Classical pathway

The classical pathway of complement consists of the glycoproteins Cl q. Cl r and Cls and C2-C9 (Figure 1.1). One molecule of Clq and two of C ls and C lr associate in the presence of Ca^'^ ions to form a large protein complex known as C1. C1 q is the molecule that interacts with most potential targets. Classical pathway activation has been mostly studied with immune complexes that contain IgG or IgM antibodies as the activator.

1.3.1 The structure of Clq, C lr and Cls

The complement classical pathway C1 enzyme complex is composed of the subunits Cl q, Cl r and Cl s. C1 q is made from 18 polypeptide chains of three types, 6 A, 6B and 6C arranged in a complex structure often described as a “bunch of tulips”. Three of these chains ( 1 A, 1B and 1C) form a collagen-like triple helix at the N-terminal end of the molecule (Reid and Porter, 1976). The six triple helices are aligned in parallel for half the collagen-like length and then diverge to terminate at the C-terminus in six globular heads (Brodsky-Doyle et al., 1976). The subcomponents Clr and Cls have homologous amino acid sequences and similar tertiary structures (Kishumoto et al., 1989). Clr and C ls interact to form a tetrameric chain, C1 s-C 1 r-C 1 r-C 1 s in which the two catalytic domains of Cl r are orientated in a head to tail manner (Arlaud et al, 1986). Cls interacts with C Ir via the Clr/Cls specific domain. The activated CTr2CTs2 complex has been found to have an asymmetric X structure (Weiss et al,

1986; Perkins and Nealis, 1989). A variety of models have been proposed for the interaction of the CTr2CTs2 tetramer with C lqto form the C1 complex (Perkins, 1989). These models

can be divided into two groups: those which propose that the CTr2CTs2 tetramer is positioned

on the outside ofClq, summarized in the “ W-model” (Perkins and Nealis, 1989) and the “02-model” (Cooper, 1985); and those proposing that the CTr2CTs2 subunits are interwoven

between the arms ofClq, summarized in the “S-model” (Schumaker et al., 1986) and the “8-model” (Colomb et al., 1989).

1.3.2 Activation

Activation of the classical pathway is initiated by the binding of C 1 q to a variety of substances (Sim and Reid, 1991). The most familiar is the formation of immune complexes of either IgG or IgM with antigens. Many other substances can activate the classical pathway, without a requirement for antibody (Sim and Malhorta, 1994). These include:

1. nucleic acid and chromatin

2. cytoplasmic intermediate filaments

3. mitochondrial membranes possibly via cardiolipin or mitochondrial proteins 4. some viruses, e.g. murine leukaemia virus (MuLV)

polysaccharide

6. gram negative bacteria via the lipid A component of the lipopolysaccharide of the cell wall.

The initial step of activating the classical pathway is the binding of Clq via the globular heads to the activator. The interaction between Clq and the Fc region of immunoglobulin (IgG or IgM) is facilitated by the close proximity of many IgG molecules (Borsos, 1989) or a conformational change in the Fab arms of IgM (Perkins et a/., 1991) to expose the Clq binding sites. It is thought that the domain types of the globular heads are likely to have differing specificities for charge groupings on the surface of the activator. Activation of complement requires multiple interactions between a single molecule ofClq and the activator, and therefore the activator is usually of high molecular mass and has a repetitive structure such as exposed lipid A on bacterial surfaces or multiple antibody molecules bound to a particulate antigen.

When two or more of the globular heads bind the activator, a conformational change in the collagenous stalks of C1 q is induced (Heinz, 1989) which increases the affinity ofClq for C lr2CTs2. C lr2CTs2 will interact with C lq only in the presence of Ca^^ ions, once C1 q

has bound to an efficient activator. The catalytic domain of one C lr autoactivates and cleaves the corresponding domain in the neighbouring C1 r molecule (Dodds et al, 1978). This in turn activates Cl shy a proteolytic cleavage (Ziccardi, 1976), The activated CTs is then able to cleave the classical pathway component C4 into C4a and C4b (Thielens et al., 1984). C4 has sequence homology to the complement components C3 and C5 and the non-complement proteins a2-macroglobulin and pregnancy zone protein. The proenzyme form of C4 is

composed of three chains: a, p and y. CTs cleaves a single peptide bond in the a-chain of C4 to produce a 9 kDa fragment C4a and a metastable fragment C4b. The C-terminal portion of the a-chain (a'-chain) of C4b remains disulphide linked to the P-chain. The larger fragment, C4b, has an exposed thiol ester in the a' chain that is able to react non-specifrcally with any available nucleophiles (Harrison etal.,\9S\). These nucleophiles may, for example, be the hydroxyl groups on sugars that form an ester bond to the carbonyl of the thiol ester or

amino groups located on a variety of surfaces, which form an amide bond. A proportion, usually less than 10%, of the C4b ends up covalently bound to the complement activator. The remainder reacts with water and diffuses away from the site of complement activation. Proenzyme C2 binds to the surface-bound C4b and, if it is appropriately positioned close to activated Cls, it is cleaved to form a dependent complex, C4b2a (Horiuchi et al.,

1991). The C4b2a complex is the classical pathway C3 convertase enzyme which is able to cleave and activate C3, a homologue of C4.

1.3.3 Mannose binding protein (MBP)

Molecules other than Clq can also participate in the activation of the classical pathway of complement (Sim and Malhotra, 1994; Holmskov 1994; Malhotrae^a/., 1994,1995). The activation of the classical pathway via complex carbohydrates is often referred to as the lectin pathway. Mannose binding protein (MBP), also known as Ra reactive factor (RaRF) and mannose- or mannan binding lectin (MBL), is able to substitute for C1 q after interaction with mannose-rich structures on yeasts, bacteria and viruses (Figure 1.1). It does not bind to normal IgG, but can activate complement on interaction with the carbohydrate groups of a glycosylation variant of IgG, which is present at elevated levels in rheumatoid arthritis (Malhotra et a l, 1995). The structure of MBP resembles that of C1 q in that it has collagenous segments and 6 globular heads. In MBP, each globular head is made up of three identical C-type lectin domains which interact with carbohydrate in a Ca^"^ dependent manner. MBP can activate C lr and Cls and molecules that are structurally similar to C lr and C ls that are termed MBP associated proteases (MASPs) (Malhotra et a l,

1994). This antibody-independent pathway may be important for immunodeficient iudividuals and the very young who have not yet developed a mature immune system.

1.3.4 C-reactive protein (CRP)

C-reactive protein (CRP) is a protein which, in mammals, is expressed during the acute phase response to tissue injury or inflammation. CRP displays several functions associated with host defense: it promotes agglutination, bacterial capsular swelling, phagocytosis and complement fixation. CRPs have also been sequenced in an invertebrate, the Atlantic horseshoe crab, where they are a normal constituent of the hemolymph. CRP can form complexes with charged groups, including the phosphate groups in choline phosphate of pneumococcal C-type polysaccharide and microbial polysaccharides (both those containing phosphocholine (PC) and not containing PC) and lipids, polyanion/polycation complexes and chromatin. Bound CRP can interact with Clq to activate the classical pathway.

The classical pathway is therefore activated by a wide range of stimuli. Clq and CRP are principally involved in recognising charge clusters including carbohydrates and lipids. In contrast MBP recognises neutral sugars.

1.3.5 Control proteins

Regulation of the classical pathway is controlled at two stages: formation of the C l enzyme complex; and control of the C3 convertase, C4b2a. Many serine proteases in the blood have relatively specific natural inhibitors that belong to the “serpin” family. Of the complement proteases, only C1 r, C1 s and possibly both MASPs are controlled by a serpin, named CT-inhibitor (CT-inh). C 1 inhibitor controls Clr2 activation through non-covalent

interactions (Arlaud et al., 1989; Ziccardi and Cooper, 1979). CT inhibitor can also covalently interact with the four serine protease active sites of the CT complex, resulting in the dissociation of the complex to form two molecules of CT inhibitor, CTr-CTs-CT inhibitor (Ziccardi and Cooper, 1979). The free collagen-like stalks of C lq are then free to bind to cell-surface Clq receptors which may then initiate phagocytosis (Malhotra et al., 1990; Malhotra and Sim, 1989; Ghebrehiwet, 1989). Hereditary or acquired lack of CT-inhibitor causes angiooedema. Factor J also controls the formation of the CT complex by inhibiting the association of CTr2CTs2 with C lq (Lopez-Trascasa et al., 1989).

Control of the C3 convertase is governed by its inherently unstable nature and cleavage by factor I. C2a spontaneously dissociates from the complex. This dissociation is enhanced by C4 binding protein (C4bp) (Gigli et a l, 1979) and decay accelerating factor (DAF) (Nicholson-Weller et al., 1982). Factor I cleaves C4b in two positions to give the fragments C4c and C4d. C4bp (Fujitae/a/., 1978; Gigli etal., 1979), membrane cofactor protein (MCP) (Seya et al., 1986) (Table 1.2) or complement receptor 1 (CRl ) (Table 1.3) act as cofactors for this factor-I mediated cleavage. In most physiological circumstances, the concentration of C4bp exceeds that of C4b generated by activation, and therefore further activation of the complement components is prevented.