l5 - complement

8/7/2019 L5 - Complement

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Key Terms

Acute-phase the phase of inflammation where the liver is activated via cytokines to secrete many proteins, such as

CRP

Complement A collective term describing a machinery of plasma proteins that react with each other too opsonise

pathogens and induce a series of responses to fight the infection

CRP C-reactive protein, a protein that is secreted by the liver during an infection, and is particularly high in bacterial

infections

Opsonisation is the process where a pathogen is marked for ingestion and destruction by a phagocyte, and involves

the binding of an opsonin, such as an antibody to the membrane of the pathogen, marking it

Introduction

Part of innate immunity however requires some input from adaptive (acquired) immunity.

Complement involves a proteolytic cascade

Many complement proteins are proteases that are secreted as pro-

enzymes (zymogens)

Proteolytic cleavage by other enzymes will activate them

This results in huge amplification of the cascadeIt also allows tight control of the process, as the enzymes will not

usually be active

It is important to have control over complement, as it is toxic when

active in the plasma, even in the presence of bacteria.

The Three Pathways


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1. Classical pathway

A heat-labile (heat sensitive) protein found in the serum that complemented heat-stable antibodies in killing bacteria

The first receptor is C1

Multimeric complex of C1q, 2 C1r and 2C1sC1q:(c1r:C1a)2

C1q has 6 globular heads involved in binding to pathogen

The heads are cross-linked with a collagen tail

C1q can bind in the following ways

a) To antibodies complexed with antigens on the pathogen surface (Fc region of antibodies)

b) To the surface of certain pathogens (antibody independent)

c) To C-reactive protein (CRP, acute-phase protein) bound to bacteria phosphocholine

C1q cannot bind antibodies in solution; they need to be bound to pathogens in order to activate complement. The

binding regions on antibodies (IgM / IgG) are only exposed when they are bound to a pathogen. IgM in solution has a

planar / star-like conformation in which the binding site is hidden. When it binds to an antigen it takes the staple / crab-

like conformation, which exposes the binding sites for complement.

Each globular domain of C1qbinds one Fc domain on the antibody

C1r and C1s are both zymogens and serine proteases

The binding of C1q to the pathogen will induce a conformational change in the complex, resulting in autocatalytic

cleavage of C1r, which in turn will catalyse the cleavage of C1s.

C1s is now the active protease

The C1 complex will only be activated if 2 or more C1q heads bind to an antibody. IgM is pentameric, so a single

bound IgM antibody is enough to activate complement. IgG is monomeric, so there has to be a number of bound IgGs

near to each other. This makes IgM much more efficient at activating complement than IgG. Monomeric IgG can bind

C1 in solution, however the binding affinity is low, and it cannot activate complement in this way.

This is a simple level of regulation that prevents complement from being activated all the time.


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Step One

a) C1q binds to an antibody, increasing its affinity for C4

C4 naturally circulates in the plasmaIt can now bind to C1

b) C1s will then cleave C4 p C4a (small) + C4b (large)

C4a is an anaphylatoxinC4a will diffuse and cause weak inflammationC4b is the opsonin, it will attackand opsonise the pathogen

Step Two

a) C2 is also cleaved by C1s p C2a + C2b

C2a binds to C4b to form a complexp C4b2a (C3 convertase)C2b diffuses and causes vasodilation

Step Three

a) C3 convertase makes C3p C3q + C3b

Many C3 molecules will be cleavedC3 is homologous to C4 in structure2a is the active enzyme, it is a serine protease / zymogen

Step Four

C3b is the major opsonin of complementC3b will covalently bind and coat the pathogen surface in a similar way to C4b, marking the pathogen for

phagocytosis

C3a initiates a local inflammatory response

2. MB-lectin pathway

Complement can be activated by bacterial recognition receptors, other than antibodies

Similar to the classical pathway

Mannose binding lectin (MBL) is an acute phase protein

Its structure is similar to that of C1q

It binds with2-6 globular CRD (carbohydrate recognition domains) to mannose residues on the surface of the pathogen

MBL associated serine proteases (MASPs) are zymogens homologous to C1, especially MASP-2


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MASP-2 catalyses the activation of C4 and C2, and formation of C3 convertase

Activation of MASP-1/ -2 is not known

We do not usually have mannose on the surface of our cells, so this process will

only be activated by bacteria

After this point, all components are shared with the classical pathway

Acute-Phase Proteins:

CRP and MBL are acute phase proteins

These proteins are secreted from the liver

Cytokines trigger secretion

Important for complement activation

Bacteria induce macrophages to produce IL-6 (a cytokine)

This will act on hepatocytes to induce synthesis & secretion of acute-phase proteins

More in later lectures

3. Alternative pathway

Complement can be activated by bacterial surfaces through an antibody-independent pathway

More complex than the previous 2 pathways

Does not need any recognition for activation, it is activated all the time, spontaneously

It is triggered by the absence of specific antibody or recognition protein binding

a) Spontaneous hydrolytic cleavage (thioester bond) of C3 in plasma produces C3(H20)

A spontaneous conformational change exposes the otherwise hidden thioester bondKnown as complement tickover

b) C3(H20) binds plasma factor B, which is homologous to C2

c) Factor B is activated through cleavage by a protease (factor D)

Factor B p Ba (unknown function) + BbBound factor B has increased affinity for factor D

d) C3(H20)Bb is a C3 convertase, it catalyses the cleavage of C3 p C3a + C3b

The complex is not membrane-bound, it is circulated

C3b will be rapidly inactivated unless it covalently attaches to nearby self and non-self surfaces

The thioester bond will be rapidly hydrolysed, resulting in inactivation


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Bound C3b from the other pathways can bind factor B, and then be cleaved by factor D to produce active C3

convertase (C3bBb)

The alternative pathway is a means to amplify the classical and lectin pathways

An issue of this would be attack ofself cells, as the alternative pathway is always running

This is why there are negative regulatory steps

Negative regulation:

C1INH (C1 inhibitor) is a serine protease inhibitor (serpin)

It functions as a suicide substrate, so when the protease is active it will attempt to cleave C1INH, resulting in

permanent inhibition of the serine protease

C1INH will bind to the active enzymes C1r / C1s / MASP-1/-2 causing them to dissociate from their complexes

Complement receptor (CR1) & decay-accelerating factor (DAF) bind to C3b

This will prevent factor B binding in turn preventing convertase formation

Factor H acts in a similar way to CR1 and DAF by preventing convertase formation

It binds to sialic acid, which is only found in host cells (never in bacteria)

Factor I cleaves and inactivates C3b in conjunction with C3b binding proteins

This process is facilitated by other co-factors such as MCP (membrane co-factor of proteolysis)

Positive regulation:

In contrast, Factor P (properdin) stabilises C3bBb on the surface of pathogens

It is a pathogen recognition receptor (PRR) it recognises something on the pathogen

The thioester motif:


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C5a Strong response

In general, they all will initiate the following responses:

Induce smooth muscle contraction o blood flow velocity, o cells reaching areaIncrease vascular permeability, to allow entry of cells / complement moleculesSimulate mast cells in submucosal tissues to produce cytokines TNF-E and histamineAct on neutrophils and monocytes to recruit them at infection site

oWork in a similar way to chemokinesoVia G-protein activationoEspecially in C5a

oMicrobicidal activity of macrophages and PMNs (polymorphonuclear neutrophils)

2. Opsonisation of pathogens

The most immediate effect

Molecules bind to the pathogen, and flag it for ingestion and degradation by phagocytes

I) Ingestion into a phagosome

II) Fusion of phagosome with a lysosome

III) Degradation of pathogen

C3b mediated phagocytosis

Both C3b & C5a are required to initiate phagocytosisComplement receptors (CR) on phagocytes recognise C3b and promote phagocytosisC5a binds to the C5a receptor on phagocytes and activates themC4b has similar activity, but it is less important

3. Killing of pathogens

Formation of the membrane attack complex (MAC)

Terminal phase of complement

A pore is formed by complement factors on the pathogen that has been marked by complement

There is a change in the osmotic potential

Lysis of the pathogen occurs

If pathogen is removed by phagocytosis then the MAC complex will not be formed

It is not favourable to lyse 1000s bacteria within the bloodp toxins will be released

Opsonisation and phagocytosis are preferable

C5b initiates assembly of later complement components: C6/7/8/9 as well as the MAC complex

It also facilitates their insertion into the cell membrane


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a) C5b binds C6 p C5b6

b) C5b6 binds C7 p C5b67

c) A conformational change then occurs exposing the hydrophobic site of C7 within C5b67

C5b67 then inserts into the lipid bilayerd) C5b67 then binds C8, exposing the hydrophobic site in C8, which also inserts into the bilayer

e) ~10-16 C9 molecules polymerise to C5b678, with their hydrophobic sites also being exposed

All of the hydrophobic C9 sites enter the bilayerf) This forms the MAC pore which causes lysis of pathogens

Pore has 10nm diameter

This will disrupt ion gradients & osmotic potentials due to the free passage of water and solutes

It also allows lytic enzymes to enter the cell

Negative regulation of MAC formation:

If the MAC complex were to form on host cells, CD59 will bind the C5b678 complex

CD59 is a GPI-anchored glycoprotein that is found on the surface of host cells

This stage is not usually reached as the complement pathway is usually inhibited earlier on in host cells

4. Clearance of immune complexes

Removal of antibody-antigen complexes (immune complexes) from the circulation

E.g. toxins / debris from dead athogens

Not enough IgGs to enable binding to FcK receptors

Complexes bind C1q and activate the complement cascade to opsonise them with C3b and C4b

These new complexes are bound by erythrocytes (RBCs) via CR1 (complement receptors)

RBCs have bound CR1s


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These RBC-bound complexes are then cleared in the spleen and liver by macrophages through binding to CR1 and Fc

receptors. The erythrocytes are not destroyed.

Insects also have a complement-like system. Not required for exam but in slides if interested.

l5 - complement

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