The complement system represents one of the most ancient cornerstones of innate immunity. It consists of over 40 soluble and membrane bound proteins. This highly integrated network, consisting of many signaling molecules and regulators, acts as an intricate immune surveillance system to discriminate between healthy host tissue, unwanted cellular debris, apoptotic cells, necrotic cells and microbial invaders.
Traditionally, activation of the complement system is achieved through three distinct initiation pathways: classical (CP), lectin (LP), and alternative (AP). These cascade-like pathways all culminate to the formation of the convertases, the major enzyme of complement activation, which converge to a common effector C3, generating opsonin C3b and anaphylatoxin C3a. Further activation leads to the formation of C5 convertases which direct the assembly of the terminal complement complex (C5b-9), a lytic pore which assembles on membranes to mediate cell destruction. Recently, a new proteolytic pathway in which thrombin acts as a C5 convertase cleaving C5 to the anaphylatoxin C5a, has been described.
The classical pathway is initiated by the calcium dependent C1 complex through its pattern recognition molecule C1q. Classical pathway is strongly initiated through binding of C1q to IgG or IgM clusters. As such, it is often referred to as the antibody-dependent pathway. However, C1q is also capable of recognizing distinct structures directly on microbial cells. In addition to endogenous pattern recognition molecules (immunoglobulins, C-reactive protein), C1q can also bind to prions, DNA, late apoptotic, and necrotic cells. C1q binding leads to conformational changes in the C1 complex allowing interaction between the C1r and C1s proteases. C1r undergoes autoproteolytic activation cleaving the zymogen of C1s. C1s is a highly specific protease which cleaves C4 into C4a and C4b, and C2 into C2a and C2b to form the classical pathway C3 convertase (C4b2b). C4b also opsonizes targets further directing C3 convertase assembly on membrane surfaces. This convertase can cleave C3 to initiate amplification and downstream functions of complement amplification.
The lectin pathway is initiated through recognition of carbohydrates on microbial surfaces by Mannose-binding lectin (MBL) or ficolin. MBL belong to the collectin family of proteins which contain a carbohydrate recognition domain and a collagen-like domain. The carbohydrate recognition domain of MBL binds to carbohydrates with 3- and 4- hydroxyl groups in the pyranose ring in a calcium dependent manner. This allows for effective recognition of carbohydrate surfaces (e.g. N-acetyl-glucosamine [GlcNAc]) on pathogens and leaves mammalian glycoproteins (e.g. galactose and sialic acid), which do not fit this steric requirement, virtually undetected. Ficolins, like MBL, contain a collagen-like stem structure, however, they also contain a fibrinogen-like domain. Serum ficolin are lectins that have similar binding specificity as MBL.
MBL-associated serine proteases (MASP) are a serine-protease superfamily (MASP1, MASP2, sMAP/MAp19, MASP3). Structurally, they are similar to the proteolytic components (C1r/C1s) of the classical pathway. Binding of MBL or ficolins to carbohydrates activates lectin-pathway MASP through mechanisms that are currently unknown. MASP2, like C1s, cleaves C4 and C2 to form the C3 convertase (C3b2b), which is common to the CP C3 convertase. In contrast, MASP1 can cleave C2 but is unable to cleave C4, suggesting that it can enhance the activation of lectin pathway triggered by MBL-MASP2 complexes but not initiate lectin pathway itself.
In contrast to the inducible lectin and classical, the alternative pathway (AP) is constitutively active and interacts with cell surfaces for constant immune surveillance. Initiation of AP begins in the fluid phase where C3 is hydrolyzed to C3H2O, exposing new binding sites to allow for rapid probing of cells in a process commonly referred to as the tick-over pathway. Complement factor B (CFB) protease binds to C3H2O and is cleaved by complement factor D (CFD), in the presence of Mg2+, releasing the complement factor B amino terminal fragment (Ba) and activating the serine protease domain (Bb), to form fluid phase C3 convertase (C3H2OBb). Fluid phase C3H2OBb cleaves C3 into C3a and C3b. C3b binds to amine and carbohydrate groups on cell surfaces and engages CFB. Once again, CFB is cleaved by CFD, in the presence of Mg2+, to form the AP C3 convertase (C3bBb). The half-life of the C3bBb complex is shortlived (T1/2 ~ 90 sec) but is stabilized 5 to 10 fold by association with properdin (CFP) to form a stable C3 convertase (C3bBbP).
Amplification and Terminal Pathway
All surface bound C3 convertase, regardless of origin, induces amplification of AP by cleaving C3 to deposit C3b in the vicinity of complement activation. In the presence of CFB and CFD, this allows for an efficient cycle of C3 cleavage and AP convertase assembly. Despite its name, the alternative pathway contributes to 80-90% of the final C5a generation, regardless of the initial triggering cascade. Amplification of complement by AP causes a rapid increase in C3 cleavage and C3b deposition onto surfaces. Deposition of C3b onto C3 convertases drives the formation of the C5 convertases (C4b2b3b/C3bBb3b) shifting substrate specificity from C3 to C5. C5 is cleaved into C5a, a powerful anaphylatoxin, and C5b, which directs a non-enzymatic assembly of the terminal pathway components C6, C7, C8, and C9 (C5b-9) to form the cell lytic terminal complement complex (TCC). Thus, activation of complement through its various initiating pathways can trigger a cascade which leading to the formation of C5b-9.
Other Activation Pathways
In addition to the three traditional pathways of complement activation, direct cleavage of C3 or C5 can also occur through a number of extrinsic proteases. In an in vitro assay, Vogt demonstrated that leukocyte elastase can cleave C5 to form biologically active C5b-6 like complex to initiate formation of TCC. Using a C3-/- mice, Huber-Lang et al, demonstrated that thrombin can cleave C5 to produce biologically active C5a/C5b in the absence of C5 convertase. Other proteases that have been known to cleave complement C3/C5 include plasmin and plasma kallikrein. These extrinsic pathways for complement activation suggest that there is cross talk between complement, innate immune cells and coagulation.