Molecular Biology of the Cell - Volume 3 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

From Cells to Multicellular Organisms
The Immune System
The Complement System

The Complement system, named for its ability to complement and enhance the action of Antibodies, serves as a primary weapon used by antibodies to protect vertebrate hosts against most bacterial infections. It consists of a system of Serum Proteins that can be activated by antigen-antibody complexes or microorganisms, triggering a cascade of proteolytic reactions that ultimately culminates in the assembly of membrane-attack complexes. These complexes punch holes in the target microbial membrane, thereby destroying The Cell. Simultaneously, proteolytic fragments released during activation amplify the defense response by diluting and widening Blood Vessels and recruiting phagocytic Cells to sites of infection. Furthermore, complement enhances the ability of phagocytes to bind, ingest, and destroy the microorganisms under attack.

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Fig. 18-39. An individual lymphocyte can be functionally linked to other lymphocytes through idiotype-anti-idiotype interactions. The scale of such an idiotypic network can theoretically be vast, since each lymphocyte interacting with an anti-A lymphocyte, as shown in the figure, can similarly react with other lymphocytes. It remains unclear to what extent anti-idiotypic interactions are important for the Regulation of Immune responses.

Individuals with a deficiency in one of the major complement components (C3) are prone to frequent bacterial infections, much like individuals with antibody deficiencies themselves. Complement-deficient patients may also develop DISEASES ASSOCIATED WITH the deposition of immune complexes (antigen-antibody complexes) in small Blood vessels of the Skin, joints, Kidneys, and Brain, where these complexes cause inflammation and tissue damage. Therefore, it is thought that complement normally promotes the clearance and dissolution of such complexes when they form during an Immune Response.

18.5.1. Complement Activation Is an Amplifying Mechanism Mediated by a Proteolytic Cascade; Cleavage of the C3 Protein Plays a Central Role [27]

Complement is composed of approximately 20 interacting proteins; some of these are reacting components (C1 through C9, Factor B, and Factor D), while the remainder are regulatory proteins. All of these are soluble proteins produced primarily by The Liver and circulating in the blood and extracellular fluid. Most of them remain inactive until triggered either directly by invading microorganisms or indirectly through the immune response. The ultimate result of complement activation is the association of the late complement components (C5, C6, C7, C8, and C9) into a large membrane-attack complex that causes lysis of the microbial cell.

Because The primary function of complement is to attack microbial cell membranes, its activation occurs mainly On the surface of the invading microorganism, where it is triggered either by antibodies bound to the cell or by Polysaccharides of the MICROBIAL Cell wall. In both cases, early complement components are activated. There are two sets of early components belonging to two distinct pathways of complement activation: C1, C2, and C4 belong to the classical pathway, which is triggered by antibody binding; Factor B and Factor D belong to The alternative pathway, which is triggered by microbial polysaccharides. The early components of both pathways ultimately converge on C3, the most crucial component of the complement system (Fig. 18-40). The early components and C3 are proenzymes sequentially activated by Limited proteolysis. When any of them is specifically cleaved, it becomes an active Serine protease capable of cleaving the next proenzyme in the cascade, and so on. Many of these proteolytic events result in the release of a small peptide fragment and the exposure of a membrane-binding site on the larger fragment. The large fragment firmly attaches to the target cell membrane via this site and drives the next step in the reaction chain. Thus, the complement activation process is largely restricted to the cell surface where it began. The small peptide fragment frequently acts independently as a diffusible signaling molecule that stimulates the inflammatory response (Section 18.5.4).

The activation of C3 via its cleavage is the central reaction of the complement cascade; this is where the classical and alternative pathways converge (see Fig. 18-40). In both cases, C3 is cleaved by an enzyme complex called C3 convertase. The two distinct pathways generate different C3 convertases, which are formed by the spontaneous association of two complement components activated earlier in the pathway. Both convertases cleave C3 into two fragments. The larger of these (C3b) binds covalently to the target Cell Membrane and recruits C5. Once bound, the C5 protein is cleaved by the C3 convertase (which now acts as a C5 convertase), thereby initiating the spontaneous assembly of the membrane-attack complex from the late components, C5 through C9.

Fig. 18-40. MAIN STAGES OF complement activation via the classical and alternative pathways. Complement activation reactions in both cases typically occur on The surface of an invading microorganism, such as a bacterium.

Because each activated enzyme cleaves many molecules of the subsequent proenzyme, the proteolytic cascade of early components acts as an amplifier: each molecule activated at THE START OF the pathway triggers The formation of numerous membrane-attack complexes.

18.5.2. The Classical Pathway Is Activated by Antigen-Antibody Complexes [27, 28]

The classical pathway is typically activated when IgG or IgM antibodies bind to Antigens on the surface of a microorganism. Antigen binding to antibodies in turn causes the constant domains of the antibodies to bind the first component of the classical pathway, C1, which is a large complex composed of three subcomponents—C1q, C1r, and C1s (Fig. 18-41). The binding of the globular "HEAD" of C1q to an IgG or IgM molecule attached to an antigen activates C1q, triggering the early proteolytic cascade of the classical pathway. However, more than one head must be engaged in this manner before activation can occur; therefore, triggering the classical pathway requires a cluster of foreign antigenic determinants (Fig. 18-41B). This requirement also serves to focus complement activation on the surface of microorganisms, where antigenic determinants are typically densely clustered. Activation of the C1q subcomponent within the C1 complex activates C1r, which acquires proteolytic activity and in turn cleaves and activates C1s. Activated C1s then cleaves C4 into two fragments, C4a and C4b (by convention, the larger fragment resulting from such proteolysis is designated b, and the smaller fragment a); C4b immediately attaches covalently to the membrane and subsequently binds C2. Once bound, C2 is also cleaved by activated C1s; the larger fragment, C2b, remains bound to C4b, forming the C4b2b complex, which Functions as the classical pathway C3 convertase. As shown in Fig. 18-42, C4b2b cleaves C3 into two fragments, C3a and C3b. The C3b fragment rapidly binds to the target membrane and recruits C5. The bound C5 is cleaved by the C4b2b complex into C5a and C5b (recall that the C3 convertase also acts as a C5 convertase). The C5b fragment associates with C6, thereby initiating the assembly of the late components to form the membrane-attack complex.

Fig. 18-41. A. The unusual Structure of C1q. It is a large protein (molecular mass ~450,000) composed of six identical subunits, each of which in turn consists of three different polypeptide chains. The C-terminal halves of all three chains in each subunit form a globular structure; the N-terminal halves have an Amino Acid Sequence typical of Collagen and are wound into a collagen-like triple helix (see Section 14.2.6). All six subunits are linked together by Disulfide Bonds between their triple-helical "tails," forming a bouquet-like structure reminiscent of tulip heads. IgG or IgM antibodies can bind to the globular heads of this structure. Thus, each C1q molecule has six antibody-binding sites. B. Binding of C1 to two IgG molecules attached to a cluster of antigenic determinants on the target cell surface. Each C1 complex consists of a single C1q molecule loosely associated with a tetramer composed of two C1r and two C1s molecules (the tetramer is shown schematically).

Fig. 18-42. Generation of C3 convertase in the classical and alternative pathways. In both cases, the C3 convertase is formed by the association of two complement components activated earlier in the proteolytic cascade. The C3 and C5 proteins are homologous. Upon binding C3b, C5 becomes structurally analogous to C3 and is cleaved by C3 convertase (acting as a C5 convertase) to yield C5b, which initiates the assembly of the membrane-attack complex. Activated complement components are often denoted with a bar above (e.g., activated C1 as C1); to simplify the text, we have omitted this notation here.

18.5.3. The Alternative Pathway Can Be Directly Activated by Microorganisms [27, 29]

Cell wall polysaccharides of microorganisms can directly activate the alternative pathway even in the absence of antibodies. Activation of the classical pathway also triggers the alternative pathway via a positive feedback loop. Thus, the alternative pathway provides a first line of defense against infection before an adaptive immune response is established, and it amplifies the action of the classical pathway once an immune response has begun.

Although we speak of microbial polysaccharides directly activating the alternative pathway, this is merely a simplified model. In reality, the pathway is activated by the C3b fragment, and The Role of the polysaccharides is to protect C3b from rapid inactivation by regulatory mechanisms that shield surrounding normal host cells from attack by activated complement (see below). The C3b that drives the alternative pathway is generated either via the classical pathway (hence the positive feedback loop) or through the spontaneous proteolysis of C3, which occurs at a low rate even when the complement cascade is not triggered.

The next step in the activation of the alternative pathway is the binding of Factor B to membrane-bound C3b. Subsequently, Factor D, which circulates in the blood in an active form, cleaves the bound Factor B to yield the active fragment Bb, resulting in the formation of C3b,Bb—the alternative pathway C3 convertase—which generates additional molecules of C3b; some of these then bind C5. As in the classical pathway, this C3 convertase can also function as a C5 convertase. The convertase cleaves membrane-bound C3 molecules and thereby initiates the assembly of the membrane-attack complex (see below, Fig. 18-43).

Damage to the membranes of normal host cells by complement activated via the classical pathway is prevented by the requirement for bound antibodies to activate C1. The alternative pathway, however, is triggered by the C3b fragment, which can bind to any membrane and, as mentioned earlier, is generated spontaneously at a low rate. Therefore, special protective mechanisms are required to shield normal host cells from such attacks. Unlike the large clusters of C3b formed via the classical pathway, most spontaneously generated C3b binds to membranes as isolated single molecules. Here, they are rapidly inactivated by a specific inhibitory protein, Factor H, which competes with Factor B for the binding site on C3b. By contrast, C3b bound to microbial surfaces is protected from this inhibitory protein (The Mechanism of protection remains unknown) by the polysaccharides of the microbial cell wall. Consequently, these C3b molecules are able to bind Factor B and thereby activate the alternative pathway.

18.5.4. Complement Activation Promotes Phagocytosis and Inflammation [27]

The C3b fragment generated by both the classical and alternative pathways possesses A number of important properties. As we have seen, it feeds into the alternative pathway to generate additional quantities of C3b and binds C5, ensuring its cleavage by C3 convertase. However, complement does more than simply assemble membrane-attack complexes; C3b plays a third crucial role by attaching to specific receptor proteins on macrophages and neutrophils, thereby enhancing the ability of these cells to phagocytose the microbe to which C3b is bound. Thus, C3b makes a vital contribution to antibacterial defense independent of complement-mediated cell lysis.

During the proteolytic cascade of complement activation, several small, biologically active protein fragments are released. Among these, C3a and C5a are the most prominent. Both of these fragments are strongly basic Peptides cleaved from the N-termini of the parent proteins; both induce smooth Muscle contraction and stimulate histamine secretion by mast cells and basophils. Histamine locally increases blood vessel permeability, allowing leukocytes and additional amounts of antibodies and complement proteins to penetrate infection sites. C5a also acts as a potent chemoattractant for neutrophils and enhances the mechanisms by which these cells destroy engulfed Bacteria. These two peptides are primarily responsible for the local inflammatory response that typically accompanies complement activation.

18.5.5. The assembly of the late complement components yields a giant membrane-attack complex [27, 30]

The assembly of the late components begins when C5 (already loosely bound to C3b on the target cell membrane) is cleaved by the classical or alternative pathway C3 convertase, generating C5a and C5b. As we have just seen, C5a is released and promotes an inflammatory response. C5b remains bound to C3b and temporarily gains the capacity to bind C6, forming C56, and then C7, to form C567. Subsequently, the C567 complex binds firmly to the membrane via C7. One molecule of C8 is then added to this complex, and the resulting C5678 complex binds 8 to 18 molecules of C9, which partially unfold and polymerize to form a transmembrane channel (Fig. 18-43). Sequence analysis of cloned DNA suggests that the C6, C7, C8, and C9 proteins are evolutionarily related to one another.

Fig. 18-43. Assembly of the late complement components into a membrane-attack complex. The binding of a C9 molecule to C5678 induces a conformational change in C9 that exposes a hydrophobic region and drives the insertion of C9 into The Lipid Bilayer of the target cell adjacent to C8. This initiates a chain reaction: the modified C9 binds a second C9 molecule, which undergoes a conformational change and inserts into the bilayer, where it can bind a further C9 molecule, and so on. In this way, a chain of C9 molecules forms a large transmembrane channel. The final C5678(9) complex can contain up to 18 molecules of C9 and has a molecular mass of approximately 2 million (each C9 molecule contains 537 amino acid residues). The C5678 complexes alone form small transmembrane pores.

When viewed by negative-staining Electron Microscopy, membrane-attack complexes exhibit a characteristic appearance: they form Water-filled pores that pierce the membrane (Fig. 18-44). For this reason, and owing to the disruption of the adjacent lipid bilayer, the membrane becomes highly permeable. Because small molecules can pass through the membrane near and through the complexes while macromolecules remain trapped inside the cell, the cell's normal osmotic regulatory mechanisms fail (see Scheme 6-1, Vol. 1). Consequently, the cell takes up water by osmosis, causing it to swell and burst. This process is so efficient that very few membrane-attack complexes (perhaps even a single one) can kill an erythrocyte. These complexes can even disrupt enveloped Viruses, which lack a large osmotic pressure gradient across their membrane and are therefore not susceptible to osmotic lysis; this likely results from general disorganization of the viral membrane.

18.5.6. The complement cascade is tightly regulated [27, 31]

Because the complement cascade acts as an amplifier and can lead to cell destruction, key active components must be inactivated shortly after their generation to prevent the attack from spreading to nearby host cells. This is achieved in at least two ways. First, specific inhibitory proteins present in the blood halt the cascade by binding to or cleaving certain components as soon as they are activated by proteolysis. For instance, some inhibitory proteins bind to the activated Components of the C1 complex and shut down their activity, whereas others cleave and thereby inactivate C3b. Without these inhibitors, all serum C3 could be rapidly depleted through the positive feedback loop of the alternative pathway.

The second major regulatory mechanism relies on the instability of many activated cascade components: unless they bind immediately to another specific component or to a nearby membrane, they are rapidly inactivated. Striking Examples of this are activated C4b and C3b. When either is generated by the cleavage of its precursor, it undergoes a series of rapid conformational changes that yield a short-lived active form. This active form features a hydrophobic region as well as a highly reactive glutamine side chain, which is exposed when an unusual thioester bond within the protein is mechanically broken (Fig. 18-45). As a result, the glutamine forms a covalent bond with a protein or polysaccharide on a nearby membrane. Because the half-life of the active forms of C4b and C3b is extremely short (less than 0.1 ms), they usually manage to bind only to membrane patches located immediately adjacent to the site of complement activation. Consequently, the complement attack is restricted to the surface membrane of the microbe and does not spread to surrounding normal host cells.

Fig. 18-44. Holes in The Plasma Membrane of an erythrocyte resulting from complement action. Electron micrographs (negative contrast). In micrograph A, a hole is viewed from above; in B, from the side. The contrast medium fills the individual channels, causing them to appear dark. (R. Dourmashkin, Immunology, 35, 205-212, 1978.)

Fig. 18-45. Proteolytic activation of C3 or C4 leads to a conformational change in the protein. This process breaks the unusual intramolecular covalent bond indicated in the figure. The rupture of this thioester bond generates a highly reactive carbonyl group between the protein side chains, which can covalently attach to another macromolecule via an ester or amide bond. However, the protein's ability to react in this manner decays with a half-life of about 60 μs; therefore, it binds only to membranes located very close to the site where complement activation was initiated. Both C3 and C4 consist of more than one polypeptide chain; the reactions shown in the figure occur in the largest chain of each protein.

How could such a complex system have evolved? It is plausible that it arose through a succession of gradual steps, with many of the most intricate components—such as the membrane-attack complex—appearing relatively late. It seems likely that the system originally evolved around the C3 component, serving to establish a covalent linkage between C3b and the membranes of foreign cells. This complex alone significantly enhances the capacity of macrophages and neutrophils to ingest and destroy microorganisms. Nevertheless, individuals who lack one of the late components and thus cannot assemble the attack complex remain protected against most bacterial infections. The sole exceptions are a few bacteria capable of surviving inside phagocytic cells; for defense against these pathogens, complement-mediated lysis is particularly crucial. Both the alternative and classical pathways are thought to have evolved from such a primitive complement system. The alternative pathway presumably arose first as a mechanism of innate, non-specific defense against infection, whereas the classical pathway developed much later, coupling C3 activation to antibody binding and thereby to specific adaptive immune responses. METABOLISM/2.html">THE CONCEPT OF an evolutionary relationship between the two pathways is consistent with the finding that many of their components are homologous, including the serine proteases C1r, C1s, C2, factor B, and factor D.

Summary

The complement system acts both independently and in concert with antibodies to protect the vertebrate Organism against infection. The early complement components are circulating blood proenzymes that are sequentially activated in an amplifying cascade of limited proteolytic reactions. This process can proceed either via the classical pathway, triggered by the binding of IgG or IgM antibodies to an antigen, or via the alternative pathway, which can be triggered directly by the cell walls of invading microorganisms. The most critical complement component is the C3 protein, which is activated by proteolytic cleavage and subsequently binds covalently to nearby membranes. Microorganisms bearing activated C3 (C3b) on their surface are readily ingested and destroyed by phagocytic cells. In addition, C3b helps initiate the assembly of the late components, which form a large membrane-attack complex that lyses invading microorganisms. Complement activation also releases a number of small soluble peptide fragments that attract and activate neutrophils and stimulate histamine secretion by mast cells, triggering an inflammatory response at the sites of complement activation. The proteolytic cascade of complement remains tethered to the membranes of the invading microorganisms that triggered it, largely because certain components, including C3b, remain active for less than 0.1 milliseconds and are therefore unable to propagate the attack to neighboring host cells.