Complement Flashcards by Amelie Wyatt

What is the effect of a deficiency in Complement proteins?

Increased susceptibility to infection

Particularly bacterial

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What are the 3 stages of the complement system?

Recognition

Amplification

Effects

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Why is the complement system regulated?

Effects of the complement system are hazardous so excess activation could lead to complement attacking self cells

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What is intrinsic regulation in the case of complement?

The cascade can only be activated under specific circumstances

Determined by the characteristics of complement cascade proteins themselves - referred to as “activators”

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What is extrinsic regulation in the case of complement?

Stops the cascade, preventing excess activation

Determined by other proteins - referred to as “regulators”

Something else stops the pathway

Proteins dampen following immune response so that it doesn’t lead to chronic inflammation

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Describe the structure of the C1 complex in the classical pathway

C1q - Collagen region and flower

C1r and C1s are associated enzymes

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What is the C1 complex activated by?

IgM

IgG

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Describe the structure of MBL in the lectin pathway

Collagen tails with recognition domains

Mannose binding lectin

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What is the lectin pathway activated by?

Sugars

MBL -> mannose

Ficolins -> NAG (N-acetyl glycoseamine)

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What happens in the recognition stage of the classical pathway (IgM)?

IgM binds to target antigen

Causes a conformational change in IgM
○ Planar -> staple
§ Expresses binding sites for C1q

C1q binds to IgM:Ag

Conformational change of C1q

Allows activation of C1r

C1r cleaves/activates C1s
○ C1r and C1s are associated enzymes

C1s can cleave/activate C4

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What happens in the recognition stage of the classical pathway (IgG)?

IgG binds to target antigen in regular patterns (PAMPs)

Spacing is important
○ Need to have enough IgG across the surface and close enough together

C1q binds to IgG n:Ag (n>1)

Conformational change of C1q

Allows activation of C1r

C1r cleaves/activates C1s
○ C1r and C1s are associated enzymes

C1s can cleave/activate C4

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What happens in the recognition stage of the lectin pathway

MBL or ficolins bind to target antigen in regular patterns
○ Bind via mannose or NAG found on pathogen surface

Spacing is important

Conformational change of MBL

Allows activation of MBL associated serine proteases (MASPs)

MASPs can cleave/activate C4

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What are some methods of intrinsic regulation?

Conformational change of IgM from planar -> staple

Spacing of IgG across pathogen surface

Conformational change of C1q required to active C1r and so on

Spacing of MBL or NAG across pathogen surface

Conformational change of MBL to activate MASPs

Pro-enzymes/zymogens -> cleaved by another protease -> conformational change and exposure of catalytic active site

Thioester domains - super reactive so if it doesn’t bind to a pathogen it is hydrolysed instantly and can no longer bind -> ensures

C4b/C3b only binds to the pathogen and not to neighbouring human cells

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What happens in the amplification stage of the complement pathway

Activated MASP-2 associated with MBL/ficolins or activated C1s cleaves C4 to C4a and C4b

C4b covalently binds to the pathogens surface

C2 is cleaved by MASP-2 to C2a and C2b, C2a binds to C4b on the pathogens surface

Forming C4b2a -> C3 convertase

One enzyme of C3 convertase (C4b2a) can cleave lots of C3 into C3a and C3b

Deposit large amounts of C3b onto the pathogen surface

Also forms C4b2a3b

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What happens in the process of opsonisation

Process of depositing C3b on the pathogen surface/target membrane (eat me signals)

C3b can be recognised by receptors on phagocytic cells

C3b forms a thioester bond

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What happens in the alternative pathway

C3b is deposited on pathogen surface by classical or lectin pathway C3 convertase
C3b binds to factor B
Factor B is cleaved by plasma protease factor D into Ba and Bb
C3bBb complex is a C3 convertase, cleaving many C3 molecules to C3a and C3b
C3bBb3b is a C5 convertase
C3 can also by hydrolysed C3(H2O) which binds to factor B
Allows it to get cleaved by factor D into Ba and Bb
C3(H2O)Bb complex is a C3 convertase which cleaves more C3 into C3a and C3b
C3b is inactivated unless bound to cell surface
Factor B binds noncovalently to C3b on a cell surface and is cleaved to Bb by factor D

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What are the 2 C5 convertases? how are they formed and what is their action?

C3b binds to both C4b2a and C3bBb, forming active C5 convertases

C4b2a3b and C3bBb3b.

The C2a part or the Bb part of the C5 convertase cleaves C5 into C5a and C5b

C5a is released = potent anaphylatoxin

C5b undergoes a conformational change which exposes the binding site for recruitment of “terminal pathway components” of the membrane attack complex (MAC)

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What is the process of MAC pore formation

Series of sequential conformational changes

C5b binds C6 and C7

C5b67 complexes bind to the membrane via C7

C8 binds to the complex and inserts into the cell membrane

C9 molecules bind to the complex and polymerise

10-16 molecules of C9 bind to form a pore in the membrane

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How does the complement system lead to immune cell recruitment

C3a (C4a) and C5a are anaphylatoxins
○ Small molecules that act as chemical messengers (travel up chemotactic gradient)
○ Attract and activate ells with their cognate receptor

They attract and activate cells expressing C3a and C5a receptors

C3aR and C5aR ae 7 transmembrane G-protein coupled receptors

Excess activation results in inflammation

Which 2 complement receptors are phagocytic receptors

CR3 and CR4

What is the process of complement mediated phagocytosis when the bacterium is coated in complement by the alternative and MBL pathways

Bacteria opsonised with C3b

2 signals are required

Bacterium coated with C3b binds to CR3

C5a needs to bind to GPCR C5a receptor to undergo phagocytosis

What is the process of complement mediated phagocytosis when the bacterium is coated with complement and IgG antibody

iC3b binds to CR3 and IgG on surface of bacterium binds to Fc receptor the bacterium is phagocytosed

Macrophage membranes fuse creating a membrane bound vesicle - the phagosome

Lysosomes fuse with these vesicles, delivering enzymes that degrade the bacterium

What are some diseases associated with complement (4) and why

Paroxysmal nocturnal haemoglobinuria

Systemic lupus erythematosus

Age-related macular degeneration

Atypical haemolytic uremic syndrome

Excessive activation or inefficient/deficient regulation can lead to disease

What are the methods of extrinsic regulation of complement

Inhibition of C3 and C5 convertases

What is the reason behind inhibiting C3 and C5 convertases to extrinsically regulate the pathway

All pathways converge on C3b deposition This point in the pathway represents the "point of no return" because of the enormous amplification that happens at thios stage Majority of regulators act on C3/C5 convertases

What are the 2 mechanisms of inhibiting C3 and C5 convertases

Decay acceleration Enzymatic inactivation by factor 1

What are the different types of substances that act to inhibit C3 and C5 convertases

Fluid phase regulators ○ C4-binding protein (C4BP) ○ Factor H (fH) ○ Factor H like (FHL-1) Membrane bound ○ Decay accelerating factor (DAF) ○ Membrane cofactor protein (MCP) ○ Complement receptor 1 (CR1)

How does decay acceleration work to inhibit C3 and C5 convertases in the complement pathway

DAF, CR1, MCP, factor H Interact with C3b and prevent the complexes forming or accelerate dissociation of the components from the complex C1R bound prevents factor B from binding They compete with binding of the next component of the pathway

What is the mechanism of enzymatic inactivation by factor 1

Regulators are cofactors for Factor 1 which is a fluid phase serine protease F1 (+cofactor) cleaved C3b into iC3b Cofactors include: CR1, MCP, Factor H, C4BP iC3b is still an effective opsonin so by cleaving to iC3b can still get effective phagocytosis but without too much terminal pathway activation

How does Factor H work to inhibit the complement pathway

Able to bind to the surfaces of our cells by the sugars found in the cells Factor H binds to carbs found of self-cells which are not found on pathogens: sialic acid or heparan sulphate, glycosaminoglycan (GAG)

How does a mutation in factor H -> atypical haemolytic uremic syndrome

FH mutation in heparin binding site can prevent FH binding and protecting cells from TP activation Allows complement activation on self cells Thrombus formation in the kidney -> kidney failure

What is the treatment for FH-mediated atypical haemolytic uremic syndrome

Anti-C5 monoclonal antibody (prevents C5 and MAC generation) Liver transplant as FH is made by the liver so healthy donor with no mutation will correct the FH defect ○ If kidney is damaged patient may also require a kidney transplant

What is the treatment for DAF or MCP mediated atypical haemolytic uremic syndrome

DAF and MCP are expressed on the surface of kidney cells on which complement is activated In this case a kidney transplant from a healthy donor without the mutation can correct the defect

Genetic variations in what are associated with age related macular degeneration?

FH and FHL-1

What is the main symptoms of paroxysmal nocturnal haemoglobinuria and how is it caused?

Dark urine caused by haemoglobin Presence of haemoglobin in urine is caused by cell lysis by MAC Patients are deficient in CD59 and DAF - these are GPI anchored proteins Caused by mutation in PIG-A - the enzyme responsible for attaching the GPI-anchor ○ So PIG-A not protecting the RBCs so RBCs burst in urine

What is the treatment for paroxysmal nocturnal haemoglobinuria?

Eculizimab

What do immune complexes consist of

Antibodies bound to antigen

What do immune complexes activate?

The classical pathway Resulting in C3b opsonisation

What is the process of immune complex clearance?

C3b is recognised by CR1 on erythrocytes Erythrocytes transport Ics to the liver and spleen to be removed from circulation

What does a deficiency in CR1 lead to?

Systemic lupus erythematosus

What is the action of C1INH (C1 inhibitor)

Dissociates C1r and C1s from the C1 complex

What is the action of C3 and C5 convertase regulators

Decay acceleration (compete for binding with C3b) Factor-1 cofactor activity (cleavage of C3b) Deficiencies include aHUS and AMD

How do pathogens evade complement (4)

Avoid recognition Disguise Intracellular pathogens Avoid eradication

How does S. aureus avoid recognition by complement

Express Protein A (SpA) which binds to the Fc portion of IgG and depletes it, therefore avoids CP initiation S.aureus also expresses an enzyme which cleaves antibodies - avoids CP initiation

How do pathogens disguise themselves to evade the complement system?

Mucus layer Change in LPS Carbohydrate capsule (G-encapsulated bacteria) Intracellular pathogens - protected by host cell membrane

How do pathogens avoid eradication using complement regulators

Blocking of C-receptors Block complement activators Enzymatic cleavage of complement activators