IMMUNOLOGY - Roit I. - Mir 2000

Chapter 4. Complement

COMPLEMENT ACTIVATION

There are three pathways (mechanisms) of Complement activation: the classical, the lectin, and The alternative pathway. All of them lead to The formation of a convertase that cleaves C3 into C3a and C3b, which is the central event of any complement cascade (Fig. 4.2).

Class="center">

Fig. 4.2. Activation of the complement via both the classical and alternative pathways results in the generation of C3 convertase, which converts C3 into C3b, this conversion being the central event of the entire cascade. In turn, C3b activates The sequence of terminal complement components (C5–C9) that form the lytic complex. In classical pathway activation, antigen first binds to specific Antibodies, and only then is C3 fixed. Antibodies are not involved in alternative activation. It begins with the covalent binding of C3b to hydroxyl groups on the cytoplasmic membrane of a microbial Cell. Activation via the alternative pathway serves as a mechanism of nonspecific innate Immunity, whereas the classical pathway represents a link between innate and acquired immunity that appeared relatively recently in phylogeny.

The convertase of the classical and lectin pathways is a combination of C4 fragments and , whereas the alternative pathway convertase is a complex of C3 with . The C3b fragment, which is cleaved from C3 by both convertases, binds to the target membrane and becomes the focus for additional C3b production—a step in the cascade known as the Amplification loop.

By additionally attaching a C3b molecule, both C3 convertases can be converted into C5 convertase, which Functions as a catalyst in the first step of the cascade leading to the Formation of the membrane-attack complex.

The classical pathway of complement activation is most commonly triggered by immune complexes

Antibody-dependent complement activation unfolds mainly via the classical pathway (Fig. 4.3); the protein C1 acts as the first enzyme complex in it.

Fig. 4.3. The table lists the main complement activators via the classical, lectin, and alternative mechanisms.

Activation is initiated by the binding of C1 to antibodies within immune complexes. The C1 enzyme complex consists of 5 molecules—one C1q, two C1r, and two C1s; their assembly is dependent on Ca²⁺ (Fig. 4.4). The first step of the classical pathway activation cascade is the binding of an antibody to at least two of the six spherical domains of the C1q molecule. This high-avidity binding involves the CH2 domains (PARTS OF THE Fc regions) of aggregated IgG molecules in a complex with the antigen. C1q molecules can also bind to the CH3 domains of an unaggregated IgM molecule, whose conformation changes from "flat" to "stapled" As a result of complex formation with the antigen.

Fig. 4.4. Each C1q subunit is Y-shaped, with both Branches of the Y terminating in spherical heads (1). The C1q molecule is formed by three such subunits joined together (2, 3). A subunit consists of six, and the entire C1q molecule of 18 polypeptide chains. The spherical heads of the subunits serve as receptors for the Fc region of IgG and form a ring around the perimeter of the C1q molecule. Pairs of C1r and C1s molecules, combined into a single structural unit (4, 5), are located across the C1q molecule (6). The catalytic sites of C1r and C1s are adjacent in the center of the ring. The mutual adhesion of all units of the C1 complex depends on Ca²⁺. [3 and 5 after Ross G.D. (see additional ref.); photos kindly provided by Dr. N. Hughes-Jones.]

It is hypothesized that multi-point binding of the spherical domains of C1q to IgG or IgM molecules incorporated into immune complexes leads to a conformational change of the entire C1 complex, inducing the autolytic self-activation of first one and then the other C1r molecule, converting them into two molecules of the active enzyme , which cleave both C1s molecules to form, respectively, two molecules of possessing Serine esterase activity.

The lectin pathway of complement activation is nearly identical to the classical pathway, but is triggered independently of antibodies. The C1q protein belongs to the family of calcium-dependent Lectins called collectins (collagenous lectins). This same protein family includes mannan-binding lectin (MBL), otherwise known as mannan-binding protein (MBP), conglutinin, and pulmonary surfactant Proteins A and D. Serum MBL can bind to terminal mannose groups On the surface of bacterial Cells, thereby acquiring The ability to interact with two mannan-binding lectin-associated serine proteases, MASP1 and MASP2, which are structurally homologous to C1r and C1s. This interaction is similar to the interaction of C1q with C1r and C1s and leads to antibody-independent complement activation via the classical pathway.

In addition, C1q binds directly—i.e., without the participation of antibodies—to certain microbes, particularly Mycoplasmas and A number of Retroviruses (but not HIV).

The action of C1 results in the Cleavage of C4 with the formation of activated C4b. Complement protein C4 contains an internal thioester bond, the Location of which is highly homologous to the thioester-containing region of C3 (see below). Upon cleavage of C4 by , two fragments are generated: C4a, which possesses weak anaphylatoxic activity, and a larger (unstable, intermediate) fragment, C4b*. (The asterisk indicates the unstable state of the molecule in which the binding site is activated.) Within a few milliseconds, C4b* is attacked by nearby nucleophilic groups. Most C4b* molecules are hydrolyzed to form inactivated iC4b. However, C4b* can form covalent bonds with amino or hydroxyl groups of cell membrane molecules, turning into surface-bound C4b.

Two C4 isotypes are known—C4A and C4B. They are encoded by tandemly arranged GENES OF THE Major Histocompatibility Complex. Activated C4A interacts predominantly with amino groups, whereas C4B interacts with hydroxyl groups, forming amide and ester bonds, respectively. Thus, C4A binds mainly to proteins, and C4B to CARBOHYDRATES.

The attachment of C2 to cell surface-bound C4b results in the formation of the classical pathway C3 convertase. Cell surface-bound C4b in turn becomes a binding site for the C2 proenzyme. Bound C2 serves as a substrate for C1s, which cleaves it with the release of C2b, while the larger fragment, C2a, remains attached to C4b, resulting in the formation of C4b2a—an enzyme complex referred to as the classical pathway C3 convertase.

The C3b protein generated by the action of C3 convertase can covalently bind to cell surface molecules. The C3 polypeptide belongs to proteins with unusual post-translational structural modifications. Closely positioned Cysteine and glutamine residues form a metastable internal thioester bond via the elimination of ammonia. The electrophilic (electron-accepting) carbonyl group (—C⁺=O) of this thioester is susceptible to attack by nucleophilic groups (electron Donors), including the amino and hydroxyl groups of approaching protein and carbohydrate molecules. Thus, C3 is capable of covalently binding to these molecules (Fig. 4.5).

Fig. 4.5. The α-chain of the C3 molecule contains a thioester bond formed by cysteine and glutamine residues. As a result of the cleavage of C3 into C3a and C3b*, this bond becomes unstable and sensitive to nucleophilic attack by —OH and —NH₂ groups (electron donors), allowing C3b to covalently bind to proteins and carbohydrates.

Proteolytic cleavage of C3a from the N-terminus of the C3 α-chain by C3 convertase leads to a conformational change in the remainder of the molecule (i.e., C3b*), rendering the internal thioester bond highly unstable. It becomes a new binding site within C3b*, capable of interacting very actively with nearby nucleophilic groups. As with C4b*, the majority of C3b* molecules undergo Hydrolysis, but some molecules bind to proteins and carbohydrates located in the immediate vicinity of the activation site. Since C3 convertase typically forms on a foreign surface or on immune complexes, C3b accumulates mainly in the same location. Subsequently, the bound C3b becomes the focus for further complement activation via the so-called alternative pathway amplification loop (see below) (Fig. 4.6).

Fig. 4.6. The binding of C1q to immune complexes triggers the catalytic autoactivation of C1r and the activation of C1s. Subsequently, cleaves C4, releasing C4a; the C4b* fragment remains in place and immediately binds to nearby proteins or carbohydrates. The Cell-surface-bound C4b then binds C2 (in the presence of Mg²⁺). C1s then cleaves C2b from the resulting complex, leaving C2a bound. (Recall that contrary to the general rule for fragment nomenclature, C2a is the larger fragment. Although proposals have been made to revise the nomenclature and adopt a clearer designation system, this book retains the traditional designations.) The complex is the classical pathway C3 convertase. Here and in subsequent diagrams throughout this chapter, all enzymatic reactions are indicated by red arrows.

Activation of complement via the classical pathway is tightly regulated. There are two regulatory Mechanisms for the classical pathway of complement activation in the fluid phase. The first is the action of the C1 inhibitor—a serine protease inhibitor (serpin) that binds and inactivates and

The second mechanism involves suppressing the formation of the classical pathway C3 convertase, . In the fluid phase, this is mediated by factor I and C4-binding protein (C4bp), which cooperatively cleave C4b. In addition, C4bp causes the dissociation of into C2a and C4b.

Classical pathway activation is also regulated by inhibiting the interaction of complement with host cell surfaces. This inhibition is carried out by complement regulatory proteins: decay-accelerating factor (DAF, CD55), complement receptor type 1 (CR1, CD35), and membrane cofactor protein (MCP, CD46). These proteins function as follows (Fig. 4.7):

✵ they inhibit the binding of C2 to C4b (DAF or CR1);

✵ they induce and accelerate the dissociation of C4b2a into C2a and C4b (DAF and CR1);

✵ they act as Cofactors, stimulating the Catabolism of C4b by factor I (MCP or CR1).

Fig. 4.7. Decay-accelerating factor (DAF) and CR1 inhibit the binding of C2 to C4b and promote The breakdown of the pre-formed complex. CR1 and membrane cofactor protein (MCP) induce the cleavage of C4b by factor I (FI). These molecules also regulate the interaction of C3b with factor B.

Spontaneous activation of complement occurs via the alternative pathway

"Ticking over" of the alternative pathway continuously maintains a low concentration of C3b* in Blood Plasma. The internal thioester bond in the native C3 molecule is susceptible to spontaneous hydrolysis, converting it into the activated form, C3i. (This continuous, low-level spontaneous activation of C3 in blood plasma is referred to as "tick-over".) The resulting C3i binds factor B to form C3iB (Fig. 4.8). [Similarly, C2 binds to C4b (Fig. 4.9).] Bound factor B is cleaved by factor D, releasing Va. The remaining complex, , represents the fluid-phase alternative pathway C3 convertase (Fig. 4.8), which cleaves C3 into C3a and C3b. Complement regulatory proteins prevent the initiation of the alternative pathway amplification loop by C3b bound to The surface of autologous cells.

Fig. 4.8. Hydrolysis of the thioester bond in the native C3 molecule converts this component into C3i [C3(H₂O)], which binds factor B in the presence of Mg²⁺. Cleavage of factor B by factor D converts the resulting complex into a fluid-phase C3 convertase, which can then directly cleave native C3 into C3a and C3b.

Fig. 4.9. Both the classical and Alternative pathways of complement activation lead to the generation of C3 convertases: and , respectively. The classical pathway begins with the activation of C1s by the antigen–antibody complex, followed by the cleavage of components C4 and C2 by activated C1s. The smaller fragments, C4a and C2b, are released, while the larger fragments form . Components C4 and C2 can also be activated by MASP (mannan-binding lectin-associated serine protease), a lectin pathway protein analogous to C1s, and MBL (serum mannan-binding lectin). In the Cytology/cytology/16.html">Early stages of the alternative pathway, C3b generated by "tick-over" activation and bound to the surface combines with factor B; factor D then cleaves a smaller fragment, Ba, from factor B. The larger fragment of B, namely Bb, remains bound to C3b, forming , a C3 convertase that cleaves additional molecules of C3 (positive feedback loop). A complement-activating surface (e.g., of microorganisms) stabilizes C3b, ensuring its binding to factor B. This promotes further alternative pathway activation. The classical and alternative pathway C3 convertases can additionally bind C3b to form enzyme complexes called C5 convertases ( and , respectively), which activate the next component of The Complement System, C5.

Because the alternative pathway C3 convertase operates in the fluid phase, most of the C3b* generated by its activity is hydrolyzed and inactivated by Water. However, upon contact with a foreign surface—such as a bacterial cell membrane—C3b* binds covalently and initiates the alternative pathway amplification loop. The overall scheme of complement component interactions during activation via the classical, lectin, and alternative mechanisms is shown in Fig. 4.9.

On the microbial cell surface, C3b is protected from proteolysis. Surfaces that strongly activate complement are termed activating surfaces (Fig. 4.12) because the C3b bound to them is protected from proteolysis. A foreign surface, such as a bacterial cell membrane, "protects" C3 because, upon binding to it, C3 exhibits a higher affinity for factor B than for factor H, presumably forming a more stable convertase. Furthermore, foreign surfaces lack host regulatory proteins that inhibit complement activation.

Although the specific structural characteristics required for a surface to be activating are not entirely clear, its carbohydrate composition appears to be of particular importance. For example, the presence of acidic sugars, notably sialic acid, seems to help protect the host's own cell membranes from excessive C3b deposition.

The initial attachment of a single C3b molecule to an "activating" surface is followed by an amplification phase, during which many additional C3b molecules are fixed at the same site. The key to the rapid accumulation of C3b is the formation of a membrane-bound C3 convertase.

The amplification loop is a positive feedback mechanism in alternative pathway complement activation. Surface-bound C3b binds factor B. The resulting C3bB serves as a substrate for factor D, a serine esterase that cleaves a small fragment, Ba, from factor B. The remaining surface complex, , dissociates very rapidly unless stabilized by the binding of properdin (P) to form the complex, which is the surface-bound alternative pathway C3 convertase.

The complex cleaves numerous new C3 molecules. Because the convertase is localized on the "activating" surface, the generated C3b* molecules will bind precisely there rather than elsewhere (Fig. 4.10).

Fig. 4.10. The C3b fragment can be generated by the action of the classical () or alternative () pathway C3 convertase. The resulting C3b binds to factor B in a Mg²⁺-dependent complex (C3bB), which is acted upon by factor D. Factor D cleaves the Ba fragment from factor B, converting the complex into the alternative pathway C3 convertase (). This convertase, in turn, acts on fresh C3 molecules to produce additional C3b. This positive feedback mechanism ("loop") functions to amplify the initial complement activation.

Note that the amplification loop also operates when C3b is fixed to a surface as a result of classical (antibody-dependent) complement activation.

Alternative pathway complement activation, including the amplification loop, is controlled by regulatory proteins. Fluid-phase alternative activation, occurring when C3b is not bound to a surface, is strictly regulated by proteins similar or identical to those that restrain classical complement activation. Factor H, which is homologous to the C4-binding protein and whose Gene is located within the RCA cluster, causes the dissociation of Bb from its complexes with C3i or C3b, and also acts as a cofactor in the catabolism of C3i and C3b mediated by factor I (Fig. 4.11).

Fig. 4.11. Factor I cleaves C3b at three sites, releasing C3c and leaving C4dg—a fragment of the C1 $\alpha$-chain—covalently attached to the surface. The first two cleavages, which yield the intermediate product iC3b, are promoted by factor H, MCP, or CR1. Cleavage at the third site is promoted by CR1.

Regulation of the amplification mechanism is critically important for the Organism. If it fails, amplification—acting as a positive feedback loop—proceeds until all C3 molecules are completely consumed. (This was first observed in a patient with a hereditary deficiency of the regulatory enzyme factor I. In the absence of factor I, the amplification loop operates until all C3 molecules in the patient's serum are converted into C3b.)

On the membranes of the host's own cells, both DAF and CR1 accelerate the dissociation of the C3bBb complex, releasing C3b. Both CR1 and MCP act as cofactors for the cleavage of C3b by factor I (see Fig. 4.7). In a fully analogous manner, DAF, MCP, and CR1 regulate The activity of (the classical pathway C3 convertase) when it is bound to cell membranes.

Thus, The Fate of surface-bound C3b is the most crucial step in the non-specific mechanism by which the complement system distinguishes "self" from "non-self". There are two possible outcomes for bound C3b.

✵ Amplification: C3b binds factor B to form a convertase that promotes the deposition of additional C3b molecules onto the same surface.

✵ Suppression: C3b is cleaved by factor I in the presence of one of three cofactors: factor H (in plasma), CR1, or MCP (surface-bound).

Which of these outcomes is realized depends on The Nature of the surface that has bound C3b (Fig. 4.12). The presence of host molecules, such as DAF, CR1, and MCP, on autologous (particularly cellular) surfaces effectively limits the formation of C3 convertases. Conversely, a foreign surface, such as a bacterial cell membrane, provides "protection" for C3b, as factor B exhibits a higher affinity for C3b on such surfaces than factor H does. As a result, the deposition of just a few C3b molecules leads to the formation of a relatively stable alternative pathway C3 convertase——an enzymatic complex that drives the binding of further C3b molecules at the same site.

Fig. 4.12. Alternative pathway complement activation depends on the presence of "protective" surfaces—that is, surfaces capable of shielding bound C3b from proteolysis. Factor B binds to C3b fixed on such a complement-activating surface; the resulting alternative pathway C3 convertase, , initiates the amplification loop. In contrast, on the surface of the host's own Cells and Tissues, C3b preferentially binds factor H and is inactivated by factor I. Consequently, the progression of alternative pathway reactions depends on whether factor B or factor H is bound. Additionally, host cell membranes express several regulatory proteins that suppress complement activation (see Figs. 4.7 and 4.14).

The terminal phase of complement activation is the formation of the membrane-attack complex

The complement activation cascade culminates in the formation of the lytic complex (the membrane-lytic or membrane-attack complex, MAC) resulting from the Enzymatic cleavage of C5—a protein homologous to C3 and C4, but lacking an internal thioester bond.

Before being cleaved by the C5 convertase, C5 selectively binds to C3b within the convertase complex. The classical pathway C5 convertase is a trimolecular complex, , in which C3b, covalently attached to C4b, exhibits a higher binding affinity for C5 than C3b bound to other cell surface molecules. The alternative pathway C5 convertase is also a trimolecular complex——in which one C3b molecule is covalently linked to another. Cleavage of C5 releases a small peptide fragment, C5a, which is a highly potent anaphylatoxin.

The membrane-attack complex is formed via the non-enzymatic assembly of C5b-9. The subsequent formation of the MAC proceeds without the involvement of Enzymes. Component C5b binds to C6 to form C5b6, which interacts with C7 to form the C5b67 complex (Fig. 4.13). Upon binding C7, the hydrophilic C5b6 converts into a hydrophobic C5b67 complex capable of spontaneously inserting into The Lipid Bilayer. Component C8 then associates with this complex, followed by the sequential addition of up to 14 C9 monomers. This generates a lytic "probe," or pore-forming molecule, the first ELECTRON MICROGRAPHS OF which were obtained by Humphrey and Dourmashkin (Fig. 4.13). Although the complex already exhibits minor lytic activity after C8 binds to C5b67, its full lytic potential depends on polymerized C9. (Note that the C5b6789 complex is commonly designated by the abbreviation C5b-9; preceding assembly intermediates may be denoted similarly, e.g., C5b-8.)

Fig. 4.13. 1. Component C5b binds C6 and C7 to form the hydrophobic C5b67 complex, a specialized region of which anchors to The Plasma Membrane near the site of complement activation. C8 then joins the complex and penetrates the membrane. Subsequently, a series of C9 molecules inserts into the membrane and polymerizes, completing the formation of the membrane-attack complex (MAC). 2 and 3. Electron micrographs of the membrane-attack complex. A funnel-like channel (3) in a lecithin liposomal membrane, generated by the insertion of the human C5b-9 complex, $\times 234,000$. (Photographs kindly provided by Prof. J. Tranum-Jensen and Dr. S. Bhakdi.)

The polymerization of hydrophobic molecules to form membrane pores is a common mechanism of cellular cytotoxicity. T lymphocytes destroy target cells by inserting pore-forming molecules called perforins into their membranes (see Chapter 9). Perforins are structurally homologous to C9; similar molecules have also been found in eosinophil granules (eosinophil cationic proteins). Certain Bacterial toxins, such as streptolysin O, are likewise pore-forming molecules.

Formation of the membrane-attack complex is regulated to prevent "reactive lysis"

The assembled hydrophobic C5b67 complex is capable of spontaneously inserting into the membranes of neighboring cells located close to the cell surface where primary complement activation is taking place. In the absence of regulation, this process of "reactive lysis" could cause damage to the host's own tissues.

The C5b67 complex can be inactivated in the fluid phase. A number of proteins can inhibit "reactive lysis" by binding to fluid-phase C5b67 before it can attach to host cell membranes. Among these proteins, S protein (vitronectin) is present in blood plasma at the highest concentration. The resulting SC5b-7 complex lacks the ability to insert into the lipid bilayer; this ability is also absent in the C5b678 complex because it binds to low-density Lipoproteins (LDLs) if the association of C8 with C5b67 occurs in the fluid phase.

Host cell Membranes Contain Proteins that protect them against MAC-mediated lysis. Erythrocytes were long ago shown to be readily lysed by heterologous complement and less readily by homologous complement. The basis for this species restriction became clear with the discovery of specialized Membrane Proteins that protect host cells from MAC-mediated lysis. Two such proteins have been studied in detail.

The first of these is CD59, a protein anchored to the membranes of many cells via a glycophospholipid tail. It binds to C8 within the C5b-8 complex and inhibits the insertion and unfolding of C9 in The cell membrane (Fig. 4.14).

Fig. 4.14. The CD59 protein binds to C8 within the C5b-8 complex, thereby blocking the recruitment of C9 and the subsequent formation of the MAC.

The second protein is homologous restriction factor (HRF), which exhibits the same activity as CD59, though it is a weaker inhibitor of C9 membrane insertion. HRF (mol. mass 65 kDa) is also membrane-bound via a glycophospholipid anchor; its Amino Acid Sequence has not yet been determined.

Notably, nucleated cells—specifically Cells of the body's own immune system—are more resistant to complement-dependent lysis than erythrocytes due to their ability to actively eliminate MAC via Endocytosis and Exocytosis of those membrane fragments into which it has inserted.