Complement Flashcards
What does heating serum do to complement?
What does it do to antibodies?
The lytic capabilities of complement are heat-labile.
Antibodies are heat-stable
Complement is a series of how many serum and cell-surface proteins?
over30
What branch of the immune system is complement a component of?
innate immune system- it provides immediate but relatively non-specific protection. It also is an effector of the humoral immune system and works closely with Ab.
What are the three key concepts of complement?
- Activation
- Amplification
- Protective/Harmfull effects so it must be turned off
How is complement activated?
It circulates as an inactive zymogen in the blood plasma but is activated by:
- Undergoing conformational change
- Proteolytic cleavage
What are the two things that can happen when the complement is activated?
What does this allow complement to do?
- It can have new chemical properties
- It can gain protease activity
This allows for a complement cascade (one protein can activate enzymes for the next step)
What is the central effect of complement activation?
to attach a form of the third component of the complex (C3) to activating surfaces like bacteria or immune complexes
What are the three parallel processes for the deposition of C3?
- Classic
- Alternative
- Lectin Pathway
What does the classic pathway rely on to begin activation?
the formation of an Ag-Ab complex
What does the alternative pathway rely on to begin activation?
It is activated directly by microbial or polysaccharide surfaces (no Ab)
What is necessary to begin the lectin pathway of complement activation?
glycoproteins
What are the three major effects of complement activation?
- opsonization
- bacterial lysis
- inflammation
When C3 attaches to bacteria, viruses, cells, or proteins, what happens to the activation of complement?
It is amplified resulting in rapid generation of local effector molecules.
It requires strict regulation.
What are the 4 major protective functions of complement?
What is the major harmful effect?
Benefits:
1. opsonize microbes
2. Lyse bacteria (MAC)
3. Cause inflammation (recruit neutrophils)
4. get rid of harmful Ag-Ab complexes
Harm:
1. If too vigorous or unregulated, can harm host tissue
What does each pathway recognize to initiate the activation of complement?
Classic pathway –> Ag-Ab complex
Alternative–> polysaccharides on microbial surfaces
Lectin–> glycoproteins
What are the 3 major steps of activation of complement?
- Recognition
- C3 attachment
- generation of effector components
What is the recognition complex called for the classic pathway?
What are the three subunits?
C1 and consists of 3 subunits:
C1q, C1s and C1r
How many polypeptide chains is C1q composed of?
18 arranged into 3 groups of six each with a collagen-like triple helix and a globular domain.
What are the four activators of the classic pathway?
IgM
IgG (3>1>2»4)
Apoptotic cells
oxidized LDL
What part of C1q binds to the Ig?
the globular domain binds to the Fc of an Ig that is bound to an antigen
How many IgM does it take to bind to C1q? Why?
Just one because it is a pentamer
How many IgG are required to bind to C1q? Why?
2 or more because they are just monomers
Which IgG molecule is the worst at binding complement?
IgG4
As a rule of thumb, which subunit of complement (alpha or beta) is going to be larger?
What is the notable exception?
beta is usually larger except for C2a»C2b
What does the binding of Ig to C1q activate?
A serine protease in C1s that cleaves C4 and C2 into alpha and beta fragments
When the C1s serine protease cleave C2 and C4 what does C4b bind to? What does C2a bind to?
C4b binds COVALENTLY to the surface of whatever the antibody is bound to, and C2a binds NON-COVALENTLY to C4b.
What function does C2a gain when C2 is cleaved?
It gains protease function and is able to cleave C3 into two fragments.
When C2a cleaves C3, what does C3b bind to? What type of interactions?
It binds COVALENTLY to the activating surface and NON-COVALENTLY to C4bC2a
What is C4bC2a called?
C3 convertase
What is C4bC2aC3b called?
C5 convertase
Which 3 molecules in the classic pathway have protease function?
- C1s
- C4bC2a
- C4bC2aC3b
Which proteins in the classic pathway have a post-translational modification called internal thiolester?
C3 and C4
What is an internal thiolester and what does it allow structures to do?
it is a bond between cysteine and a glutamic acid that is unstable and high energy.
After proteolysis of C3 and C4 it exposes the thiolester to bind covalently to the activating surface of immune complexes, pathogens and cell surfaces
What is the “central factor” of the Alternative pathway? What is this factors role?
It is C3 and it ramps up the activation loop
Which of the three parallel complement pathways is Ab independent?
the Alternative pathway- it is activated by polysaccharides and components of bacteria and viruses
Lectin Pathway- activated by proteins that bind carbohydrates (glycoproteins)
What are the 5 activators of the alternative pathway?
- Repeating polysaccharides
- Endotoxins
- Virally infected cells
- Yeast cell walls
- IgA complexes
What is the first step of the alternative pathway?
The C3 molecule spontaneously attaches to a pathogen surface
In the Alternative pathway, after C3 binds to the pathogen surface what happens?
Factor B binds to C3
What happens to the C3B complex bound to a pathogen surface in the AP?
It is cleaved by Factor D to C3Bb
What makes factor D different from complement molecules in terms of circulation?
Factor D circulates in active form whereas molecules of complement circulate as zymogen
After cleavage what does the complex become?
C3bBb (another form of C3 convertase)
What are the two forms of C3 convertase?
C4b2a and C3bBb
What are the two forms of C5 convertase?
C4b2a3b and C3bBbC3b
Where are the two main areas of the body where you would see the Lectin pathway?
Respiratory and GI tract
What is the recognition molecule of the Lectin Pathway?
What structure is it structurally similar to?
Mannan Binding Lectin- it is structurally similar to C1q of the classic pathway (collagen-like triple helices bound to globular lectin domain)
What are the activators of the Lectin pathway?
- Mannose-containing microbial carbohydrates on yeast/bacteria
- Agalactosyl IgG
What structures on the MBL are homologous to C1r and C1s?
MASPs (Mannose binding lectin Associated Serine Proteases) 1 and 2
What do MASPs do>?
Cleave C2 and C4 to form C3 convertase and C5 convertase the same way as the classical pathway (C4bC2a and C4bC2aC3b)
What are the 4 major effector functions of complement?
- MAC to lyse bacteria
- Opsonization
- Clearance of Immune Complexes
- Anaphylatoxin
What is a MAC?
Membrane Attack Complex that forms a channel or pore to disrupt ionic and osmotic gradients to lyse the bacteria or host cell (typically that contains virus or intracellular pathogens)
What two bacteria REQUIRE complement for their clearance?
Neisseria gonorrhea
Neisseria meningitidis
What are the steps in MAC lysis of cells?
- C5 convertase cleaves C5 into C5a and C5b
- C5b binds to C6 and C7
- C5b67 has increased affinity for lipids and binds to the lipid bilayer of the cell membrane
- C8 binds to C5b67 and starts to perturb the membrane
- C5b678 bind C9 and has a conformational change that creates a 100A pore
What stage of the MAC complex is able to bind to the lipid bilayer of the cell membrane?
C5b67
What stage of the MAC complex is able to start perturbation of the cell membrane?
C5b678
What stage of the MAC complex undergoes a conformational change to self-polymerize and form the pore?
C5b6789
How many C9 molecules are on the MAC complex? Why?
What does it allow the complex to do?
3-18 but usually around 12.
It forms because of self-polymerization and allows for the formation of the pore
How large is the pore formed by the MAC complex?
What does this allow to happen?
100 Angstroms- disrupting cell homeostasis letting ions, water, lysozymes across the membrane
Which complement components are opsonins? Why?
C3b and C4b because they covalently bind to activating surfaces to mark cells microbes and immune complexes for uptake by phagocytes
How many receptors are there for the C3 and C4 opsinins?
What are the roles of these receptors?
4 -CR1, CR3 and CR4 have the major role of phagocytosis
CR2 is on B cells for amplification of activation
In addition to the C3 and C4 opsinins, what other complement molecule can perform opsonization? With what receptor on what cell?
C1q with the C1qR on leukocytes, endothelium, epithelium and platelets
What is the only C3 and C4 complement receptor that allows for opsonization in RBC as well as PMN, monocytes, Kupffer cells, etc?
CR1
Which complement receptor is a B-cell activator?
CR2
Why is the formation of Ag-Ab complexes dangerous in circulation?
They can deposit in tissue of the capilllaries, glomeruli, alveoli, or synovium activate complement and cause inflammation and tissue damage
How does the body clear immune complexes?
- C3b and C4b bind covalently to the Ag-Ab complex
- RBC with CR1 binds C3b/immune complex
- The immune complex is transfered to hepatic FcR in the liver and the bond between the IC and RBC is broken
- RBC returns to circulation
What complement fragments are anaphylatoxins?
What is their function?
C3a, C4a, C5a are potent inflammatory mediators
How many AA are C3a, C4a, and C5a?
77, 77, 73
What do anaphyatoxins bind to?
What 4 things are activated?
G protein receptors that activate:
- mitogen-associated protein complexes
- Phosphatydlinosital kinase
- PKC
- Calcium flux (intracellular)
Which is the most potent anaphylatoxin?
What does it bind to and what is its concentration?
C5a binds to C5aR and is less than 5nm in concentration
What do C3a and C4a bind to? How much less potent are they than C5a?
C3aR
They are 10x less potent than C5a
What are the general effects of anaphylatoxins?
- Chemotaxis to attract myloid lineage cells to site of infection
- inflammation
- cytokine production
- oxidative burst
- Vascular permeability etc etc etc
Why does there need to be strict regulation of complement activation?
because left unchecked, there would be a depletion of complement components in the serum, and excess anaphylatoxin and MAC
What is a way the body prevents complement activation on host tissue?
The host has regulatory molecules but the microbial surfaces lack regulatory proteins
What are the three major ways complement is regulated and controlled?
- Regulation of C1s
- Regulation of C3 convertase by regulators of complement activation (RCA)
- Regulation of MAC formation
What is the major inhibitor of C1s?
C1 esterase inhibitor (C1-INH) which inhibits the serine protease activity of C1s, C1r, kallikrein, and coagulation factors.
What does C1-INH inhibit?
C1s, C1r protease activity
Kallikrein
Factor XIa, XIIa, and plasmin (coagulation factors)
What pathway does C1-INH inhibit?
Classic pathway at discrete sites to keep C1s from acting systemically
What condition is associated with C1-INH deficiency?
Hereditary Angioedema- it acts as autosomal dominant with even a heterozygous deficiency being deleterious
The primary control of complement occurs at the level of _____ and _____ because they are common to all three pathways.
C3 and C5 convertases
What are the two mechanisms by which RCAs work?
- Cofactors for a serum protease FACTOR I that cleaves C3 to inactive fragments
- Bind to C3 to physically disrupt the convertase destroying enzymatic activity
What does Factor I do?
It works with RCA cofactor to cleave C3 into inactive fragments (C3bi and C3dg that remain attached to the activating surface)
RCA is a cofactor with Factor H to regulate which pathway?
the alternative pathway- fluid phase
RCA is a cofactor with C4bp to regulate which pathway? What phase is it?
classical - fluid phase
RCA is a cofactor with Membrane cofactor protein (MCP, CD46) where and in what pathway?
On the cell surface in all pathways
RCA is a cofactor for CR1 (CD35) where and in what phase?
The cell surface in all pathways as an opsinin receptor
RCA is a cofactor for what five things?
Which ones are pathway specific?
- Factor I
- Factor H- alternative
- MCP, CD46)
- CRI (CD35)
- CD4bp- classical
What 5 ways can RCA accelerate decay?
- Disrupts non-covalent C3b with C2a and Bb
- Factor H- alternative
- C4bp- classical
- CR1 (CD35) - cell surface all pathways, opsinin receptor
- Decay Accelerating Factor (DAF. CD55)
Where does DAF operate and in what complement pathway?
Cell surface in all pathways
Which RCA cofactors operate in the fluid phase (plasma)
Factor H, CD4bp
What RCA cofactors operate on the cell surface?
CR1 (CD35), DAF (decay), MCP (cofactor)
What are the three regulators of MAC formation?
- Vitronectin- binds C5b-7 trimer and makes it hydrophilic instead of lipophilic (cant attach to membrane)
- Clusterin- prevents C5b-9 that forms in the serum from binding to cells
- CD59- binds to C8 in the C5b-8 complex and prevents the subsequent incorporation and binding of C9
Where are CD59 molecules located?
Attached to the RBCs, leukocytes, endothelial cells, neuronal cells by glycophosphotidyl-inositol anchor
What does CD59 do?
Prevents MAC from forming by binding to C8 and not allowing C9 to bind to C5b-8
What is clusterin and what does it do?
It prevents MAC formation by binding to C5b-9 in serum and not allowing it to attach to cells
What is vitronectin and what does it do?
It prevents MAC formation by binding C5b-7 and making it hydrophilic so it can’t attach to cell membranes
Almost all forms of _______________ and _____________ have been associated with complement activation.
Tissue damage, inflammation
What does a defect in C3 or anything earlier in the classical pathway (C1,C2,C4) or alternative pathway (B, D) cause?
Recurrent infections especially in the respiratory and GI tract
People with defects later in the complement pathway (C5-C9) present with what?
recurrent infections from neisseria bacteria if they are HOMOZYGOUS only.
Heterozygous people have half the level of complement which is enough to give 100% protection
What would a defect in Factor H or Factor I lead to?
Uncontrolled activation of complement.
glomerulonephritis- because complement gathers in the kidney
What syndrome is associated with a deficiency in factor H?
Hemolytic uremic syndrome
What is autoimmune hemolytic anemia?
when IgG or IgM antibodies on RBCs activate complement and lead to hemolysis.
Happens with transfusions or autoantibodies
What 2 lab tests can detect if C3 is bound to RBC or if Ig is attached?
Coombs or DAT (Direct Anti-globulin Test)
What is immune complex disease?
When soluble Ag in the blood binds Ab that overwhelm complements ability to clear it and they deposit in capillaries, alveoli, glomeruli, activate complement and cause inflammation and tissue damage in these sites
What is sub-lytic MAC deposition?
When MAC deposits on host tissue in amounts higher than normal but not enough to lyse.
Causes intracellular signalling to activate neutrophils and platelets.
What do endothelial cells release when there is sub-lytic MAC deposition?
IL-1, vWF and TF which causes cell adhesion to blood vessels and thrombosis
What is the major cause of excessive anaphylatoxin productions?
What is the major result?
sepsis where there is massive amount of complement due to massive amounts of bacteria and there is a lot of C3a and C5a .which cause hypotension, thrombosis and edema
