The Complement System: Pathways and Activation (Part 4- Antibody Basics)
Welcome to the 4th part of the 13-part series on Antibody basics.
Part 1: Introduction to Antibodies and their basic structure
Part 2: Detailed Structure of Antibodies- Immunoglobulin Domains, CDRs and FRs
Part 3: Antibody classes and their functions
Introduction to the complement system
The complement system is a part of the immune system that enhances the ability of the antibodies and phagocytic cells to clear microbes and damaged cells from the body. The word “complement” is used because the complement system enhances or complements our immunity.
Jules Bordet first discovered the complement system as a heat-labile component of plasma that causes the killing of bacteria. Jules Bordet showed that the sheep antiserum caused the lysis of bacterium Vibrio cholerae, and upon heating the antiserum, its bacteriolytic activity was destroyed. But the ability of the heated antiserum to lyse the bacteria was restored by adding the fresh serum that contained no antibodies directed against the bacterium.
The serum containing no antibodies was otherwise unable to kill bacteria by itself. Thus, Bordet observed that the bacteriolytic activity of antiserum was mediated by two components:
1. Specific antibacterial antibodies which were heat resistant
2. And the other component which was heat sensitive.
Later, scientist Paul Ehrlich coined the term ‘complement’ for the heat-sensitive component, defining it as the activity of the blood serum that completes the action of antibodies. The complement system refers to a series of more than 20 proteins circulating in the blood and tissue fluids. The complement proteins are synthesized mainly by liver hepatocytes, although significant amounts are also produced by tissue macrophages, blood monocytes, and epithelial cells of the gastrointestinal and genitourinary tracts.
Most of the complement proteins that circulate in the serum are in functionally inactive forms as proenzymes or zymogens, and they come into effect only when activated. In response to the recognition of microorganisms, the complement proteins get sequentially activated in an enzyme cascade which means the activation of one protein enzymatically cleaves and activates the next protein in the cascade.
Nomenclature of complement proteins
Complement proteins are designated by capital letter C followed by a number from 1 to 9, for example, C1, C2, C3, till C9. The numbers in the names of complement proteins C1-C9 represent the order of their discovery. Some complement proteins are also designated by letter symbols, like Factor B and Factor D. Also, the complement proteins circulate in serum in inactive forms, and they come into effect only once activated. For the activation, the complement protein is cleaved, and the resulting peptide fragments are denoted by adding a lower suffix a and b. In most cases, the smaller fragment resulting from the cleavage of complement protein is designated as “a,” and the larger fragment is designated as “b”.
For instance, C3 on activation produces two peptide fragments C3a and C3b. In this case, C3a is the smaller fragment, and C3b is the larger fragment.
Note that among all the complement proteins, C2 is an exception because the cleaved fragment C2a is the larger fragment and C2b is the smaller fragment.
The larger fragment of the complement proteins binds to the target sites and contributes to the activation of other complement proteins in the cascade. At the same time, the smaller fragment of the complement proteins diffuses from the site and can initiate localized inflammatory responses by binding to the specific receptors. The larger complement fragments of different complement proteins interact with one another to form functional complexes leading to activation of the complement system.
Pathways of complement activation
The complement system can be activated by three pathways: the classical pathway, the alternative pathway, and the lectin pathway.
Classical pathway
Complement activation by the classical pathway begins with the formation of the Ag-Ab complex. In other words, the complement activation occurs upon binding of Ab to the Ag on a target such as a bacterial cell. The classical complement system is generally activated by IgM and certain IgG subclasses such as IgG1, IgG2, and IgG3.
There are primarily four complement proteins that participate in activating the classical complement pathway- C1, C2, C3, and C4. All these proteins are present in a functionally inactive form in plasma.
The numbers in the names of complement proteins C1, C2, C3, and C4 do not reflect the order in which they react; instead, they represent the order of their discovery. The formation of the Ag-Ab complex induces the conformational change in the Fc region of the IgM Ab that exposes a binding site for the C1 component of the complement system.
C1 protein is a macromolecular complex consisting of one molecule of C1q and two molecules each of C1r and C1s held together to form the C1qr2s2 complex stabilized by Ca2+. C1q molecule comprises 18 polypeptide chains that associate to form six collagen-like triple-helical arms and the globular heads.
The globular heads of C1q bind to the exposed C1q binding sites in the CH2 domain of the Ab molecule. Each C1 molecule must bind by its C1q globular heads to at least two Fc sites for a stable C1-Ab interaction to occur. Therefore, when attached to the Ag on a target, pentameric IgM assumes staple conformation in which at least three binding sites for C1q are exposed.
On the contrary, an IgG molecule contains only a single C1q binding site in the CH2 domain of the Fc region. Therefore, for a stable C1-IgG interaction to occur, at least two IgG molecules should be in close proximity on a target surface to provide two attachment sites for C1q.
Because of this reason, a single molecule of pentameric IgM bound to a red blood cell is sufficient to activate the classical complement pathway and lyse the cell. In contrast, some 1000 molecules of IgG are required to ensure that two IgG molecules are close enough to each other on the cell surface to initiate C1q binding.
1. 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 cleaves C1s to convert it into an active serine protease enzyme C1s. So now, the active C1s has two substrates C2 and C4.
2. C4 complement protein has three polypeptide chains- α, β, and 𝛾. When active C1s hydrolyses a small fragment C4a from the amino terminus of the α chain of C4, the resulting C4b fragment becomes active.
The binding site for C2 on C4b is now exposed, and C4b binds to the target surface in vicinity of C1. Then C2 proenzyme binds to the exposed binding site on C4b, followed by cleaving C2 into C2a and C2b by C1s active enzyme. The smaller fragment C2b diffuses away.
3. The resulting C4b2a complex is called C3 convertase, which can convert C3 complement protein into an active form. A single C3 convertase can generate over 200 molecules of active C3b.
Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b, which is now called C5 convertase. C5 convertase can further activate C5 complement protein by cleaving C5 into C5a and C5b.
C5a diffuses away, and C5b attaches to C6 and initiates the formation of the membrane attack complex.
Alternative pathway
The second pathway by which the complement system can be activated is the alternative pathway. Like the classical pathway, the alternative pathway also culminates in the formation of C5b, which then initiates the formation of a membrane attack complex to lyse the target cell. But it does so without the need for antigen-antibody complexes to activate the complement system. Because no Ab is required, the alternative pathway is considered a component of the innate immune system. This complement activation pathway involves four proteins- C3, Factor B, Factor D, and properdin.
Generally, this pathway is initiated by cell surface components that are foreign to the host, such as lipopolysaccharides from gram-negative bacteria cell walls, teichoic acids from gram-positive bacteria cell walls, and zymosan from fungal and yeast cell walls. Additionally, some viruses, parasites, virus-infected cells, tumor cells, etc., can also activate the alternative pathway of the complement system. In the classical pathway, C3 is cleaved into C3a and C3b by C3 convertase. But in the alternative pathway, the thioester bond of serum C3 undergoes slow spontaneous hydrolysis to yield C3a and C3b.
Now, this C3b component can bind to foreign surface Ags and not just foreign antigens; it can even bind on the host’s own cells. But the membrane of mammalian cells has high levels of sialic acid, which enables the rapid inactivation of C3b molecules on the host cells. Thus, the binding of C3b molecules on host cells rarely leads to activation of the complement system. On the other hand, foreign antigenic surfaces like bacterial and yeast cell walls, viral envelopes, etc., have low sialic acid levels; therefore, C3b bound to them remains active and can activate the complement system.
1. The C3b present on the surface of foreign cells binds to another serum protein called factor B, stabilized by Mg2+.
2. Binding to C3b exposes a site on factor B that makes factor B accessible to be cleaved by another serum protein called factor D. Factor D cleaves Factor B, generating two fragments. The smaller fragment Ba diffuses away, and the other larger fragment Bb makes a complex with C3b, and the complex C3bBb has C3 convertase activity.
3. This activity is analogous to the C4b2a complex of classical pathways. C3bBb complex has a half-life of 5 minutes unless the serum protein properdin binds to it, stabilizing the C3bBb complex, thus extending its half-life to 30 minutes. The C3bBb generated can activate unhydrolyzed C3 to generate more C3b. As a result, initial steps of the alternative pathway are again activated and amplified from binding of C3b to foreign cell surface to the formation of C3bBb to generate more C3b.
4. C3b generated makes a complex with C3bBb resulting in the formation of C3bBb3b, and this complex has C5 convertase activity analogous to C4b2a3b complex in the classical pathway.
C5 convertase can further activate C5 complement protein by cleaving C5 into C5a and C5b. The resulting C5a component diffuses away while the C5b component attaches to C6 and initiates the membrane attack complex formation.
Lectin Pathway
The third pathway by which the complement system can be activated is the lectin pathway. As the name indicates, the lectin pathway of complement activation is initiated by the lectins. Lectins are the proteins that can recognize and bind to the specific carbohydrate residues on the target cells. The lectin that activates the complement system binds to mannose residues; therefore, this pathway is also named the MBLectin pathway or Mannan binding lectin pathway. Like the alternative pathway, the lectin pathway does not depend on Ab for its activation.
1. The lectin pathway is activated when mannose-binding lectin (MBL) binds to the mannose residues on glycoproteins or carbohydrates on the surface of microorganisms like Salmonella, Neisseria, Listeria strains, Candida albicans, etc. Additionally, the mannose-binding lectin protein has activity analogous to the C1q protein of the classical pathway.
2. After MBL binds to the surface of a pathogen, MBL associated serine proteases bind to mannose-binding lectin. These MBL associated serine proteases are MASP-1 and MASP-2.
3. The active complex formed by the MASP-1 and MASP-2 proteins causes cleavage and activation of C4 and C2 proteins. MASP-1 and MASP-2 have activity similar to C1r and C1s of the classical pathway. C4 is cleaved into C4a and C4b; and, C2 is cleaved into C2a and C2b.
C4b2a complex has C3 convertase activity, which cleaves and activates C3.
5. Further, like the classical pathway, the C4b2a3b complex is formed, with C5 convertase activity resulting in the formation of C5b. C5b attaches to C6 and initiates the formation of the membrane attack complex.
Membrane Attack Complex
The end result of all the three complement pathways: classical, alternative & lectin pathway is the production of an active C5 convertase which cleaves C5 into smaller fragment C5a, which diffuses away, and the larger fragment C5b, which binds to the surface of the target cell. Now, this C5b component provides a binding site for the subsequent components C6, C7, C8, and C9, which interact sequentially to form a macromolecular structure called Membrane Attack complex abbreviated as MAC. MAC creates a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse freely across the membrane, thus disrupting the cell membrane of the target cells, which in turn leads to cell lysis and death of these cells.
1. The C5b component is highly labile and becomes inactive within 2 minutes unless C6 binds to it and stabilizes its activity.
2. All the complementation reactions, till the binding of C6 to C5b, take place on the hydrophilic surface of the target membrane. But as soon as C5b6 binds to C7, the resulting complex undergoes a hydrophilic amphiphilic structural transition to expose the hydrophobic site on the C5b67 complex, which mediates its binding to the phospholipid bilayer of the target cells. As a result, the C5b67 gets inserted into the phospholipid bilayer of the target cell.
3. Binding of C8 to the inserted C5b67 complex also induces a conformational change in C8 such that C8 also undergoes a hydrophilic-amphiphilic structural transition. It exposes its hydrophobic region, which mediates the insertion of C8 into the phospholipid bilayer of the target cell. After insertion of C8, the C5b678 complex creates a small pore of diameter 10 Å in the target cell.
4. The final step of membrane attack complex formation involves the binding and polymerization of C9 molecules to the C5b678 complex. Around 10–17 molecules of C9 can bind and get polymerized by a single C5b678 complex. During polymerization, C9 molecules also undergo a hydrophilic-amphiphilic transition, thus exposing their hydrophobic region, which facilitates the insertion of C9 molecules into the target cell’s membrane. The whole C5b678(9)10–17 complex is known as the membrane attack complex.
5. The complete membrane attack complex forms a channel with a pore size of 70–100 Å in diameter in the target cell, through which small ions and molecules can diffuse freely. Membrane attack complexes create such pores all over the microbial cell membrane. These pores, in turn, compromise the membrane integrity of the pathogen. The creation of these pores leads to the loss of essential electrolytes from the microbial cell. Simultaneously these pores also allow the inflow of extracellular fluid like water, ions, and other small molecules into the microbial cell, because of which the microbial cell can not maintain its osmotic stability and eventually gets lysed and killed. So, this is the complete mechanism of how the complement proteins kill pathogens or other target cells such as tumor cells.
Refer to Part 5 for knowing about the regulation and functions of the Complement System.
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