Complement System Part I - Molecular Mechanisms of Activation and Regulation

Abstract

Complement is a complex innate immune surveillance system, playing a key role in defense against pathogens and in host homeostasis. The complement system is initiated by conformational changes in recognition molecular complexes upon sensing danger signals. The subsequent cascade of enzymatic reactions is tightly regulated to assure that complement is activated only at specific locations requiring defense against pathogens, thus avoiding host tissue damage. Here, we discuss the recent advances describing the molecular and structural basis of activation and regulation of the complement pathways and their implication on physiology and pathology. This article will review the mechanisms of activation of alternative, classical, and lectin pathways, the formation of C3 and C5 convertases, the action of anaphylatoxins, and the membrane-attack-complex. We will also discuss the importance of structure-function relationships using the example of atypical hemolytic uremic syndrome. Lastly, we will discuss the development and benefits of therapies using complement inhibitors.

Keywords: alternative complement pathway; anaphylatoxins; classical complement pathway; complement regulatory proteins; complement system proteins; endothelial cells; membrane-attack-complex; structure–function relationships.

Figure 1

Complement activation in physiological conditions. (A) The alternative pathway is permanently active due to spontaneous transformation of bio-inactive molecule C3 to bioactive C3(H₂O). This allows generation of C3b, which is rapidly inactivated by FH and FI in fluid phase or is covalently bound to the surface and then inactivated on host cells. (B) Classical and lectin pathway recognition molecules bind to apoptotic cells and together with C3b from the alternative pathway induce a low level of complement activation. Apoptotic cells are not lysed, but rapidly cleared by phagocytes in an immunologically silent manner. Host cells and plasma contain multiple regulatory proteins to assure that complement activation will be limited in physiological conditions.

Figure 2

Complement during infection with a pathogen. The permanent activity of the alternative pathway allows it to immediately identify pathogens that are not specifically protected against complement. Danger-associated molecular patterns on its surface of pathogens are recognized by C1q, MBL, and ficolins allowing classical and lectin pathway activation, C3 convertase, C4b2a generation, and C3 cleavage. Opsonization due to covalent binding of C3b to the target is accelerated by the amplification loop of the complement pathways. The effector functions resulting from this complement activation are: pathogen lysis by C5b-9 membrane attack complex, opsonization and phagocytosis of the pathogen, activation of host immune and non-immune cells by complement anaphylatoxins, inflammation, stimulation of an adaptive immune response, and antibody generation. Secreted antibodies will bind to the pathogen and create immune complexes that will be recognized by C1q and will activate the classical pathway. Altogether these mechanisms contribute to pathogen elimination.

Figure 3

Classical and lectin pathway activation. (A) Activation of the classical pathway. Multiple adjacent IgG molecules are needed to bind C1q. IgG interacts with its target antigen forming specific circular structures. A single FAB binds to the antigen, while the other does not. The movement of the Fc domain exposes the C1q-binding sites allowing complementarity with the six globular domains of C1q (gC1q). The number of engaged IgG molecules will determine the compatibility of the immune complex with C1q and hence the strength of classical pathway activation. C1q circulates in plasma-associated with the serine proteases C1r and C1s, forming inactive C1 complex. After binding, the target C1q undergoes a conformational change to increase the angle between its collagenous stalks (CLR). This conformational change activates C1r, which in turn activates C1s. (B) Activation of the lectin pathway. MBL recognizes mannose containing sugars on pathogens. MBL circulates associated with serine proteases MASP-1 or MASP-2. Upon target binding, juxtaposition of MASP-2 and MASP-1 containing MBL complexes is required for MASP-1 to activate MASP-2.

Figure 4

Mechanism of C1 classical pathway activation and regulation. (A) Structural changes in C1q are necessary to induce auto-activation of C1r and activation of C1s. Upon binding of the inactive, closed C1 complex, electrostatic interactions with a target surface may alter the electrostatic field of the domain. This will induce a rotation of gC1q, leading to opening of the angle between the CLRs. A part of the binding site on gC1q apex will be lost, but new links will be formed with the side surface of the B-chain. (B) Concomitant with the structural changes in C1q, C1r₂C1s₂ complex will pass from closed, inactive eight-shaped conformation to an active, S-shaped conformation, allowing C1r auto-activation and further C1s activation by C1r. (C) The C1 inhibitor is a serpin that binds covalently to the active site of C1r and C1s, blocking their function. It also dissociates C1r₂C1s₂ from C1, releasing free C1q. C1 inhibitor also inhibits the lectin pathway by binding to MASP-1 and MASP-2. (D) MBL can bind to MASP-3, MAp44, or MAp19, which cannot cleave C4 and C2.

Figure 5

Platforms for complement activation. (A) Properdin is released from activated neutrophils and is bound to the cell membrane where it recruits C3b to form the alternative pathway C3 convertases. C5a then activates additionally neutrophils and they secrete more properdin. This installs a vicious cycle of neutrophil and complement activation. (B) Properdin released from neutrophils or in the plasma binds to activated platelets promoting C3(H₂O) recruitment and complement activation. (C) Stimulation of endothelial cells with C3a, heme, or other agonists induces expression of P-selectin. P-selectin contains CCP domains and binds C3b, promoting formation of C3 convertases that generate more C3a to stimulate cells.

Figure 6

Alternative pathway C3 convertase. (A) Structure and domain organization of the central complement component C3 and its cleavage fragments C3b and C3a. C3b is shown in two orientations to illustrate the surfaces containing the ANA domain and the opposite surface, carrying FB and FH binding sites. (B) Steps of formation of the alternative pathway C3 convertase. C3b is shown in green, FB in magenta, FD in yellow, C3a is in violet, and the substrate molecule C3 – in light green. For these molecules, the available crystal structures were used for the visualization. The C3bBbC3 complex is visualized based on molecular modeling. Properdin, for which a crystal structure is not available, is depicted in orange.

Figure 7

Formation of the C3 convertase by the classical and lectin pathways. (A) C1s or (B) MASP-2 will cleave C4 into bioactive fragment C4b that bind covalently to the surface of cells and interacts with C2. The small fragment C4a is released. Following, the same enzyme will cleave C2 to generate the classical pathway C3 convertase C4b2a. (C) C4b2a will interact with C3 cleaving it and releasing the bioactive fragments C3a and C3b. C3b will bind covalently to the surface and allow formation of alternative pathway C3 convertases C3bBb via the amplification loop. The C3a generated is a pro-inflammatory anaphylatoxin. The C4 molecule is presented in red, with brow colored the ANA domain, which will become C4a after cleavage and in green – the TED domain, which will become C4d after cleavage. The crystal strictures of C4, C4b, and C2a were used for the representation. The CP C3 convertase C4b2a is modeled based on the structure of the AP convertase C3bBb, with which it shares high homology.

Figure 8

Regulation of the classical and lectin pathways C3 convertase. To avoid overactivation, the CP and LP tightly regulate signaling. (A) If a C4b2a C3 convertase is formed, it will be rapidly dissociated by DAF and/or CR1 depending on the cell type. Bound C4b will be inactivated by FI in presence of cofactors such as CR1 and/or MCP. C4d will remain bound to the surface and C4c will be released. (B) C4BP can act in fluid phase as well as on the cell surface. C4BP has an octopus structure and interacts with several C4b molecules. It dissociates the C3 convertase and serves as a cofactor for FI in the cleavage of C4b to inactive fragments C4c and C4d. The structures of the complexes of C4b with the regulatory proteins have not been resolved yet, therefore the proteins are depicted in proximity one to another, represented by their known structures, but no complex could be reliably modeled.

Figure 9

Regulation of the alternative pathway. (A) FH as a master regulator of C3b in the fluid phase and on the cell surface. FH binds to C3b in fluid phase preventing novel convertase formation. FH may bind to C3b and GAGs on the cell surface and the architecture of the complex depends on the level of activation of the cell and the density of deposited C3 fragments. Resting cells have only a few C3b molecules that are deposited and FH binds to them with the regulatory domains CCP1-4. CCP7 and CCP20 interact with GAG on the membrane. Alternately, CCP19 may bind to the TED domain of C3b allowing CCP20 to interact with GAGs. If the cell is activated and C3b and C3d (or two C3b molecules) are deposited in close proximity, FH may bind to two of these molecules, allowing GAG binding by CCP20. (B) On resting cells, C3b will immediately be inactivated to iC3b by the action of FI and the assistance of cofactors (FH, MCP, CR1). iC3b cannot bind FB and forms C3 convertases. Only the cofactor CR1 allows FI to execute a second cleavage generating C3c (released in the fluid phase) and C3dg, which remains bound to the cell. C3dg is rapidly transformed to C3d by tissue proteases. (C) If the host cell is activated, the complement control will not be sufficient to prevent any complement deposition and C3 convertases could be formed. To avoid cell damage, these convertases need to be dissociated. Multiple complement regulators such as DAF, CR1, and FH decay the C3bBb complex formed on host cells. Remaining C3b will be inactivated by FI, using FH, MCP, or CR1.

Figure 10

Domain organization and mechanism of CFHRs. CFHR1, 2, and 5 carry dimerization N-terminal domains allowing them to form homo and heterodimers. These CFHRs, particularly CFHR1, are downregulators of FH, competing with FH for C3b and binding on the cell surface. This allow C3 convertases and MAC formation.

Figure 11

C5 convertase formation. A C5 convertase is generated when a C3b molecule binds covalently in the vicinity or directly to a C4b or C3b, already engaged in a C3 convertase. This new enzyme loses its capacity to cleave C3 and starts to cleave C5. The binding site of the second C3 molecule is unclear, but it has been suggested to bind to the TED domain of C4d and to the CUB domain of C3b. Since the atomic coordinates of the two C5 convertases have not been published yet, this figure represents the current model for their organization. The CP C3 convertase is modeled here on the basis of the structures of C4b, C2a, and the AP C3 convertase C3bBb. The second C3b molecule is depicted in a schematic representation in blue to be distinguished from the C3b molecule interacting with FB to form the C3bBb complex. The Bb and C2a fragments are depicted in magenta and violet, respectively. They are partially visible behind the C3b and C4b molecules.

Figure 12

The terminal complement pathway. The C5 convertase cleaves an inert molecule of C5 into a potent anaphylatoxin, C5a, and a bioactive fragment C5b. C5b interacts with C6, C7, C8, and multiple copies of C9 to form the membrane attack complex C5b-9 (MAC). C5b-8 inserts into the membrane and C9 polymerize to form a pore inducing Ca flux and pathogen lysis. Host cells are protected from lysis by expression of CD59, which prevents the insertion and by clusterin and vitronectin, which bind to C8 and prevents insertion in the membrane. If MAC is bound to the membrane, host cells can internalize it or remove it by ectocytose.

Figure 13

Complement anaphylatoxins. C3a and C5a have a four and three helical bundle topology. Mouse C5a (in the square) is different from its human counterpart, because it has four helical bundle structure. These anaphylatoxins bind to G protein-coupled receptors C3aR and C5aR and stimulate pro-inflammatory signaling pathways.

Figure 14

Complement receptors. CR1 is composed of CCP domains and is expressed primarily by immune cells and erythrocytes. Apart from being cofactor of FI, CR1 is also a complement receptor facilitating immune complex clearance and phagocytosis. CR1 interacts with C3b. CRIg has immunoglobulin-like structure in its C3b recognition domain. CRIg binds to C3b and iC3b and is expressed on macrophages and Kupffer cells. Immune cells also express CR3 and CR4 containing integrin domains that bind to iC3b (and C3d for CR3) on different binding sites on iC3b molecule. CRIg, CR3, and CR4 facilitate phagocytosis and modulate the activation state of cells. CR2 is expressed primarily on B-cells and recognizes C3d using the first two CCP domains. It serves as a co-stimulatory molecule for the B-cell receptor upon binding C3d-opsonized pathogen.

Figure 15

Understanding aHUS using structure–function relationships. (A) The role of complement alternative pathway in the physiopathology of aHUS. On healthy endothelial cells, deposited C3b is rapidly inactivated by regulatory molecules including FH, MCP, and FI. For FB binding and C3 convertase formation, FB is dissociated by FH and DAF preventing excessive host tissue damage. Mutations in the complement regulators FH, MCP, and FI can result in inefficient complement regulation. Mutations in the components of the C3 convertase (C3 and FB) induce the formation of overactive C3 convertase or a convertase that is resistant to regulation. In both cases, the complement cascade is activated on glomerular endothelial cell surface leading to endothelial damage, thrombosis, erythrocyte lysis, and aHUS. (B) FH mutations in the CCP19–20 region are mapped on the structure of the C3d–C3d–FH19–20 complex. A model GAG bound to FH CCP20 is indicated in gray. FH disease-associated mutations that decrease only C3b-binding are indicated in orange and mutations decreasing both C3b and GAG binding are in magenta. Reduced C3b and/or GAG binding will cause inefficient endothelial cell protection and complement overactivation. (C) C3 mutations found in aHUS patients. The majority of the mutations (in red) are not randomly distributed, but mapped to the FH binding sites on C3b. These mutations correlated with decreased FH and MCP binding allowing characterization of the MCP binding site, which overlaps with the FH binding site in CCP-3–4.