Lecture 25 - Complement Activation 1 Flashcards
Where is complement located?
- In the serum
* In the inactive form (pro-enzyme)
In general, how is C’ activated?
Proteolysis
What are the generalised effector functions of C’?
- Lysis of cells (MAC formation)
- Opsonisation (Enhanced phagocytosis)
- Inflammation (recruitment of lymphocytes)
- B cell activation
Which cells can by lysed by C’?
• Bacteria
• Infected cells
• Transplanted cells
etc.
How many different C’ proteins are there?
More than 30
Where are C’ proteins produced?
- Hepatocytes
- Monocytes & Macrophages
- Some epithelial cells
Is C’ a major component of the blood?
Yes
They make up 10% of the ‘globulin’ fraction (i.e. the protein component) of the blood
Describe the generalised process of the complement cascade
→ Trigger
S → T
Active T converts:
U → V
Active V converts:
W → X
Active X converts:
Y → Z
What is the nomenclature of C’ protein?
C1, C2 etc.
Products of a cleaved protein: • 'a': smaller fragment • 'b': larger fragment e.g. C3 → C3a + C3b
Physically, where does activation of the C’ system occur?
On the surface of cells (pathogens or others)
How are host cells protected from C’?
Cell surface proteins
• Present on healthy host cells
• Absent on microbes
These proteins regulate C’ activation to minimise host damage
Soluble C’ components are often…
Inactive
Describe the mechanisms of C’ activation
- Classical pathway
• Ag-Ab complexes
• IgM & IgG - Alternative pathway
• Ab independent
• C’ directly binds to bacterial surfaces - Lectin pathway
• Mannan binding lectin (MBL)
Which pathway of C’ activation is evolutionarily older?
Alternative pathway
What are the steps in C’ activation?
- Initiation
• 3 different pathways of activation
• Formation of a C3 convertase complex - Early steps
• Cleavage of C3
• Formation of the C5 a convertase complex
3. Late steps • Cleavage of C5 • Effector mechanisms: - lysis - MAC formation - inflammation
What is the outcome of initiation of C’ activation?
Formation of C3 convertase
The different pathways create different convertases, but they are homologous
What are the common steps in C’ activation?
These steps are the same, regardless of which pathways has been initiated
- C3 cleavage
C3 → C3a + C3b - C5 cleavage
C5 → C5a + C5b
What type of molecule is C3a?
Describe its function
Inflammatory mediator
Recruitment of inflammatory cells
What type of molecule is C3b?
Describe its function
Opsonin
- Binds to the microbe surface
- Phagocytes bind C’ receptors, and engulf the pathogen
What are the different mechanisms of C3 cleavage?
Specific molecules act as convertases
- Classical / lectin pathway
• C4bC2a - Alternative pathway
• C3bBb
Describe the different mechanisms of C5 cleavage
The C3 convertases join with C3b to form the C5 convertases
- Classical / lectin pathway
• C4b2aC3b - Alternative pathway
• C3bBbC3b
Describe the following steps of the alternative pathway:
• Initiation
• Early steps
• Late steps
“Tickover”
– Initiation –
- Natural low-level hydrolysis of C3
C3 → C3a + C3b
If there are microbes present:
2. C3b binds to microbial surfaces
– Early steps –
- ‘B’ binds C3b on microbial surface
- B cleaved by Factor D
B → Bb + Ba - C3b + Bb come together to form a C3 convertase
- Properdin binds and stabilises C3bBb (the C3 convertase) on the microbial surface
- C3 convertase cleaves C3:
C3 → C3a + C3b
– Late steps –
- C3b binds C3 convertase to form C5 convertase
C3bBb + C3b → C3bBbC3b - Convertase cleaves C5
C5 → C5a + C5b
How does C3b bind to microbes?
C3b: Thioester bonds
Microbial surface: amino and hydroxyl groups
What cleaves ‘B’?
Factor D
What is the structure of the C3 convertase in the following pathways:
• Classical
• Lectin
• Alternative
Classical
• C4bC2a
Lectin
• C4bC2a
Alternative:
• C3bBb
What is the structure of the C5 convertase in the following pathways:
• Classical
• Lectin
• Alternative
Classical:
• C4bC2aC3b
Lectin
• C4bC2aC3b
Alternative:
• C3bBbC3b
What is the function of Factor D?
Cleaves B:
B → Bb + Ba
When B is bound to C3b
What is the function of Factor B?
Inactive form
Factor B binds to C3b on the microbe surface in the alternative pathway
Factor B is then cleaved by Factor D:
B → Bb + Ba
What is Factor Bb?
Active form of Factor B
Formed by cleavage of Factor B by Factor D
Forms part of the C3 convertase
What is the function of Properdin?
Binds and stabilises C3 convertase on the microbial surface in the alternate pathway
→ stabilisation of the C3 convertase
How is the alternative pathway regulated?
- Factor I & Factor H
• C3b is rapidly degraded by these factors - Complement regulatory proteins
• When C3b binds healthy host cells it is rapidly inactivated by these proteins
What degrades C3b?
Factor I and Factor H
What is the “Pathogen sensing complex”?
C1 complex
Involved in initiation of the Classical pathway
Describe how C1q is activated
Activated by binding: 1. IgM or IgG • binds heavy domain • Ig has bound pathogens already • Must bind at least two Ig heavy chains
- CRP
• CRP binds bacterial surfaces at phosphocholine residues
Describe the classical pathway
• Initiation
• Early steps
• Late steps
– Initiation –
- C1q binds CRP or Ig on bacterial surface
- C1r cleaves C1s to form an active serene protease (C1s)
– Early steps –
- C4 binds Ig-bound C1q
- Activated C1s cleaves C4
C4 → C4b + C4a - C4b binds covalently to the surface of pathogen
- C4b binds C2
- C2 cleaved by C1s
C2 → C2a + C2b - C2a associates with C4a to form C3 convertase:
C4bC2a - Cleavage of C3 by C4bC2a
C3 → C3a + C3b
– Late steps –
- C3b that has just been cleaved binds to C4bC2a to form C5 convertase:
C4bC2aC3b - Cleavage of C5 by C4bC2aC3b
C5 → C5a + C5b
Which Ig best activates C1q?
In order of efficacy:
IgM
IgG1, IgG3
What is the structure of the C1 complex?
Composed of: • C1q (6 strands) - recognises foreign stuff - collagen-like tails - globular heads
• C1r(2)
• C1s(2)
- serine proteases involved in activation of the complex
What are the Ig requirements for C1 complex activation?
- C1 complex must bind at least two Igs
* Ig must be bound to the bacterial surface
Why must Ig be bound to the surface of the pathogen to activate the C1 complex?
Soluble IgM (pentamer): • Inaccessible Fc
Soluble IgG:
• Fc portions not adjacent
Antigen bound IgM:
• There was a conformational change
• Fc exposed
Antigen-bound IgG:
• Fc portions adjacent
What allows C1 to bind IgM when bound to the surface?
Binding stimulates a conformational change in IgM
Fc portion is now exposed
Which part of the C1 complex actually binds Ig?
C1q globular heads
What is active C1s?
How does it become activated?
Active serine protease
Activation:
- Binding of C1q to Ig
- C1r cleaves C1s
- C1s now active
What are the homologous comounds for: • Factor B • Factor Bb • C4bC2a • C4bC2aC3b
Factor B = C2
Factor Bb = C2a
C3 convertase:
C3bBb = C4bC2a
C5 convertase:
C3bBbC3b = C4bC2aC3b
What type of molecule is C5a?
Describe its function
Powerful inflammatory mediator
Recruits inflammatory cells
Describe the lectin pathway
– Initiation –
- MBL binds sugars on the surface of microbes with lectin domains
- MASP associated with MBL cleaves C4
C4 → C4a + C4b - C4b binds covalently to surface of pathogen
- C2 binds C4b and is cleaved:
C2 → C2a + C2b - C2a associates with C4b to form the C3 convertase
- C3 cleaved by C4bC2a
C3 → C3a + C3b
– Late steps –
- C3b associates with C4bC2a to form C5 convertase:
C4bC2aC3b - Cleavage of C5 by C4bC2aC3b
C5 → C5a + C5b
Where is MBL normally found?
What is it similar to?
In the serum
Similar to C1q
What is MASP?
MBL-associated serine protease
What is the homologous factor to C1s in the lectin pathway?
MASP (MBL-associated serine protease)
Describe how C3b can feedback positively on itself
C3b produced by any of the three pathways at the cleavage stage can interact with Factors B and D
→ more C3b produced through the alternative pathway
Is C’ heat stable or heat labile?
Heat labile
Only active at 37°
How is amplification achieved in the C’ cascade?
A signal molecule will activate many of the next molecule in the cascade, and so on and so forth
Which C’ pathway is formally an effector mechanism of humoral immunity?
Classical pathway
Because it involves Ag-Ab immune complexes
Describe the structure of C1q
6 collagen-like tails
6 globular heads
• bind Fc on Ab that have bound a pathogen
What can C1q bind?
- Ab that have bound pathogens
* CRP
How does CRP bind pathogens?
CRP binds phosphocholine residues on bacterial surfaces
What is the structure of MBL?
(Mannose binding lectin)
MASP1 and 2
• Serine proteases
Collagen like domain
Lectin domains:
• Globular heads
Where is MBL found normally?
In the serum
What does MBL bind?
Which bit of MBL binds?
Sugars found on the surface of microbial cells (and not human cells)
The Lectin domains (globular heads) bind the sugars
Compare the molecule that performs cleavage of C4 in the following pathways:
• Classical
• Lectin pathway
Classical: C1s
Lectin: MASP
Is there any cross over between the Classical/lectin pathways and the Alternative pathway?
Yes
C3b produced through the Classical / Lectin pathways can go back and feed the Alternative pathway
What happens to the cleavage products of C5?
C5b: Formation of MAC
C5a: Inflammation
Describe the process of formation of the MAC
- Cell bound C5b from common cleavage step
- C5b binds C6 and C7
- C5bC6C7 inserts into the cell membrane
- C8 binds, one of its subunit inserts into membrane
→ transient lysis of cells
- C5bC6C7C8 causes polymerisation of C9
→ formation of MAC
What are the physical properties of C6 and C7?
Hydrophobic
What is the structure of C8?
Trimer
How big is the pore of the MAC?
100 Angstroms
Made up of poly-C9
How does the MAC bring about cell death?
- Osmotic rupture
* Triggers apoptosis
What is the function of C5bC6C7C8?
Transiently able to lyse cells (to some degree)
More importantly, leads to the polymerisation of C9 to form the MAC
Discuss the role of CRP in the C’ cascade
CRP bound to microbial surfaces can trigger the classical pathway (like IgM and IgG)
- CRP bound to phosphocholine residues on microbial surfaces
- C1q binds CRP and is activated
- C1(r2s2) activated to become active serine protease
- C4 binds C1q bound to CRP
- C1s cleaves C4
- C4b binds covalently to microbe surface
- C2 binds C4b
- C2 cleaved by C1s to form C2a
– C3 convertase formed – - C4bC2a cleaves C3 to form C3a and C3b
- C3b binds C4bC2a to form C5 convertase
- C5 cleaved to form C5a and C5b
→ effector functions
Describe how the following bind to the surface of the pathogen: • MBL • C3b • C4b • CRP
MBL:
• Lectin binding domains bind to mannose (sugars) on the pathogen surface
C3b:
• Thioester on C3b binds hydroxyl and amino groups on pathogen surface
C4b:
• Binds covalently to pathogen surface
CRP:
• Phosphocholine on pathogen
