A. A transition occurs from a flat planar molecule to a staple form, which exposes binding sites for the first component of the complement system, C1 (see Fig. 3.9).
COMPLEMENT ACTIVATION PATHWAYS HAVE EVOLVED TO LABEL PATHOGENS FOR ELIMINATION
Summary of the activators of the classical, lectin, and alternative pathways
Fig. 4.3 Summary of the activators of the classical, lectin, and alternative complement activation pathways. microorganisms immunoglobulins viruses other other classical pathway immune complexes containing IgM, IgG1, IgG2, or IgG3
HIV and other retroviruses, vesicular stomatitis virus Mycoplasma spp. polyanions, especially when bound to cations PO43– (DNA, lipid A,
cardiolipin) SO42–
(dextran sulfate, heparin, chondroitin sulfate) lectin
pathway
HIV and other retroviruses many Gram-positive and Gram-negative organisms arrays of terminal mannose groups alternative pathway immune complexes containing IgG, IgA, or IgE (less efficient than the classical pathway)
some virus-infected cells (e.g. by Epstein –Barr virus) trypanosomes, Leishmania spp., many fungi many Gram-positive and Gram-negative organisms dextran sulfate, heterologous erythrocytes, complex carbohydrates (e.g. zymosan) bacteria
Rapid Reference Box 4
alternative pathway – the activation pathways of the complement system involving C3 and factors B, D, P, H, and I, which interact in the vicinity of an activator surface to form an alternative pathway C3 convertase (C3bBb).
amplification loop – the alternative complement activation pathway that acts as a positive feedback loop when C3 is split in the presence of an activator surface.
anaphylatoxins – complement peptides (C3a and C5a) that cause mast cell degranulation and smooth muscle contraction.
bystander lysis – complement-mediated lysis of cells in the immediate vicinity of a complement activation site that are not themselves responsible for the activation.
C1–C9 – the components of the complement pathways responsible for mediating inflammatory reactions, opsonization of particles, and lysis of cell membranes. C3 convertases – the enzyme complexes C3bBb and C4b2a
that cleave C3.
classical pathway – the pathway by which antigen–antibody complexes can activate the complement system, involving components C1, C2, and C4, and generating a classical pathway C3 convertase (C4b2a).
complement – a group of serum proteins involved in the control of inflammation, the activation of phagocytes, and the lytic attack on cell membranes.
complement receptors (CR1–CR4) – a set of four cell surface receptors for fragments of complement C3.
decay accelerating factor (DAF) – a cell surface molecule on mammalian cells that limits activation and deposition of complement C3b.
lectin pathway – a pathway of complement activation, initiated by mannan-binding lectin (MBL), that intersects the classical pathway.
membrane attack complex (MAC) – the assembled terminal complement components C5b–C9 of the lytic pathway that becomes inserted into cell membranes.
zymogen – pro-enzyme that requires proteolytic cleavage to become active; the enzymatically active form is
Overview of the complement activation pathways
Fig. 4.4 The proteins of the classical and alternative pathways are assigned numbers (e.g. C1, C2). Many of these are zymogens (i.e. pro-enzymes that require proteolytic cleavage to become active). The cleavage products of complement proteins are distinguished from parent molecules by suffix letters (e.g. C3a, C3b). The proteins of the alternative pathway are called ‘factors’ and are identified by single letters (e.g. factor B, which may be abbreviated to fB or just ‘B’). Components are shown in green, conversion steps as white arrows, and
activation/cleavage steps as red arrows. The classical pathway is activated by the cleavage of C1r and C1s following association of C1qr2s2with classical pathway activators (see Fig. 4.3),
including immune complexes. Activated C1s cleaves C4 and C2 to form the classical pathway C3 convertase C4b2a. Cleavage of C4 and C2 can also be effected via MASP-1 and MASP-2 of the lectin pathway, which are associated with mannan-binding lectin (MBL). The alternative pathway is activated by the cleavage of C3 to C3b, which associates with factor B and is
cleaved by factor D to generate the alternative pathway C3 convertase C3bBb. The initial activation of C3 happens to some extent spontaneously, but this step can also be effected by classical or alternative pathway C3 convertases or a number of other serum or microbial proteases. Note that C3b generated in the alternative pathway can bind more factor B and generate a positive feedback loop to amplify activation on the surface. Note also that the activation pathways are functionally analogous, and the diagram emphasizes these similarities. For example, C3 and C4 are homologous, as are C2 and factor B. MASP-1 and MASP-2 are homologous to C1r and C1s, respectively. Either the classical or alternative pathway C3 convertases may associate with C3b bound on a cell surface to form C5 convertases, C4b2a3b or C3bBb3b, which split C5. The larger fragment C5b associates with C6 and C7, which can then bind to plasma membranes. The complex of C5b67 assembles C8 and a number of molecules of C9 to form a membrane attack complex (MAC), C5b–9.
C4 C3 C4a C3a C2b C3a Ba C2 C5 C5b C6 C7 C8 C9 C3 C3b C3b C3b C5a C4b C1qr2s2 C1qr2s2 C4b2 C5b67 surface site C5b–9 B D C3bB C4b2a C3bBb MBL MASP–1, MASP–2 MBL MASP–1, MASP–2 classical pathway activators alternative pathway activators C3bBb C4b2a proteases lectin pathway activators
The first component of the pathway, C1, is a complex molecule comprising a large recognition unit termed C1q and two molecules each of C1r and C1s, the enzymatic units of the complex (Fig. 4.5). Assembly of the C1 complex is Ca2+-dependent, and the classical pathway is
therefore inactive if Ca2+ions are absent.
C1 activation occurs only when several of the head groups of C1q are bound to antibody
C1q in the C1 complex binds through its globular head groups to the Fc regions of the immobilized antibody and undergoes changes in shape that trigger autocatalytic activation of the enzymatic unit C1r. Activated C1r then cleaves C1s at a single site in the protein to activate it.
Because C1 activation occurs only when several of the head groups of C1q are bound to antibody, only surfaces that are densely coated with antibody will trigger the process. This limitation reduces the risk of inappropriate activation on host tissues.
C1s enzyme cleaves C4 and C2
The C1s enzyme has two substrates – C4 and C2 – which are the next two proteins in the classical pathway sequence. (Note that the complement components were named chronologically, according to the order of their discovery, rather than according to their position in the reaction.) C1s cleaves the abundant plasma protein C4 at a single site in the molecule:
• releasing a small fragment, C4a; and
• exposing a labile thioester group in the large fragment
C4b.
Through the highly reactive thioester, C4b becomes covalently linked to the activating surface (Fig. 4.6).
C4b binds the next component, C2, in a Mg2+-
dependent complex and presents it for cleavage by C1s in an adjacent C1 complex:
• the fragment C2b is released; and
• C2a remains associated with C4b on the surface.
C4b2a is the classical pathway C3 convertase
The complex of C4b and C2a (termed C4b2a – the classical pathway C3 convertase) is the next activation enzyme. C2a in the C4b2a complex cleaves C3, the most abundant of the complement proteins:
• releasing a small fragment, C3a; and
COMPLEMENT ACTIVATION PATHWAYS HAVE EVOLVED TO LABEL PATHOGENS FOR ELIMINATION
Structure of C1
Fig. 4.5 Electron micrograph of a human C1q molecule demonstrates six subunits. Each subunit contains three polypeptide chains, giving 18 in the whole molecule. The receptors for the Fc regions of IgG and IgM are in the globular heads. The connecting stalks contain regions of triple helix and the central core region contains collagen-like triple helix. The lower panel shows a model of intact C1 with two C1r and two C1s pro-enzymes positioned within the ring. The catalytic heads of C1r and C1s are closely apposed and conformational change induced in C1q following binding to complexed immunoglobulin causes mutual activation/cleavage of each C1r unit followed by cleavage of the two C1s units. The cohesion of the entire complex is dependent on Ca2+.
(Electron micrograph, reproduced by courtesy of Dr N Hughes-Jones) C1s C1r C1r C1s C1q intact C1
Activation of the C3 thioester bond
Fig. 4.6 Theα chain of C3 contains a thioester bond formed between a cysteine and a glutamine residue, with the elimination of ammonia. Following cleavage of C3 into C3a and C3b, the bond becomes unstable and susceptible to nucleophilic attack by electrons on -OH and -NH2groups,
allowing the C3b to form covalent bonds with proteins and carbohydrates – the active group decays rapidly by hydrolysis if such a bond does not form. C4 also contains an identical thioester bond, which becomes activated similarly when C4 is split into C4a and C4b.
C3/C4 C3b/C4b C3a/C4a C3b/C4b O O O S C S H C H C S O _ O +
• exposing a labile thioester group in the large fragment
C3b.
As described above for C4b, C3b covalently binds the activating surface.
C4b2a3b is the classical pathway C5 convertase
Some of the C3b formed will bind directly to C4b2a, and the trimolecular complex formed, C4b2a3b (the classical pathway C5 convertase), can bind C5 and present it for cleavage by C2a:
• a small fragment, C5a, is released; and
• the large fragment, C5b, remains associated with the C4b2a3b complex.
Cleavage of C5 is the final enzymatic step in the classical pathway.
The ability of C4b and C3b to bind surfaces is fundamental to complement function
C3 and C4 are homologous molecules that contain an unusual structural feature – an internal thioester bond between a glutamine and a cysteine residue that, in the intact molecule, is buried within the protein.
When either C3 or C4 is cleaved by the convertase enzyme, a conformational change takes place that exposes the internal thioester bond in C3b and C4b, making it very unstable and highly susceptible to attack by nucleophiles such as hydroxyl groups (-OH) and amine groups (-NH2) in membrane proteins and carbohydrates.
This reaction creates a covalent bond between the complement fragment and the membrane ligand, locking C3b and C4b onto the surface (see Fig. 4.6).
The exposed thioester remains reactive for only a few milliseconds because it is susceptible to hydrolysis by water. This lability restricts the binding of C3b and C4b to the immediate vicinity of the activating enzyme and prevents damage to surrounding structures.
The alternative and lectin pathways provide non-specific ‘innate’ immunity
The lectin pathway is activated by microbial carbohydrates
The lectin pathway differs from the classical pathway only in the initial recognition and activation steps. Indeed, it can be argued that the lectin pathway should not be con- sidered a separate pathway, but rather a route for classical pathway activation that bypasses the need for antibody.
The C1 complex is replaced by a structurally similar multimolecular complex, comprising the C1q-like recog- nition unit mannan-binding lectin (MBL) and several
MBL-associated serine proteases (MASPs), which
provide enzymatic activity. As in the classical pathway, assembly of this initiating complex is Ca2+-dependent.
C1q and MBL are members of the collectin family of proteins characterized by globular head regions with binding activities and long collagenous tail regions with diverse roles (see Fig. 6.23).
MBL binds the simple carbohydrates mannose and N- acetyl glucosamine present in the cell walls of diverse pathogens, including bacteria, yeast, fungi, and viruses. Binding induces shape changes in MBL that in turn
induce autocatalytic activation of the MASPs. These enzymes can cleave C4 and C2 to continue activation exactly as in the classical pathway.
The lectin pathway is not the only means of activating the classical pathway in the absence of antibody. Mitochondria and other products of cell damage can directly bind C1q, triggering complement activation and aiding the clearance of the dead and dying tissue.
Alternative pathway activation is accelerated by microbial surfaces and requires Mg2+
The alternative pathway of complement activation also provides antibody-independent activation of complement on pathogen surfaces. This pathway is in a constant state of low-level activation (termed ‘tickover’).
C3 is hydrolyzed at a slow rate in plasma and the product, C3(H2O), has many of the properties of C3b,
including the capacity to bind a plasma protein factor B
(fB), which is a close relative of the classical pathway
protein C2. Formation of the complex between C3b (or C3(H2O)) and fB is Mg2+-dependent, so the alternative
pathway is inactive in the absence of Mg2+ ions. (The
differences in the ion requirements of the classical and alternative pathway are exploited in laboratory tests for complement activity.)
The C3bBb complex is the C3 convertase of the alternative pathway
Once bound to C3(H2O) or C3b, fB can bind and activate
a plasma enzyme termed factor D (fD). fD cuts fB in the C3bB complex:
• releasing a fragment, Ba;
• while the residual portion, Bb, becomes an active protease.
The C3bBb complex is the C3 cleaving enzyme (C3 convertase) of the alternative pathway. C3b generated by this convertase can be fed back into the pathway to create more C3 convertases, thus forming a positive feedback amplification loop (Fig. 4.7). Activation may occur in plasma or, more efficiently, on surfaces.
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