an extended, rodlike structure (9, 11, 15). Images of human MBP produced with rotary shadowing electron microscopy reveal globular heads connected to central stalks of length 13.2 ± 2.0 nm that are broadly consistent with estimated length based on the structure of synthetic collagen peptides (8, 27). 4-Hydroxyproline and glucosylgalactosyl-5-hydroxylysine residues have been identified within the consensus sequences Pro-Gly-Xaa and Lys-Gly-Xaa in MBPs (8, 11, 15, 27). As in vertebrate collagens, hydroxyproline residues are believed to stabilise the collagen triple helix (28). The role of glycosylated hydroxylysine residues is not known. However, substitution of two residues in the first part of the collagenous domain caused structural changes that decreased the complement-fixing activity of rat MBP (29), implying that these residues might be important for folding and oligomerisation of MBP subunits. There are two sites of potential flexibility in MBPs: a region within the collageous domain, referred to as the hinge and a region at the junction between the collagenous domain and the neck, called the swivel (Fig. 1). An interruption to the Gly-X-Y repeats of the collagen consensus sequence causes flexibility at the hinge (12, 16). Measurements based on images observed by rotary shadowing electron microscopy are consistent with the hinge being the point at which individual subunits diverge from each other in the
436 · R. WALLIS
Figure 1. Structural organisation of MBP and MASP-2. An MBP trimer is shown. Dimers, trimers and tetramers of subunits have similar organisations. A schematic representation of MASP-2 dimer is depicted. The disulfide bond that links the serine protease to the N-terminal portion is shown. MASP-1 and MASP-3 have the same domain organisations as MASP-2. CCP: complement-control protein module.
bouquet-like structures (8). The swivel is believed to be flexible because polypeptides in the collagenous domain are staggered while the a-helices that form the neck are in register (15). Thus, each of the three polypeptides must adopt a different configuration at the junction between the two domains. Although the structure of this region in MBP is not known in detail, the corresponding junction in class A macrophage scavenger receptors is highly flexible (30). The swivel might be important for orienting the CRDs when MBP binds to a ligand (31). Recognition of exogenous sugar ligands by MBP
Recognition of a foreign cell by MBP is the event that triggers complement activation by the lectin pathway. MBP binds to a wide variety of sugar structures but still discriminates between exogenous and endogenous ligands. Binding selectivity is achieved as a result of the sugar specificity of the CRDs combined with the requirement for multiple interactions in order to form a stable complex (7). Monosaccharide recognition by CRDs of MBP
The CRDs of MBP bind to monosaccharides such as mannose, fucose and N-acetylglucosamine (32). These structures occur only rarely at the terminal positions of oligosaccharides on mammalian glycoproteins and glycolipids but they are present in high-density arrays on many bacterial, fungal and parasitic cells. Crystal structures of CRDs of rat MBPs in complex with mannose-containing sugars reveal that the sugar-binding site is localised around one of two Ca2+ sites of the CRD (33, 34). Equatorial hydroxyl groups at the 3- and 4-OH positions of the sugar residue serve as coordination ligands for the Ca2+. Additional coordination ligands are provided by asparagine and glutamic acid residues in the CRD that also form hydrogen bonds with the 3- and 4-OH groups of the
Structure and function of mannose-binding protein · 437
mannose residue. Relatively few additional interactions provide binding energy. The limited nature of the contacts between sugar and protein enables MBP to recognize a broad range of target ligands. Thus, sugars such as fucose and N-acetylglucosamine, which have equatorial OH-groups equivalent to those of the 3- and 4-OH groups of mannose are also ligands (32). However, galactose and related sugars that have an axial 4-OH group have a different stereochemistry and consequently are not recognized by MBP. High-avidity binding to sugar structures on foreign cells
Biophysical analysis has revealed that individual CRDs of MBP bind to monosaccharide ligands with affinities typically in the mM range (35). Interactions between multiple CRDs and a target ligand provide the binding energy that is necessary for MBP to trigger complement activation. Crystal structures of fragments comprising the CRDs and neck region of rat and human MBPs reveal that the three CRDs of each subunit are maintained in a fixed geometry through hydrophobic contacts between the CRD of one polypeptide and the upper part of the a-helix that forms the neck region in the adjacent polypeptide (36, 37) (Fig. 2). The sugar binding sites are too far apart (53 Å and 45 Å in rat and human proteins) for multivalent interactions with a single high-mannose oligosaccharide. Because sugars terminating in mannose are relatively rare on the surfaces of mammalian cells, MBPs cannot make the multiple contacts necessary for activation. However, foreign cells such as bacteria, fungi and parasites that are covered in mannoselike sugars will be targets for binding and hence complement activation by MBP.
Figure 2. Structure of the C-terminal portion of a trimeric subunit of rat serum MBP in complex with carbohydrate ligands. The picture was created using the coordinates from the crystal structure of a trimeric fragment of rat serum MBP and the coordinates of the structure of an isolated CRD in complex with a mannose-containing oligosaccharide (33, 37).
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Figure 3. Lateral interactions between the CRDs of adjacent trimers in the crystal structure of a trimeric fragment of rat serum MBP in complex with a mannose-containing oligosaccharide. Adjacent trimers are packed laterally in sheets in the crystal lattice and are crosslinked by sugars (shown in black). The sugar structures presented by adjacent trimers resemble ligands projecting from a cell surface. Each trimer is crosslinked to three neighbours in the lattice (38).
In a recent crystal structure of a C-terminal fragment of rat serum MBP in complex with a high-mannose oligosaccharide, each CRD makes lateral contacts with a CRD in an adjacent subunit within the crystal lattice (38) (Fig. 3). The MBP fragments are arranged in sheets, in which the sugar-binding sites of subunits project in the same orientation, as would be expected when an MBP molecule binds to the surface of a microorganism. Lateral interactions between the CRDs of different subunits would be predicted induce a fixed conformation at the hinge and swivel regions when MBP binds to a target cell. These changes probably trigger auto-activation of MASP-2, thus initiating complement fixation (see below). MASP binding and complement activation by MBP Interactions between MBP and MASPs
Naturally occurring mutations within the first part of the collagenous domain of MBP cause structural changes that disrupt interactions with MASPs (26, 39), suggesting that MASPs bind near to the hinge of MBP subunits. This conclusion is consistent with analysis of chimeras of rat serum and liver MBPs, in which high-level complement-fixing activity of serum MBP was found to be associated with the first part of the collagenous domain and the cysteine-rich domain (11).
Structure and function of mannose-binding protein · 439
Figure 4. Comparison of the proposed structural organisation of the MBP/MASP complex with models of the C1 complex of the classical pathway. The proposed structure of a complex between an MBP trimer and a MASP-2 dimer is based on the known biophysical properties of the interacting proteins, in which the N-terminal three domains of each MASP protomer bind to a separate MBP subunit (40). Two alternative models of the C1 complex are shown. C1r is shaded in dark grey and C1s is shaded in light grey. The larger spheres correspond to the catalytic domains of the serine proteases and the smaller spheres represent the N-terminal domains.
The N-terminal CUB and EGF domains of MASPs mediate binding to MBP (26, 40). Both CUB modules probably interact directly with MBP. Hydrodynamic analysis indicates that the CUB-1-EGF-CUB-2 domains adopt an extended conformation. The most likely arrangement for the complexes is one in which each protomer of a MASP dimer extends along the N-terminal portion of the collagenous domain of an MBP subunit (Fig. 4). It is of interest to consider whether the MASP-binding site extends beyond the hinge of MBP subunits. The first part of the collagen-like region of rat MBP comprises five Gly-X-Y repeats (12). Based on the crystal structure of a collagen-like peptide, the average unit height for a single repeat is 8.4 Å, so the overall length of the collagenous domain up to the hinge would be 42 Å (27). From crystal structures, CUB domains are globular with dimensions of 42 Å × 27 Å × 23 Å, while EGF domains have a more extended ellipsoidal conformation with dimensions of 32 Å × 15 Å × 15 Å (41, 42). Because EGF-like domains have a loop-through topology, the CUB modules would be linked to opposite ends of the long axis of the EGF-like domain. The possibility that the MASP binding site extends beyond the hinge region of MBP cannot be ruled out. However, based on the dimensions of the domains, it would be possible for the N-terminal MASP modules to adopt an extended conformation and still be configured in such a way that both CUB domains contact only the first part of the collagenous domain of MBP.
440 · R. WALLIS Stoichiometry of MBP/MASP complexes for complement activation
The stoichiometry of MBP/MASP interactions has been determined using hydrodynamic analysis of reconstituted complexes assembled from recombinant components (40). MBP dimers form complexes with single MASP dimers, while trimers and tetramers of MBP subunits bind to up to two MASP dimers. Each protomer of a MASP dimer probably interacts with a separate MBP subunit. Because MBP dimers are able to fix complement in the absence of trimers and tetramers of subunits, complexes consisting of MASP2 dimers bound to MBP dimers must be sufficient to activate complement. This one-step activation mechanism contrasts with activation of the classical pathway, in which binding of C1q to immune complexes triggers auto-activation of C1r that in turn cleaves and activates C1s (43). The mechanism by which MBP interacts with MASPs allows a description of why dimers, trimers and tetramers of MBP activate complement but single subunits have relatively low activity: dimers of MBP subunits, as well as larger oligomers, form stable complexes with at least one MASP-2 dimer and therefore fix complement effectively. In contrast, single MBP subunits bind only weakly to MASPs and have very low complementfixing activities. Biophysical analysis indicates that binding of a second MASP dimer to an MBP trimer or tetramer is not cooperative (40). Thus, it is likely that activation of each MASP dimer occurs independently when more than one MASP dimer is bound to the same MBP oligomer. Rat MASP-1 and MASP-2 bind to different MBP oligomers with different binding affinities (40). MASP-2 binds to dimers, trimers and tetramers with comparable affinities, while MASP-1 binds preferentially to trimers and tetramers of subunits. Because the CUB-1, EGF-like and CUB-2 modules that interact with MBP are identical in MASP1 and MASP-3, it is likely that MASP-3 has similar MBP-binding properties to MASP1. Thus, MBP dimers probably circulate in complex with MASP-2 whereas trimers and tetramers bind to all three MASPs. MASPs-1, MASP-2 and MASP-3 compete for binding sites on MBP oligomers (40). MBP trimers and tetramers and the larger oligomers of human MBP could potentially form complexes with two different MASPs simultaneously. While MASP-2 auto-activates (26, 44), the activation mechanisms of MASP-1 and MASP-3 are not known. MASP-2 might activate MASP-1 or MASP-3 in a fashion analogous to the way in which C1r activates C1s. However, the zymogen form of MASP-2 does not interact with MASP-1 and it is more likely that MASP-1, MASP-3 or both zymogens also auto-activate. MAP19 binds to MBP with lower affinity than the full-size MASP because it lacks the second CUB domain that is directly involved in the interaction (25, 40). Because it also lacks the protease domain, it is unable to activate complement directly. MAP19 could potentially form a complex with an MBP trimer, tetramer or larger MBP oligomer together with an intact MASP. Thus, it might form part of an MBP/MASP complex that activates complement. Alternatively, it could compete for MASP-binding sites on MBP, thereby regulating the activation process.
Structure and function of mannose-binding protein · 441
Discussion Comparison of MBP/MASP complexes with the C1 complex of the classical complement pathway
It is of interest to compare the structural organisation of the MBP/MASP complexes with the arrangement of the C1 complex of the classical complement pathway. C1q is a hexamer of trimeric subunits assembled from three different polypeptides, each of which contains a collagenous domain at the N-terminal end and a globular C-terminal domain (43). C1q forms bouquet-like structures that resemble MBP oligomers in images obtained by rotary-shadowing electron microscopy (45). C1r and C1s form a heterotetramer in which the catalytic domains of C1r interact with each other to form a homodimeric core (46). A single C1s polypeptide interacts with each protomer of the C1r dimer, in a Ca2+-dependent manner, through interactions involving the N-terminal CUB and EGFlike domains of each component (47). The tetramer interacts with C1q in a Ca2+-dependent manner through the N-terminal domains of C1s (48). The configuration of the complex between C1r2C1s2 and C1q is not known, although a number of different models have been proposed based on biochemical and biophysical properties of the complex (49). For example, it has been suggested that the tetramer forms a compact figure-of-eight shaped conformation, in which the catalytic domains form a central core that is located inside the cone formed from the six collagenous stalks of C1q and the N-terminal domains of C1r and C1s loop around the rod-like stalks (Fig. 4). However, neutron-scattering data are more consistent with a model in which each protomer of C1r2C1s2 interacts with a separate subunit of C1q (50). The overall structure of C1r2C1s2 resembles a ‘W’ in which each stalk represents a separate protomer and the two central stalks comprise the C1r dimeric core (Fig. 4). Because there are six subunits in C1q and only four protomers in the C1r2C1s2 complex, C1r2C1s2 binds on one side of C1q, in a manner very similar to that proposed here for MASP dimers binding to MBP subunits. Human MBP can bind and activate C1r2C1s2 complexes in vitro, suggesting that the interaction between C1r2C1s2 and C1q is similar to the interaction between MBP and MASP-2 (8). Interestingly, in these studies, MBP tetramers were the smallest subunits able to activate C1r2C1s2, whereas MBP dimers are sufficient to activate MASPs. The difference probably arises because MASPs are dimers and consequently bind with high affinity to a pair of MBP subunits, whereas C1r2C1s2 is a tetramer and requires a minimum of four subunits to form a stable complex. Mechanism of activation by the lectin pathway of complement
The molecular mechanism of complement activation by MBP is poorly understood. The sugar-binding sites on the CRDs are a considerable distance away from the MASP-binding site, so it is likely that binding to the surface of a microorganism causes a conformational change in MBP that initiates MASP activation. It is theoretically possible that ligand binding causes a structural change in each subunit that is transmitted from the CRDs in a C- to N-terminal direction through the collagenous domain to the MASP. However, sugar binding does not induce a structural change in the CRD, so it is hard to envisage how information could be transmitted along the polypeptide (33, 51). It seems more likely that multivalent ligand interactions involving CRDs of different subunits initiate a global conformational change in MBP, resulting in a displacement in
442 · R. WALLIS
Figure 5. Proposed mechanism of complement activation by MBP. In the proposed model, lateral interactions between CRDs are stabilised by CRD/carbohydrate interactions when MBP binds to the surface of a microorganism. Binding imparts a fixed geometry as a result of a locking the conformation at the hinge and swivel regions of MBP. These changes in turn induce a conformational change in MASP-2 that leads to auto-activation.
the relative positions of subunits (Fig. 5). This hypothesis would explain why a dimer of MBP subunits is the smallest oligomer that activates complement efficiently. The model would also explain why MASPs are dimers, because each protomer interacts with a separate MBP subunit. The arrangement of subunits seen in the crystal structure shown in Figure 3 might represent the type of configuration that occurs when MBP binds to a target cell. Binding of CRDs to sugar-rich surfaces would induce lateral interactions between CRDs of separate subunits. In order to accommodate these interactions, the conformations of the swivel and hinge would become fixed with a relatively acute angle between the collagen stalks (Fig. 5). Because MASPs bind immediately adjacent to the hinge regions of MBP, they would be able to detect the conformation of the stalks, leading to activation of the protease domain. A similar molecular mechanism might cause activation of the classical pathway when C1q binds to immune complexes. Concluding remarks Detailed analysis of the interactions of MBP with its oligosaccharide ligands has afforded insight into a way in which the lectin pathway selectively targets pathogenic microorganisms. Recent characterisation of MBP/MASP complexes has provided a basis for understanding the downstream steps that lead to pathogen neutralisation. Structural and functional analysis will likely provide a more complete understanding of the mechanism of complement activation in the near future. Increasing evidence underlines the impor-
Structure and function of mannose-binding protein · 443
tant role of the lectin branch of the complement cascade in the immune system. Understanding the molecular details of complement fixation will provide a useful starting point for the development of therapeutic agents aimed at modulating immune function. Acknowledgements
I am very grateful to KURT DRICKAMER for critical reading of the manuscript, for ideas with regard to figures and nomenclature and for helpful discussion. I also thank Hadar Feinberg for providing the image for Figure 3. Funding is provided by Wellcome Trust grant 041845.
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