Molecular Defences - Complement Flashcards
What is the complement system?
- group of (30+) plasma + cell surface proteins ; normally inactive
- 9 central components of complement cascade - c1 -> c9
- certain triggers lead to activation of complement pathways
- part of both innate + humoral immunity
- for generation of products that mediate effector functions of complement -> help to get rid of microbes
There are 3 core requirements of the complement system. What are they + can you explain them?
- Amplification: compl activation involves cascade of enzymatic cleavage of complement proteins - each activated protease generates multiple activated proteases in following step
- Stabilisation: prods of complement proteolysis attach covalently to microbial surfaces (or Abs bound to microbes) - complement proteins are inactive when in fluid form; stable activation when deposited on microbes
- Specificity: regulatory proteins inhibit complement activation on healthy host cells; absent from microbes - compl activation on microbial surfaces whilst minimising complement-mediated damage to host cells
A role of the complement system is to eliminate microbes. What 3 things do complement proteins do to achieve this?
- opsonisation of pathogens (facilitates phagocytosis)
- inflammation (recruitment + activation of leukocytes)
- lysis of microbes (Membrane Attack Complex, MAC)
These are important functions
Aside from eliminating microbes, what else do complement proteins do?
- to eliminate apoptotic cells / debris
- to promote clearance of Ag:Ab complexes
- to promote B cell activation
Complement is involved in disease pathogenesis
There are 3 main pathways of complement activation. What are these 3 pathways?
- Classical pathway: discovered first, C1 interacts w antibodies (IgM, IgG) bound to microbes, effector mechanism of humoral (adaptive) immunity (works together w Abs)
- Alternative pathway: discovered later (but phylogenetically older), direct recognition of microbial structures (belongs to innate immune response)
- Lectin pathway: MBL (mannose binding lectin) recognises terminal mannose residues on microbes (innate immunity), ficolin recognises residues on microbes (innate)
Even though there are different triggers for initiation of each pathway, they have similar consequences as they all generate the same thing(s). What do they generate?
generation of proteolytic enzymatic complexes
- C3 convertase: cleaves c3 -> c3a + c3b
- C5 convertase: cleaves c5 -> c5a +c5b
a = smaller product, b = larger product
C3 cleavage is crucial for all complement functions!
Alternative pathway: What happens here that allows C3b to covalently attach to the surface of microbes/cells?
- spontaneous cleavage of C3 in plasma (C3 tickover)
- C3 contains a reactive thioester bond (hidden)
- C3 cleavage induces conformational change in C3b
- exposes thioester bond
- exposed bond reacts w amino or hydroxyl groups on surface of microbes to form ester bonds
- -> covalent attachment of C3b to surface of microbes/cells
- in absence of covalent attachment, C3b remains in fluid phase; rapidly inactivated by hydrolysis -> further complement activation is stopped
Alternative pathway: What other site is also exposed along with the thioester bond in C3b?
Binding site for factor B is also exposed
Alternative pathway: What happens after C3b becomes stabilised to the microbial surface?
- covalently tethered C3b binds factor B
- bound factor B cleaved by factor D (plasma protease)
- generates Bb (large) + Ba (small) fragments
- Bb remains attached to C3b
- C3b-Bb complex is the alternative pathway generating C3 convertase
- C3 convertase main function to cleave more C3 molecules -> amplification of complement activation
- newly generated C3b deposits on microbial surface
- C3a is a soluble fragment; mediates biological activities
Alternative pathway: What happens following amplification of C3 convertase, generating more C3b?
- C3 convertase (C3b-Bb) cleaves additional C3 molecules
- forms more C3b + C3a
- some C3b molecules bind to C3 convertase -> generates C3b-Bb-C3b
- C3b-Bb-C3b is the alternative pathway C5 convertase
- C5 convertase cleaves C5 + initiates late steps of complement activation
Alternative pathway: What are the late steps of the pathway, following generation of C5 convertase?
- C5 convertase cleaves C5 into C5b (large) + C5a (small)
- C5a is soluble fragment; has several biological activities
- C5b remains bound to C5 convertase
- C5b recruits C6 + C7
- C7 is hydrophobic -> inserts into lipid membranes
- C7 recruits C8 (3 chains; 1 inserts into membrane)
- C5b-C8 complex recruits C9 -> C9 polymerises -> pores
- C5b-C9 is called MAC (membrane attack complex)
- lysis of microbe by MAC; entry of H2O via C9 pores
Classical pathway: What initiates the classical pathway? Ensure to describe all the components + requirements involved
- initiated by binding of C1 to antigen-bound IgG or IgM
- C1 - multimeric protein complex of C1q, C1r, C1s
- C1q binds to antibodies (IgM, IgG1, IgG3)
- C1r + C1s are proteases
- C1 does not bind to soluble (free) antibody molecules
- C1 binds only to antibodies that are bound to Ag
Classical pathway: Describe the structure of the C1 complement component
- C1 protein made up of C1q, C1r, C1s
- C1q hexamer: 6 globular heads connected via collagen-like arms to central stalk (‘bunch of tulips’)
- C1r (x2) + C1s (x2) form a tetramer
Classical pathway: How does C1 bind to antibodies then?
- globular heads of C1q bind specifically to Fc regions
- each Fc region has only 1 C1q binding site
- C1q must bind at least 2 Ig Fc regions to be activated
- only antibodies bound to Ag fulfil this requirement
- multiple IgGs are brought together only when bound to Ag
- free (circulating) IgM is pentameric + in planar configuration; C1q can’t access Fc regions of free IgM
- when IgM binds to Ag -> ‘staple’ configuration; unmasks C1q binding site of Fc regions -> binds C1q
Classical pathway: What occurs following the binding of two or more globular heads of C1q to Ag-bound atnbiodies?
- activates C1r
- C1r cleaves + activates C1s
- C1s cleaves C4 -> C4b (large) + C4a (small, soluble)
- C4 has similar structure to C3 (internal thioester bond)
- C4b binds covalently to the surface of the antigen
- recruitment of C2
- C2 cleaved by C1s into C2a (larger) and C2b (smaller; no bio function)
- C4b-C2a complex is classical pathway C3 convertase
Classical pathway: After the classical pathway C3 convertase is generated, what happens next?
- C3 convertase cleaves more C3 molecules
- newly generated C3b deposits on microbial surface
- C3a is a soluble fragment; mediates biological activities
- some C3b binds to C3 convertase complex -> C4b-C2a-C3b
- C4b-C3a-C3b is the classical pathway C5 convertase
- C5 convertase cleaves C5 + initiates late steps of complement activation
Classical pathway: What are the late steps?
Same as alternative pathway
Lectin pathway: The lectin pathway is initiated in absence of antibodies and triggered by microbial carbohydrate recognition of PRRs. What PRRs are involved and what do they bind to?
- MBL (mannose binding lectin) - binds to mannose residues on microorganisms
- ficolins - bind to N-acetylglucosamine residues on microbes
both have similar structure to C1q, they are in a complex with MASP1 and MASP2 (MBL-associated serine proteases)
Lectin pathway: What happens?
- binding of MBL to mannose residues on microorganisms
- binding of ficolin to N-acetylgucosamine residues on microbes
- activates MBL-Associated Serine Proteases (MASP1+2)
- MASPs cleave C4 and then C2
- then proceeds similarly to classical pathway
What are functions of complement?
- opsonisation (c3b, c4b) -> promotes phagocytosis
- microbe lysis (MAC)
- clearance of immune complexes (c3b)
- B cell activation
- inflammation (phagocyte activation) (c3a, c4a, c5a)
- inflammation (chemotaxis) (c5a) -> microbe destruction by leukocytes
Which complement proteins trigger acute inflammation, how?
- c3a, c4a, c5a trigger acute inflammation
- bind to mast cells -> degranulation -> histamine release
- also known as anaphylatoxins (mast cell degranulation similar to anaphylaxis)
- c5a also acts on neutrophils -> chemotaxis + reactive oxygen species
- C5a increases permeability of endothelial cells + neutrophil adhesion to endothelium
Why is it important that complement activation is tightly regulated?
- prevent complement activation on healthy cells
- limit duration of complement activation on microbes or Ag:Ab complexes
What are the 3 mechanisms of complement regulation?
- inhibit formation of C3 convertase
- break down/inactivate C3/5 convertase
- inhibit MAC formation
What are some fluid phase regulators?
- present in plasma, body fluids
- factor H (alt path)
- C1 inhibitor (C1INH)
- C4 Binding Protein (C4BP) (class/lectin path)
What are some membrane-bound regulators?
- CD46 (MCP)
- CD55 (DAF)
- CD59 (protectin)
- CR1 (complement receptor 1)
present on surface of healthy cells
How does C1 INH work?
- inhibits formation of C3 convertase:
- C1 INH dissociates C1r and C1s from C1q
- C1 INH blocks proteolytic activity of C1r and C1s
How do the following regulate?
- DAF, CR1
- C4BP
- Factor H
- MCP
- DAF, CR1: displace c2a from c4b2a C3 convertase, displace Bb from alternative pathway C3 convertase
- C4BP: binds c4b + displaces c2a from c4b2a C3 convertase
- Factor H: binds C3b + displaces Bb from alternative pathway C3 convertase
- MCP: binds C3b, C4b -> co-factor for Factor I degradation of C3b and C4b
How is covalently bound C3b degraded?
- by plasma Factor I
- MCP, factor H, C4BP, CR1 are co-factors for Factor I
How does CD59 (protectin) regulate complement?
- present on surface of healthy cells
- binds to C5-C8
- inhibits recruitment + polymerisation of C9
- inhibits MAC formation
How do complement regulators uphold specificity of the complement system?
- DAF, MCP, CR1, CD59 - present on surface of healthy cells + absent on microbes
- healthy host cell surface is rich in sialic acid residues
- favours binding of factor H over factor B
- selective inhibition of complement activation on host cells
- preferential activation of complement on microbes
