The Complement System: Regulation and Functions (Part 5- Antibody Basics)
Welcome to the 5th part of the 13-part series on Antibody basics.
Previous parts: Part 1, Part 2, Part 3, and Part 4
Refer to Part 4 to know what is the Complement System, its pathways and how they are activated to form Membrane Attack Complex or MAC to kill pathogens or other target cells such as tumor cells.
Regulation of the complement system
Undoubtedly the complement system serves as an essential and effective defense mechanism to destroy the invading microorganisms. But there are many elements of the complement system that are capable of attacking not only the foreign cells and pathogens but also the host cells. It leads to the lysis and killing of host cells. For instance, the C5b67 complex, if it gets released from the target cells, poses a threat to nearby healthy cells by lysing and killing them. Thus, several regulatory proteins in the body restrict the complement activity to the target cells only.
The regulation of the complement system occurs at three stages:
1. The first stage is regulation before the assembly of C3 convertase
2. The second stage is regulation after the assembly of C3 convertase
3. And the third stage is regulation at the assembly of the Membrane attack complex.
1. Regulation before the assembly of C3 convertase
(a) Prevents activation of C2 and C4 proteins:
The first regulatory protein in the classical pathway of complement activation that prevents the assembly of C3 convertase is the glycoprotein C1 inhibitor designated as C1Inh. As the name indicates, the C1 inhibitor inhibits the activity of the C1 protein. As discussed in Part 4, C1 in serum is a macromolecular complex consisting of C1q, and two molecules of C1r and C1s are held together in a complex stabilized by Ca2+. When C1q binds to Fc sites of Ab, a conformation change is induced in C1r that converts C1r into an active serine protease C1r, which then cleaves C1s to convert it into an active serine protease enzyme C1s. Now the active C1s cleaves the substrate C4 into C4a and C4b. The smaller fragment C4a diffuses away while, the larger fragment C4b becomes active.
Then the C4b binds to the C2 proenzyme, followed by cleaving C2 by C1s active enzyme. The resulting C4b2a complex is called C3 convertase, which can convert C3 complement protein into an active form.
This step is highly regulated so that C3 convertase is activated only when the body sees any foreign pathogen or some damaged or tumor cells that are required to be destroyed by the immune system. In the absence of regulation, the spontaneous activation of C3 convertase can even kill the host cells.
To regulate this step of the classical pathway of complement activation, the regulatory protein C1 inhibitor (C1Inh) forms a complex with C1r2s2, which in turn causes the C1r2s2 to dissociate from C1q.
The dissociated C1r2s2 loses the ability to activate C2 and C4 proteins. The inactivated C2 and C4 proteins then lose their ability to form C3 convertase, thus inhibiting the classical pathway of complement activation.
(b) Prevents assembly of C3 convertase:
Classical pathway: It has already been discussed in Part 4 that the reaction catalyzed by the C3 convertase enzyme of classical, alternative, and lectin pathways is the major amplification step in complement activation, generating hundreds of molecules of C3b.
C3b generated can bind to nearby healthy cells, mediating their damage either
· by causing their phagocytosis by C3b receptors bearing phagocytic cells
· or by causing their lysis by initiating the membrane attack complex formation.
As already discussed in the alternative pathway of complement activation (Part 4), the membranes of mammalian cells have high levels of sialic acid. The presence of sialic acid leads to the rapid inactivation of C3b molecules on the host cells; thus, the binding of C3b molecules on host cells rarely leads to the activation of the complement system.
The destruction of healthy host cells by C3b is further limited by a family of regulatory proteins, known as regulators of complement activation, abbreviated as RCA. The RCA proteins include C4b binding protein abbreviated as C4BP, complement receptor type I abbreviated as CR-1, and membrane cofactor protein abbreviated as MCP. Each of these regulatory proteins can bind to C4b and prevent the assembly of C3 convertase. Once these regulatory proteins are bound to C4b, another protein factor I cleaves C4b into C4d and C4c. As a result, the cleaved C4b is unable to associate with C2a to form a C3 convertase.
Alternative pathway: In the case of the alternative pathway, a similar regulatory sequence prevents the assembly of C3 convertase C3bBb. CR1, MCP, or factor H binds to C3b and prevents its association with factor B. Once any of these regulatory proteins are bound to C3b, factor I cleaves C3b into iC3b and C3f fragments. Thus, the cleaved C3b is unable to associate with Factor Bb to form C3 convertase.
2. Regulation after assembly of C3 convertase
The second stage of regulation of the complement system is after assembly of the C3 convertase. Several regulators of complement activation can also act on assembled C3 convertase causing it to dissociate. These include already mentioned RCA proteins C4BP, CR-1, and factor H and, in addition, decay-accelerating factor abbreviated as DAF. These proteins bind to the assembled C3 convertase (C3bBb) and then dissociate the complex. Once the dissociation of C3 convertase occurs, factor I cleaves C3b into iC3b and C3f fragments. The cleaved C3b fragments then lose the ability to associate with Bb proteins to form active C3 convertase.
On the other hand, in the case of the classical pathway, when RCA proteins bind to the assembled C3 convertase (C4b2a), they dissociate the C3 convertase. The released C4b is then cleaved by Factor I into C4c and C4d fragments. As a result, the cleaved fragments lose their ability to associate with C2a to form C3 convertase.
3. Regulation at the assembly of Membrane Attack Complex (MAC)
(a) The third stage of regulation of the complement system is at the level of membrane attack complex. If the C5b67 complex of MAC gets released from target cells, it can pose a threat to nearby healthy cells as it can mediate their lysis. But the regulatory proteins prevent this event from occurring. A regulatory serum protein known as S protein binds to the released C5b67 complex. It induces hydrophilic transition in the complex, thus preventing the insertion of the C5b67 complex into the membrane of the nearby cells.
(b) The other regulatory proteins, homologous restriction factor, abbreviated as HRF, and membrane inhibitor of reactive lysis abbreviated as MIRL, protect cells from non-specific complement-mediated lysis. These proteins bind to C5b678 and prevent the assembly of poly-C9, which in turn blocks the formation of MAC. It is how our body regulates the activation of the complement system to kill specific target cells and prevent the non-specific killing of host cells.
Other functions of complement proteins
Though the complement system has an enormous role in the lysis and killing of pathogens and damaged cells, various peptides produced during complement activation play other essential functions.
1. Opsonization: First is opsonization which is facilitated by C3b larger fragments. Opsonization is the process by which pathogens, or other target cells, are marked for ingestion and are then eliminated by the phagocytes like macrophages and neutrophils. The molecules that promote opsonization are called opsonins.
C3b is the major opsonin of the complement system, although C4b and iC3b also have opsonization activity. Phagocytic cells express complement receptors CR1, CR3, and CR4 that bind to C3b, C4b, and iC3b, respectively.
Antigen coated with C3b molecules binds to phagocytic cells bearing CR1 receptors. Once bound, phagocytosis of C3b coated Ag occurs by the phagocytic cells. This process is called opsonization.
2. Inflammation: The second role of complement components is in inflammation. The cleaved products of complement components can mediate inflammation and secretion of immunomodulatory molecules. For instance, the smaller fragments C3a, C4a, and C5a resulted from the cleavage of C3, C4, and C5 proteins, respectively, are called anaphylatoxins. These bind to receptors on mast cells and basophils and then induce degranulation. As a result, histamine and other pharmacological active mediators are released, mediating a local inflammatory response.
Anaphylatoxins can also cause smooth muscle contraction and increased vascular permeability, which results in the influx of fluid that carries Abs and phagocytic cells to the site of antigen entry.
3. Clearance of immune complexes: The third important function of complement components is to clear immune complexes from circulation. C3b molecules can attach to the Fc regions of the antigen-bound antibodies. The immune complexes coated with C3b are then able to bind to the CR1 receptors on erythrocytes. The erythrocytes carry these immune complexes to the spleen and liver. In the spleen and liver, these immune complexes are removed from the red blood cells, which are then phagocytosed by phagocytic cells such as macrophages and neutrophils, thus preventing their deposition in the tissues.
In the case of complement deficiencies, immune complexes don’t get cleared from the circulation and instead get deposited into tissues, thus leading to tissue damage. Moreover, the complement deficiencies can also predispose an individual to autoimmune diseases.
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