Classical Complement Pathway
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Classical Complement Pathway

Complement is a system of circulating enzymes that is part of the bodys response to illness or injury. The complement system plays an essential role in host defence against infectious agents and in the inflammatory process. It consists of about thirty plasma proteins that function either as enzymes or as binding proteins. In addition to these plasma proteins, the complement system includes multiple distinct cell-surface receptors that exhibit specificity for the physiological fragments of complement proteins and that occur on inflammatory cells and cells of the immune system. There are also several regulatory membrane proteins that function to prevent autologous complement activation and protect host cells from accidental complement attack. The complement system can be activated by three different pathways: the classical, the lectin, and the alternative pathway (Ref.1).

The classical pathway of complement activation has always been regarded as the major effector for antibody action. The Classical pathway of complement activation is triggered when the complement component C1 binds to Fc region of an antibody that is part of an antibody: antigen complex. C1 is a complex of three proteins, one of which- C1Q specifically recognizes and binds to the Fc region of the antibody, whereas the other two-C1R and C1S are inactive proteases. Free antibodies cannot activate complement in solution because of the structural requirements of binding to C1Q . The complement cascade is initiated when antibody is bound to multiple sites on a cell surface, normally that of a pathogen. IgM (Immunoglobulin-M) is the isotype that is most efficient at activating complement. The other human isotypes that activate the complement system are IgG1 (Immunoglobulin-G1) and IgG3 (Immunoglobulin-G3) and, to a lesser extent, IgG2 (Immunoglobulin-G2). Pentameric IgM has five Fc regions, each of which can provide a binding site for one of the six binding sites of C1Q. Multipoint attachment of C1Q to IgM is required for a stable interaction; this can readily be satisfied by a single molecule of each type. IgG can also bind to C1Q, but it has only one Fc region; consequently, atleast two molecules of IgG bound to microbial surface are required to bind one molecule of C1Q (Ref.2).

C1R and C1S are serine proteases, which are activated when C1Q binds to an antibody Fc region. On binding to antibody, one molecule of C1R is induced to cleave itself, thereby becoming enzymatically active. It then cleaves and activates the second C1R molecule and both C1S molecules. Activated C1S is the protease that binds, cleaves, and activates the next two components of the classical pathway, the serine proteases C4 and C2. C4is a large globular protein that is encoded and synthesized as a single polypeptide. As part of the intracellular folding and post- translational modification of C4, this single polypeptide is cleaved into three chains called alpha (93kD), beta (75kD) and gamma (33kD). When a C4molecule interacts with the activated C1S proteases, it is cleaved at a specific site in the alpha chain, releasing a smaller fragment of the alpha chain called C4A. The larger fragment formed by the cleavage reaction, C4B, develops a labile binding site allowing it to attach to antigens nearby. Only when the cleavage of C4 takes place in the presence of acceptor sites for C4B are C4B sites generated, for the further participation of C4 in the complement cascade. In the absence of such acceptor sites for C4B, the labile binding site on C4B is no longer available, and C4becomes C4B incapable of furthering the complement cascade (Ref.3).

Besides cleaving C4, the activated C1S protease also binds and cleaves C2, another inactive serine protease, into an enzymatically active C2B fragment and a smaller C2A fragment. On being released from C1, the C2B fragment forms a complex with a C4B fragment covalently bonded to the pathogen. This C4B, C2B complex, called C3 convertase of the classical pathway, is a surface associated protease whose function is solely to cleave and activate the complement component C3, a reaction central to complement function in the immune response. Upon cleavage of the C3 complement component, two fragments are produced; a small fragment C3A which is released and appears to be important in many inflammatory responses, as increased serum levels of C3A were found as a sign of complement activation in various inflammatory skin diseases particularly in psoriasis, and a larger fragment, C3B, which becomes covalently bound to the cell or bacterial surface and appears to be of great importance in the process of opsonisation. After the binding of the C3B component to the C4B component, the C3 convertase forms a trimolecular complex C4B-C2B-C3B, which has enzymatic activity and is called C5 convertase. Complement component C5 is structurally similar to C3 and C4 but lacks the thioester bond and has a different function (Ref.4).

The C5 convertase is capable of coordinating with, and cleaving, the next component of the cascade, C5. A single cleavage in the C5 molecule leads to the formation of two fragments: C5A, a small fragment that has important biological activity but does not associate with the cell surface; and C5B, a large fragment that binds to the cell surface via a labile binding site. C5A has been found to be an inflammatory mediator that can act on target cells through a family of receptors linked to GI proteins (Guanine Nucleotide-Binding Inhibitory Protein). C5B is critical in initiating the lytic sequence of reactions, and plays an important role in directing the association of further late-acting components of the complement system which interact to produce a lesion in the bacterial surface leading to bacterial death .The function of C5B is to initiate the formation of a MAC (Membrane-Attack Complex), which can make holes in the membranes of bacterial pathogens and eukaryotic cells. When C5 is cleaved by C5 convertase and nascent C5B is produced, self-assembly of the MAC follows. C5B and C6 form a stable and soluble bimolecular complex which binds to C7 and induces it to express a metastable site through which the nascent trimolecular complex (C5B-7) can insert itself into membranes, when it occurs on or in close proximity to a target lipid bilayer. Insertion is mediated by hydrophobic regions on the C5B-7 complex that appear following C7 binding to C5B-6. Membrane-bound C5B-7 commits MAC assembly to a membrane site and forms the receptor for C8 . The binding of one C8 molecule to each C5B-7 complex gives rise to small transmembrane channels that may perturb target bacterial and erythrocyte membranes. Each membrane-bound C5B-8 complex acts as a receptor for multiple numbers of C9 molecules and appears to facilitate insertion of C9 into the hydrocarbon core of the cell membrane. The concentration of C9 in human serum is such that there are only about two molecules of C9 for every C8 molecule; therefore C5B-9 complexes generated on target membranes display a degree of heterogeneity with regard to C9 content. Binding of one molecule of C9 initiates a process of C9 oligomerisation at the membrane attack site, after at least 12 molecules are incorporated into the complex, a discrete channel structure is formed. Therefore the end product consists of the tetramolecular C5B-8 complex (approximately 550kD) and tubular poly-C9 (approximately 1100kD). This form of the MAC, once inserted into the cell membranes, creates complete transmembrane channels leading to osmotic lysis of the cell. The transmembrane channels formed vary in size depending on the number of C9 molecules incorporated into the channel structure. Whereas the presence of poly-C9 is not absolutely essential for the lysis of red blood cells or of nucleated cells, it may be necessary for the killing of bacteria. Activation of the classical pathway promotes opsonophagocytic killing, serum bactericidal activity, and removal of immune complexes. Deficiencies of the classical pathway components have mainly been reported in association with immunological diseases such as SLE, glomerulonephritis and anaphylactoid purpura (Ref.5).