Complement

Question 1

What does the complement system do?

Answer 1

• Activation involves a cascade of cleavage enzymes
• Amplication = each activated protease can generate multiple activated proteases in the following step
• Products of complement proteolysis covalently bond to microbial surfaces (or Abs bound to microbes/ other Ags)
• Stabilisation = complement proteins are inactive when in fluid form, then stably activate when on microbes
• Regulatory proteins inhibit complement activation on healthy host cells, pathogens dont have these = specificity

Question 2

What are the 5 main complement roles?

Answer 2

• To eliminate microbes
• To eliminate apoptotic cells/ debris (so dont cause inflammation)
• To promote clearance of immune complexes
• To promote B cell activation
• Also involved in disease pathogenesis - excess complement causes disease

Question 3

How can complement kill microbes?

Answer 3

• Opsonisaion of pathogens (facilitates phagocytosis)
• Inflammation - recruitment and activation of neutrophils and monocytes
• Lysis of microbes (MAC)

Question 4

What are the 3 complement activation pathways?

Answer 4

• Classical pathway - C1 interacts with IgM/G bound to microbes to be activated (adaptive)
• Alternative pathway - triggered by direct recognition of microbial structures (innate)
• Lectin pathway - Mannose binding lectin regonises terminal mannose residues on microbes (innate); ficolin recognises residues on microbes (innate)
• All have different triggers for initiation, but all generate C3 convertase (C3 to C3a/b), which can generate C5 convertase.

Question 5

What happens in the alternative pathway to make C3 convertase?

Answer 5

• Spontaneous cleavage of C3 into C3b and C3a
• C3 cleaves causes a conformational change in C3b, exposing a thioester bond, which can covalently bind to microbial surface (otherwise it is hydrolysed) = stabilisation
• C3b then binds factor B, which is then cleaved by factor D - leaving Bb (big) and Ba (small)
• This C3bBb complex is the alternative pathway C3 convertase - which cleaves more C3 molecules = activation

Question 6

How is C5 convertase made in alternative pathway?

Answer 6

• C3 convertase will cleave more C3
• These new C3b molecules bind to the microbial surface to survive
• Some C3b molecules will bind to the C3 convertase
• This generates C5 convertase (C3bBbC3b)
• C5 convertase cleaves C5 and initiates the late steps of complement activation

Question 7

What is the late stage of the alternative pathway?

Answer 7

• C5 convertase recruits and cleaves C5 -> C5a and C5b
• C5a is soluble - has biological activities
• C5b remains bound to C5 convertase
• This complex recruits C6,7,8 - builds a complex
• C8 binds into the membrane of the molecule
• This then recruits C9, which forms a pore into the microbial surface, allowing water and ions to enter the microbe and causes lysis after swelling
• C5-9 = Membrane attack complex

Question 8

What initiates the classical pathway?

Answer 8

• C1 binds to antigen-bound IgG/M
• C1 is multimeric - C1a, C1r and C1s
• C1q binds to the Abs (6 globular heads connected to central stalk)
• C1r/s are proteases - cleave other complement components (2x each, forms tetramer)
• C1 can only bind to Ab molecules if they are bound to a microbe

Question 9

How does the C1 bind to the Fc regions of IgM/G?

Answer 9

• C1q globular heads must bind to at least 2 Fc regions in order to activate pathway
• Globular heads can accomodate for different angles as are flexible
• All the heads are in the same plane, but changes conformation, bending its heads to bind to the Ag, exposing the domain that can interact with C1q

Question 10

How is C3 convertase made in classical pathway?

Answer 10

• Binding of 2 or more globular heads of C1q to Ag-bound abs -> activates C1r
• C1r cleaves and activates C1s
• C1s cleaves C4-> C4b/a
• C4b binds covalently to microbe surface to survive
• this recruits C2, which is cleaved by C1s into C2a (larger) and C2b (smaller)
• This is now the C3 convertase = C4bC2a complex

Question 11

How does the classical pathway generate C5 convertase?

Answer 11

• C3 covertase cleaves more C3
• New C3b deposits on microbial surface
• Some C3b binds to C3 convertase complex -> C5 convertase (C4b2a3b)
• C5 convertase then cleaves C5 and initiates late steps

Question 12

What is the late phase of classical pathway?

Answer 12

• Identical
• C5 convertase cleaves C5
• C5b binds to C5 convertase
• This C5b recruits C6,7,8. 8 can then bind to the surface and with C9, form the MAC -> lysis

Question 13

How does the lectin pathway activate complement?

Answer 13

• Doesnt need Ab presence - triggered by microbial carbohydrate recognition by PRRs
• PRRs are mannose binding lectin and ficolin - have similar structure to C1q
• Binding of MBL to mannose/ ficolin to N-acetylglucosamine residues -> activates MASP1 and 2
• MASPS leave C4 to generate C4b and then C2
• Then proceeds identically to classical

Question 14

C1 vs MBL and ficolin

Answer 14

• Have globular heads with collagen stems
• Also contain proteases called MASP (similar to C1r/s)
• Mannose residues are much more common on microbial surfaces than our cells, and are arranged differently, allowing MBL to distinguish between them
• same for ficolin

Question 15

What are the 6 ways that cleavage of C3 and C5 can help us?

Answer 15

• Microbe lysis via MAC
• C3b will bind to old RBCs and allow their degradation
• C3b,C4b - opsonisation on microbe promoting phagocytosis
• C3a,C4a,C5a are important in activating phagocytes and mast cells in inflammation
• C5a is involved in chemotaxis and increased permeability of enodethlium - help microbe destruction by leukocytes and ROS
• B cell activation

Question 16

Why do we have to regulate complement activation?

Answer 16

• Prevent complement activation on healthy cells

- Limit duration of complement activation on microbes or Ag-Ab complexes

Question 17

How do we regulate complement?

Answer 17

• Inhibit C3 convertase production
• breakdown/ inactivate C3/C5 convertase
• Inhibit MAC formation

Question 18

What are fluid phase regulators?

Answer 18

• Present in plasma and body fluids
• Factor H is an inhibitor of alternative pathway, properdin is an activator
• C1 inhibitor (C1INH), C4 binding protein (C4BP) - classical/lectin pathway

Question 19

What are membrane bound regulators?

Answer 19

• CD46 (membrane cofactor protein), CD55 (complement decay accelerating factor), CD59 (protectin), Complement receptor 1 (CR1)

Question 20

What does C1 INH do?

Answer 20

• inhibits formation of C3 convertase
• binds to C1r and s, and strips them off of C1q.
• This blocks their proteolytic activity, meaning that it cannot activate the complement pathway

Question 21

What do DAF and CR1 do?

Answer 21

• Displace C2a from C4b2a C3 convertase

- Displace Bb from C3bBb C3 convertase

Question 22

What does C4BP do?

Answer 22

• Binds C4b and displaces C2a from C4b2a C3 convertase

Question 23

What does factor H do?

Answer 23

• Binds C3b and displaces Bb from alternative pathway C3 convertase

Question 24

What does MCP do?

Answer 24

• binds C3b, C4b -> co-factor for Factor I degradation of C3b and C4b

Question 25

How is C3b degraded?

Answer 25

- Plasma factor I causes a bit of the protein to be cleaved | - Serum proteases cleave the remainder into C3dg (still bound) and C3c (floats off)

Question 26

What does CD59 (protectin) do?

Answer 26

- present on surface of healthy cells - binds to C5-C8 and inhibits recruitment and polymerisation of C9 -