The Complement System (Sistem Komplemen)

Ari Y

Sekolah Tinggi Farmasi Bandung

2013 Ari Y - STF Bandung'13

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The complement system is a complex system of serum proteins which interact in a cascade, many of the early components are serine proteases which activate each other sequentially.

The complement system refers to a series of proteins circulating in the blood and bathing the fluids surrounding tissues.

The proteins circulate in an inactive form, but in response to the recognition of molecular components of microorganism, they become sequentially actived working in a cascade where in the binding of one protein promotes the binding of the next protein in the cascade.

Jules Bordet (1870-1961)

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Nomenclature: - Symbol: C, called C1, C2, etc up to C9 - Where components are proteolytically cleaved, the products are referred to a or b as C2a + C2b etc. Alternative Pathway specific components are given letters as names (factor B, factor D, etc). There are 3 complement pathways that make up the complement system: - the classical complement pathway, - the lectin pathway, and - the alternative complement pathway. The pathways differ in the manner in which they are activated and ultimately produce a key enzyme called C3 convertase:

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• The classical activation pathway is triggered when a molecule of C1 binds to an antibody-antigen complex. • The "tips" of the antibody (the Fab portion) have shapes that are

complementary to epitopes - portions of microbial proteins and glycoproteins found on the surface of the microbe. The Fc portion of IgG and IgM can activate the classical complement pathway by enabling the first enzyme in the pathway, C1, to assemble.

• C1 is a complex of C1q, C1r and C1s subunits;

• C1q has binding sites for Fc portion of antibody.

• Activated C1r cleaves C1s to generate the active C1s protease which in turn cleaves first C4 (to C4a +C4b) and, after C2 has bound to C4b, C2 generate C4bC2a, the classical C3 convertase.

• C4b contains a highly reactive (i.e. short-lived) thiol-ester bond which is capable of forming a covalent attachment to nearby proteins and carbohydrates on immune complexes, cell and bacterial surfaces.

The Classical Complement Pathway

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The reactions are as follows:

a. Typically to activate the classical complement pathway is IgG or IgM is made in response to an antigen.

A protein called C1q first binds to the Fc portion of antigen-bound IgG or IgM after which C1r and C1s attach to form C1, the first enzyme of the pathway

b. The activated C1 now enzymatically cleaves C4 into C4a and C4b. The C4b then binds to adjacent proteins and carbohydrates on the surface of the antigen and then binds C2.

The activated C1 cleaves C2 into C2a and C2b forming C4b2a, the C3 convertase

Now the classical complement pathway is activated.

C3 convertase can now cleave hundreds of molecules of C3 into C3a and C3b

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c. Some molecules of C3b bind to C4b2a, the C3 convertase, to form C4b2a3b (see Fig. 6), a C5 convertase that cleaves C5 into C5a and C5b (see Fig. 7).

d. C5b binds to the surface of the target cell and subsequently binds C6, C7, C8, and a number of monomers of C9 to form C5b6789n, the Membrane Attack Complex (MAC) (see Fig. 8).

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• The alternative pathway does not require a specific antibody to commence.

• Initiation of the AP relies on the generation of an activated C3 molecule.

• There are various ways in this initiating activated C3. These include:

• proteolysis by enzymes derived from bacteria

• blood clotting enzymes,

• injury.

• However activation of C3 can occur spontaneously.

• This process proceeds constitutively at a slow rate.

• The activated thiolester will normally be rapidly hydrolysed in solution.

The Alternative Complement Pathway

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Cascade

• AP is initiated by the spontaneous hydrolysis of C3, which is abundant in the blood plasma.

• AP occurs through the spontaneous cleavage of the thio-ester bond in C3 to form C3(H2O)

• This change in shape allows the binding of plasma protein Factor B which allows Factor D to cleave Factor B into Ba and Bb.

• Bb remains part of the C3(H2O) to form C3(H2O)Bb, this complex is also known as a fluid-phase C3 convertase.

• This convertase, although only produced in small amounts, can cleave multiple C3 proteins into C3a and C3b.

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• The C3-convertase of AP consists of the activated B and D factors, forming an unstable compound that can become stable after binding properdin, a serum protein.

• After the creation of C3 convertase, the complement system follows the same path regardless of the means of activation (alternative, classical, or MBL).

• Binding of another C3b-fragment to the C3-convertase of the alternative pathway creates a C5-convertase analoguous to the MBL or classical pathway. •The C5-convertase of the alternative pathway consists of C3bBbC3b also referred to as C3bBb (instead of C4b2b3b in the other pathways)

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• The Alternative Pathway becomes self-generating by a positive feedback loop, those activated C3b molecules which are not quenched by hydrolysis binding to nearby surfaces, recruiting factors B and D and generating the C3bBb complex which in turn cleaves more C3 etc.

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Regulation

• Since C3b is free and abundant in the plasma, it can bind to either a host cell or pathogen surface.

• To prevent complement activation from proceeding on the host cell, there are several different kinds of regulatory proteins that disrupt the complement activation process:

• Complement Receptor 1 (CR1 or CD35) and DAF (decay accelerating factor also known as CD55) compete with Factor B in binding with C3b on the cell surface and can even remove Bb from an already formed C3bBb complex

• The formation of a C3 convertase can also be prevented when a plasma protease called Factor I cleaves C3b into its inactive form, iC3b.

• Factor I works with C3b-binding protein cofactors such as CR1 and Membrane Cofactor of Proteolysis (MCP or CD46)

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• Another complement regulatory protein is Factor H

• which either competes with factor B,

• displaces Bb from the convertase,

• acts as a cofactor for Factor I, or

• preferentially binds to C3b bound to vertebrate cells (because

of affinity to sialic acid residues)

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The Lectin Pathway

• Mannose binding lectin (MBL), also named mannose- or mannan-binding protein (MBP), is an important factor in innate immunity.

• MBL belongs to the class of collectins in the lectin superfamily.

• It is produced in the liver as a response to infection, and is part of many other factors termed acute phase proteins.

• MBL recognizes carbohydrate patterns, found on the surface of a large number of pathogenic micro-organisms, including bacteria, viruses, protozoa and fungi.

• Binding of MBL to a micro-organism results in activation of the lectin pathway of the complement system.

• people deficient in MBL experience a substantial increase in infections during the early years of childhood.

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• The Mannan-binding lectin pathway (also known as the Ali/Krueger Pathway) is homologous to the classical complement pathway.

• This pathway uses a protein similar to C1q of the classical complement pathway, which binds to mannose residues and other sugars in a pattern that allows binding on multiple pathogens.

• MBL is a protein belonging to the collectin family that is produced by the liver and can initiate the complement cascade by binding to pathogen surfaces.

• MBL is a 2-6 headed molecule that forms a complex with MASP-I (Mannan-binding lectin Associated Serine Protease) and MASP-II, two protease zymogens.

• MASP-I and MASP-II are very similar to C1r and C1s molecules of the classical complement pathway and are thought to have a common evolutionary ancestor.

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• When the carbohydrate-recognizing heads of MBL bind to specifically arranged mannose residues on the phospholipid bilayer of a pathogen, MASP-I and MASP-II are activated to cleave complement components C4 and C2 into C4a, C4b, C2a, and C2b.

• C4b and C2a combine on the surface of the pathogen to form C3 convertase (C4b and C2a), while C4a and C2b act as chemoattractants.

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MBL has been show to bind to:

• yeasts such as Candida albicans

• viruses such as HIV and influenza A

• many bacteria including Salmonella and Streptococci

• parasites like Leishmania

• The subsequent complement cascade catalyzed by C3 convertase results in creating a membrane attack complex, which causes lysis of the pathogen that MBL bound to.

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C3 is THE key component of Complement

The central component of the complement system is C3.

• C3 is an abundant serum protein (1.2mg/ml) which contains an unusual internal thiolester bond.

• In native C3 this bond is stable but this thiolester bond can become highly reactive as a result of conformational changes in the C3 protein structure.

• Generally the activation of C3 comes about as a result of proteolytic cleavage of the C3 molecule into 2 biologically active fragments (a and b).

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• The C3 convertases are powerful amplifiers of complement activation, up to 1000 C3 molecules are cleaved to C3b by each molecule of active C3 convertase.

• Each C3 convertase can be converted into a C5 convertase by the binding of a C3b molecule; this subsequently splits C5 into C5b and C5a.

• C5b C6,7,8, and C9 MAC

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THE BIOLOGICAL EFFECTS OF COMPLEMENT

1. Opsonisation

2. Inflammation

3. Lysis

4. Immune complex clearance

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1. Opsonisation

The C3b and, to a lesser degree, C4b molecules are opsonins.

That is they coat foreign organisms either by the AP or those already bound by antibody.

Opsonisation of particles greatly enhances their phagocytosis by means of binding to specific complement receptors

2. Inflammation The C5a and, less potently, the C4a and C3a fragments

are important inflammatory activators inducing vascular permeability, recruitment and activation of phagocytes.

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3. Lysis

C5b binds and recruits C6 and C7 to the target surface.

C7 and subsequently C8 change conformation to expose hydrophobic domains which insert in the lipid bilayer.

The C5b678 complex catalyses the polymerisation of the final component C9 which forms a transmembrane pore of ~ 10nm diameter causing lysis of the cell.

This macromolecular assembly is known as the Membrane Attack Complex (MAC).

4. Immune complex clearance. Complement has a very important role in

solubilising and causing removal from the circulation of immune complexes.

It does this by the binding of C4b and C3b, covalently bound to the immune complex, to CR1 complement receptors on red blood cells which transport the complexes to the liver and spleen where they give the complexes up to phagocytes for destruction.

Ari Y - STF Bandung'13

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Components of the complement pathways carry out 6 beneficial innate defense functions

These include:

a. triggering inflammation

b. chemotactically attracting phagocytes to the infection site

c. promoting the attachment of antigens to phagocytes (enhanced attachment or opsonization)

d. causing lysis of Gram-negative bacteria and human cells displaying foreign epitopes; and

e. serving as a second signal for activating naive B- lymphocytes;

f. removing harmful immune complexes from the body

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REGULATION OF COMPLEMENT

ACTIVATION

Regulatory mechanisms act at 3 key points.

A. C1.

C1 inhibitor (C1 INH) acts in 2 ways.

It binds free C1 in serum ([C1 INH] > [C1]) and inhibits spontaneous activation of C1;

it is released on activation by immune complexes.

It also limits the activation of C4 and C2 by binding and inhibiting the C1r and C1s proteases.

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B. C3 convertases.

The lifetime of the complexes is reduced by decay accelerating factors.

Some of these are present on host cell surfaces such as Decay Accelerating Factor (DAF) and CR1, which act on both the classical and alternative C3 convertases.

Others are serum proteins, C4 binding protein (C4bp) and factor H act respectively on C4b2b and C3bBb.

All these molecules promote the dissociation of the C3 convertases by binding to the covalently bound component (C4b or C3b) displacing the associated cofactor.

In addition C4bp, factor H, CR1, and another protein (MCP) catalyse the permanent inactivation of C3b and C4b via proteolytic cleavage by factor I (generating iC3b etc.).

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C. C9.

The polymerisation of C9 is inhibited by 2 host cell surface proteins, CD59 and Homologous Restriction Factor (HRF).

• Factor I continues to cleave iC3b and leaves the small, stable C3d fragment bound to the immune complex or pathogen surface via the covalent thiol-ester bond.

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COMPLEMENT RECEPTORS

CR1 • CR1 binds C3b and C4b.

• CR1 is found on erythrocytes, where it plays a vital role in removing immune complexes from circulation.

• It is also found on macrophages and neutrophils and can trigger phagocytosis (only after activation of cells by other mediators including C5a).

CR2 • CR2 binds iC3b and C3d and is found on B cells where it plays an

important role in activating class switching and memory formation.

The presence of bound C3d molecules enhances the response to antigen by about 20-fold for each molecule of C3d bound (at least up to 3).

In this way the innate immune system guides the antibody response onto the most 'dangerous' antigens and greatly lowers the threshold of antigen required to generate a response.

- This effect is mediated by CR2 crosslinking with surface IgM.

CR2 recruits a signalling chain which both amplifies signals and triggers a distinct signalling pathway.

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CR3 and CR4

CR3 and CR4 are related receptors which bind iC3b and

are found on monocytes/macrophages and neutrophils.

They trigger phagocytosis of opsonised particles, either in

concert with Fc receptors or independently.

Phagocytosis of microorganisms via CR3, and via

CR1+FcR, is the major defense mechanism against bacterial

and fungal infection.

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The Beneficial Results of The Complement Activation

• trigger inflammation: C5a>C3a>c4a;

• chemotactically attract phagocytes to the infection site: C5a;

• promote the attachment of antigens to phagocytes via

enhanced attachment or opsonization: C3b>C4b;

• serves as a second signal for the activation of naive B-

lymphocytes: C3d;

• cause lysis of gram-negative bacteria and human cells

displaying foreign epitopes: MAC; and

• remove harmful immune complexes from the body: C3b>C4b.

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COMPLEMENT DEFICIENCIES

Genetic deficiencies of almost all the components of the complement system have been found in humans.

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Terima Kasih

Ari Y - STF Bandung'13