The Complement System

The complement system is comprised of a group of around 30 plasma proteins that can become activated and then interact in a powerful cascade that culminates in the production of a potent immunological complex capable of rapid and widespread target

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IgD$ IgG3$ IgG1$ IgA1$ IgG2$IgG4$ IgE$ IgA2$ IgM$

3’$ V$ D$ J$

5’$

V$ D$ J$ IgG3$IgG1$IgA1$IgG2$IgG4$ IgE$ IgA2$ $$$$$$$$ IgM$ IgD$ Excision$ DNA$repair$ V$ D$ J$ IgG3$ IgM$ IgG3$ B=cell$ DNA$ $mRNA$ $AnAbody$ Specificity$

cell lysis. However, in addition, as part of the complement cascade various components can fulfill a range of immune effector functions. There are three routes by which the complement system can be initiated, the classical pathway, the alternative pathway, and the mannose binding lectin (MBL) pathway. Complement factors are given the letter C followed by a number, with the factors named in their order of discovery rather than sequence of reaction. Thus the sequence of reaction is C1, C4, C2, C3, C5, C6, C7, C8 and C9. Cleaved products are designated a lower case letter following the number with the letter “a‟ for the smaller product and letter “b‟ for the larger product (the exception to this is factor C2 where the larger fragment is factor C2a and the smaller fragment C2b). The larger fragments have specific enzymatic activity resulting in cleavage of subsequent complement proteins. Alternative pathway factors are given letters instead of numbers, for example factor B and factor D. In the MBL pathway, the initial enzymes to be activated are called MASP-1 and MASP-2 (mannose-binding-lectin associated serine proteases). An overview of the pathways of complement activation are shown in figure 1.17. As figure 1.17 shows, although the pathways are each inititated differently they do converge into a common final pathway culminating in the formation of the C5-C9 membrane attack complex (MAC). The key to each pathway is to reach the point at which the C3 cleaving molecular complex is formed.

Figure 1.17: Simplified overview of the three pathways of complement activation. All three pathways converge onto the final common pathway. Classical and MBL pathways cleave C4 and subsequently C3. Protein complexes responsible for protein cleavage are marked with an overline.

The MBL pathway is initiated when MBL associates with two serine proteases called MASP-1 and MASP-2. The resulting complex is able to bind extracellular sugar moieties on the surface of pathogens, which in turn activates MASP-2 to cleave the C4 and C2 complement proteins to generate C4b2a [75]. C4b2a is the key C3 convertase molecule in the MBL pathway (figure 1.17).

Classical'

Pathway' MBL'_Pathway' Alterna4ve'_Pathway'

C1qrs' C1qrs'__' MASP:1/2' MASP:1/2'___' C4' C4b' C2' C4b2a'___' C3' C5' C5a'&'C5b' C5b:C9' C4b2a3b'______' C3bBb'___' C3a'&C3b' C3bBb3b'______' C3bB' Factor'B' Factor'D' C6,'C7,'C8,'C9'

The classical pathway is initiated by an antibody-antigen interaction and involves the C1q hexameric complex C1qrs (figure 1.17). When the C1qrs complex is cross-linked with at least two immunoglobulin Fc portions of pathogen bound antibody it induces a conformational change in the C1qrs complex resulting in autolytic cleavage of C1r and C1s. Cleaved C1s results in the formation of an active serine protease which in turn cleaves C4 generating C4b, which binds covalently to proteins or carbohydrate on the surface of the pathogen. To prevent an auto-reactive response unbound C4b is quickly inactivated. Pathogen bound C4b then binds to the C2a fragment of the C2 molecule, which is first cleaved by activated C1s, to form the C4b2a complex, or C3 convertase.

The alternative pathway was discovered after the classical pathway and is an alternative means of complement activation. The activation of the alternative pathway does not depend on the presence of proteins such as immunoglobulins or MBL, and is more likely to be initiated following interaction with immune complexes. As shown in figure 1.17 the classical and MBL pathways are activators of the alternative pathway as they are able to generate C3b. The alternative pathway begins with a low rate of spontaneous hydrolysis of plasma C3 to form C3(H2O). C3(H2O) has an altered conformation which binds to factor B. Which in turn is then cleaved by factor D to release Ba and form a fluid-phase C3(H2O)Bb complex. The C3(H2O)Bb complex is a C3 convertase and cleaves C3 to C3a and C3b most of which is rapidly inactivated. Some C3b molecules attach to pathogen or host cell surfaces and bind factor B. Factor B is cleaved by factor D to form Ba and C3bBb. The bound C3bBb molecule is called the alternative pathway C3 convertase and functions in much the same way as C4b2a in the classical and MBL pathways.

This step is also a key amplification stage with C3 being by far the most abundant complement protein. Cleavage by C5 convertase results in C5b which binds C6 to form C5b6 which in turn binds C7, and the resulting complex is then able to insert itself into the cell wall following a conformational change which exposes a hydrophobic residue on the C7 protein. C8 then binds to the C5b portion of the C5b67 complex and recruits and polymerises up to 16 molecules of C9 to form the MAC. The MAC has a hydrophobic external surface which allow it to insert itself into the lipid bilayer of the cell membrane. A hydrophilic inner channel with a diameter of around 100 Å then allows the free passage of water and other solutes resulting in rapid and potent cellular destruction [76].

In addition to the formation of MAC, the cleaved products of all pathways of complement activation, in particular following C3 and C5 cleavage, fulfill a variety of immunological functions augmenting the potency of the complement system. Some of these are summarized in table 1.6.

Table 1.6: Immunological roles of various complement cascade proteins.

Complement)Protein Immunological)Effect Main)Target

C3a Mast)cell)degranulation Mast)cell

C3b Opsonisation Pathogen)surface

Immune)complex)clearance Red)blood)cell

C3d Mast)cell)degranulation Mast)cell

B?cell)activation B?cell

C4a Mast)cell)degranulation Mast)cell

C4b Opsonisation Pathogen)surface

C5a Mast)cell)degranulation Mast)cell