THE CLASSICAL COMPLEMENT PATHWAY
The classical pathway of complement activation is a mediator of the specific antibody response. It is triggered by antigen-bound antibody molecules. It is the binding of a specific part of the antibody molecule to the C1 component that initiates this pathway. This initial enzyme, C1, is a complex formed through a calcium-dependent association between two reversibly interacting subunits, C1q and C1r2s2 (2,3). Approximately 70% of C1 is at all times present in this complex form (4). C1 occurs in serum as a proenzyme which tends to undergo auto activation (5) but which is strictly controlled by C1 inhibitor (C1-In) (6).Upon the binding of C1 to immune complexes by virtue of the affinity of C1q for immunoglobulins (specifically IgM and IgG) (7), the controlling action of C1-In is overcome (6) and C1q effects activation of C1r2s2. C1q possesses no intrinsic catalytic activity, but when any of several activators bind to the C1q subcomponent of C1, the homologous C1r and C1s subcomponents are converted into catalytically active species, namely C1r* and C1s*, triggering the first step of the classical pathway of complement activation (8). Thus, on binding to immune complexes through C1q, the subunits of C1 become firmly associated and autoactivation commences even in the presence of the Cl-In. Initially, a conformational change in C1r occurs, followed by proteolytic activation which results in the cleavage of all four polypeptide chains of C1r2s2 (9). The two activated C1s subunits are then able to catalyse the assembly of the C3 convertase, C4b2a, which has been formed from C2 and C4.
THE C3 CONVERTASE
The initial step in the assembly of C4b2a, is the cleavage of the complement component C4. The component C4 is composed of three polypeptide chains, alpha (93kD), beta (75kD), and gamma (33kd) (10). C4 is a very sensitive substrate of C1s*. It is composed of two isotypes, C4A and C4B. These two isotypes both show clear differences in function, chemical reactivity and antigenicity despite the high sequence identity between their respective genes (about 99%)
(11). It seems that gene duplication may thus have conferred on C4 the ability to react with a wide range of different substances. C4 undergoes cleavage with the loss of a small fragment, C4a. The larger fragment formed by the cleavage reaction, C4b, develops a labile binding site allowing it to attach to antigens nearby (12). Only when the cleavage of C4 takes place in the presence of acceptor sites for C4b are C4b sites generated, for the further participation of C4 in the complement cascade. In the absence of such acceptor sites for C4b, the labile binding site on C4b is no longer available, and C4 becomes C4bi incapable of furthering the complement cascade (13).
The formation of a haemolytically active C4b site represents the binding site for C2, the second natural substrate of C1s*. C2, like C4 is encoded for within the major histocompatibility complex (MHC) (14) . Cleavage of C2 results in the formation of two fragments; a small fragment (C2b) which does not proceed further in haemolytic and opsonic processes, and a large fragment (C2a) which goes on to become part of the C3 convertase enzyme. Upon cleavage by C1s*, the C2a fragment becomes firmly associated with C4b, a reaction dependent on the presence of magnesium ions, and the C3 convertase, C4b2a, is generated (15). The formed C4b2a complex is now able to cleave the next component of the cascade, C3. In contrast to the C4bC2 complex, the newly formed C4b2a complex is no longer dependent on magnesium ions. The enzymatic site of the C3 convertase is located in the C2a molecule and has substrate specificity for C3. This unstable enzyme undergoes a time and temperature-dependent decay, lasting only several minutes, unless there is a sufficient quantity of C3 in the vicinity of the cell-bound complex to mediate the next site in the sequence. Decay is associated with the release of the C2a fragment, in a functionally inactive form, into the fluid phase. The remaining C4b site is now able to take up new native C2 and a new C4b2a enzyme can be formed. Upon cleavage of the C3 complement component, two fragments are produced; a small fragment C3a which is released and appears to be important in many inflammatory responses, as increased serum levels of C3a were found as a sign of complement activation in various inflammatory skin diseases particularly in psoriasis (16), and a larger fragment, C3b, which becomes covalently bound to the cell or bacterial surface and appears to be of great importance in the process of opsonisation. After the binding of the C3b component to the C4b component, the C3 convertase, C4b2a, becomes the C5 convertase, C4b2a3b (17).
THE C5 CONVERTASE
The C5 convertase is capable of co-ordinating with, and cleaving, the next component of the cascade, C5. The change in substrate specificity of C4b2a is accomplished by the "activation" of C5 by C3b and not by modulation of the enzyme (18). High-affinity C5 binding sites have been demonstrated on C3b4b dimers in which C3b was linked to the a chain of C4b through an ester bond (19). It is possible that this is the prevalent mode of C3b association with C4b2a within the C5 convertase structure on target cells. By implication, C5 may bind to both C3b and C4b before it is cleaved by the enzyme. A single cleavage in the C5 molecule leads to the formation of two fragments: C5a, a small fragment that has important biological activity but does not associate with the cell surface; and C5b, a large fragment that binds to the cell surface via a labile binding site. C5b is critical in initiating the lytic sequence of reactions, and plays an important role in directing the association of further late-acting components of the complement system which interact to produce a lesion in the bacterial surface leading to bacterial death (20). C5a has been found to be an inflammatory mediator which can act on target cells through a family of receptors linked to Gi proteins (guanine nucleotide-binding inhibitory protein) (21). Further activity of C5 is involved with the membrane attack complex which is discussed later.
REGULATORY PROTEINS
Both C3 and C5 convertase enzymes are regulated and rigidly controlled by fluid-phase and membrane-regulatory proteins. The following regulatory proteins function in both activation pathways with the exception of C4bp which is only involved in the classical pathway. The spontaneous decay-dissociation of these enzymes is enhanced by C4bp (C4b binding protein) (22), DAF (decay-accelerating factor) (23), CRI (C3b receptor) (24), and MCP (membrane cofactor protein) (25). C4bp and CR1 allow the degradation of C4b by Factor I (a glycosylated, disulphide-linked, two-chain serine protease of high substrate specificity found in serum and plasma as an active enzyme (26,27)). The normal concentration of the C4bp in serum is greater than the single-site dissociation constant of the C4bp-C4b interaction (28). Thus, under most conditions the concentration of the C4bp greatly exceeds the small amounts of C4b generated by the activation of the classical pathway and C4b is therefore inactivated. The only activity that DAF expresses is directed towards C3b, Bb (see alternative pathway components) and C4b2a. It prevents the assembly of the complexes and it disassembles the formed enzymes. DAF has no cofactor activity for I action on C3b or C4b and it does not function as a receptor, although it does possess a measurable affinity for C3b but not C4b (29). DAF can be inhibited by C3b located on the same cell, where just a few thousand molecules per cell have a significant effect. Finally, solubilised MCP has potent I-cofactor activity for fluid-phase C3b or C3b bound to solubilised molecules, but very weak cofactor activity for cell- or particle-bound C3b.
