Complement

Question 1

What are the three pathways of complement?

Answer 1

Classical
Manose binding lectin pathway
Alternative pathway

Question 2

What initiates Classical pathway?

Answer 2

Antigen-antibody complexes. Must be in complex (not free) Complex can be soluble or with a surface

Question 3

What initiates lectin pathway?

Answer 3

A particular patterns of carbohydrate on the surface of fungi and bacteria

Question 4

What allows the the Alternative pathway to proceed (not initiates)?

Answer 4

Pathogen surfaces. This pathway is activated all the time; ‘it ticks over’ and waits for a signal.

Question 5

What is the result of Complement activation (regardless of which pathway)?

Answer 5

1) Releases mediators and allows recruitment of inflammatory and immunocompetent cells to the site of activation
2) Opsonization (RED FLAG SIGNAL PLACED) of pathogens by removal by phagocytes.
3) Killing of pathogens specifically.

Question 6

Classical pathway/protein casade

Answer 6

First molecule is C1q. C1q is always complexed with two other molecules C1r and C1s (both enzymes). C1q globular heads actively bind to complexes.
Binding activates C1r and C1s.
C1s becomes next enzyme in cascade which cleaves C4 (in blood) into 2 molecules called 4a (floats off) and 4b.
C4b binds to same surface as C1q.
C2 cleaved by C1s too into C2a and C2b (floats off). C2a binds to C4b.
This makes C4b2a which is crucial at the start of activation as it is the ACTIVE C3 CONVERTASE (in all pathways.)

Active C3 convertase recruits C3 from the blood and cleaves it into 3a (floats away) and 3b which binds to C4b2a. to give C4b2a3b. This complex cleaves lots of C3 (1 molecule cleaves 1000 C3)

Question 7

Summarise complement in general

Answer 7

It is a Protein cascade. One molecule activates another which activates another. Enzyme fills up with more and more cleavage; one interaction leads to thousands of interactions.

Question 8

List the name and functions of the molecules of the classical pathway

Answer 8

C1: C1q- binds o pathogen or the antibody bound to pathogen so C1r activated.

C1r cleaves C1s to active protease. C1s cleaves C4 and C2.

C4b covalently binds to pathogen and oponizes it. C2 binds to C4 for cleavage by C1s. C4a is a peptide mediator of inflammation.

C2a is an active enzyme that cleaves C3 and C5. 2b is precursor of vasoactive C2 kinin.

C3b binds to pathogen surface and act as a main opsonins (flags for removal) and amplifies via the alternative pathway. 3a is a peptide mediator of inflammation.

Question 9

Lectin pathway

Answer 9

Cascade same as Classical pathway. However, the initiator is different.

Initiator is a manose binding lectin. Looks similar to C1q bu instead binds to carbohydrates in a particular way on the surface of bacteria and fungi. It has two associated molecules called MASP1 and MASP2 enzymes.

Manose binding lectin assoicated searing protein/proteases.

Initiator is less flexible than C1q (which has arms that move independently in order to bind to unlinear, folded, charged complexes.

Manose binding lectin must see the manose and fructose residues in a particular orientation (correct spacing.) Therefore it is a soluble pattern recognition molecule ( like TOLL)

Specific binding motif for specific set of pathogens.

Question 10

What molecule is related to manose binding lectin?

Answer 10

Ficolins. These also have MASP 1 and 2 and binds particular pattterns.

Question 11

How does Alternative pathway work?

Answer 11

The C3 molecule is constantly activated in solution; it is activated for a very short time. It is activated through its interaction with water to produce hydrolysed C3 with an activated bond. The bond has milliseconds before it becomes unactivated again.

If it does bind to bacteria. The next molecule is Factor B which non-covalently binds to C3b. Factor B is then cleaved by first enzyme called factor D into Ba and Bb.

Bb binds to C3 to produce C3(H20)Bb, a C3 convertase. As a convertase it cleaves more C3 into 3a and 3b.

Factor P (properdin) stabilizes complex of C33b is becomes active C3 convertase.

Question 12

Describe activation of C3.

Answer 12

It has a thiolester bond which opens up when activated/hydrolysed; if it doesnt bind to bacteria then it is deactivated by a component in serum. Therefore unstable when activated.

Question 13

What options is there in Alternative binding and its activation.

Answer 13

Binds to lots of surfaces due to thiolester bond; it will destroy whatever it bind to including host cells. There needs to be a control; regulatory molecules ensure no activation of complement on host cell surface.

Pathogens don’t have control molecules. Factor P stablises C3bBb complex, asa C3 convertase it recruits ancleaves more C3b on pathogen surface leading to oposonization and activation of terminal complement.

Question 14

What is Zero transplant issue?

Answer 14

Transplants across species. Even if genetically altered, the control molecules of complement are different in different species. No control, complement will destroy transplant.

Question 15

Describe control on host cell.

Answer 15

Regulatory proteins are CR1, H, MCP, DAF. These bind to C3b individually (one DAF to one C3b. CR1, H, DAF therefore displace Bb.

Factor I forms a complex with regulatory protein and C3b and cleaves to leave an inactive C3b

Question 16

List the molecules and functions of alternative pathway.

Answer 16

C3: C3b: binds to pathogen surface, binds to B for cleavage by D, C3bBb is C3 convertase.

Factor B: Ba: small fragment. Bb: an active enzyme of C3 convertase.

Factor D: plasma serine protease that cleaves B when it is bound to C3b in to Ba and Bb.

Properdin (P): Plasma protein that stabilises the C3bBb convertase on bacterial cells.

Question 17

What is complement amplification by ALTERNATIVE pathway?

Answer 17

Central molecule in ALL pathways is deposited C3. The pathways work together to make more deposited C3 as the alternative pathway acting as an amplification loop.

Question 18

Describe terminal complement

Answer 18

Pathways are all identical after the C3 deposition stage; C3b binds to both C4b2a and C3bBb to give active C5 convertases..

C5 recruited and binds by/to both convertases into 5a and 5b (by Bb or 2a.)

5b is the initiator for the assembly of the membrane attack system (direct killing of the target function of complement)

C6 binds to C5b to form the receptor molecule for C7. C7 inserts partly into the target membrane.

C8 binds into C5b67 complex and inserts all the way through the membrane and initiates C9 polymerisation.

Once 10-16 C9 molecules bind a pore ring is formed in the membrane.

Hole is large (10 nm)

Overall the cell (fungi, bacterial, parasite) is covered in pore holes produced this way. Lots of C5 recruitment and therefore lots of pores made. It will now die.

Question 19

List the molecules and their functions involved in terminal complement.

Answer 19

C5: C5a; small peptide mediator of inflammation. C5b initiates assembly of membrane attack system.

C6 binds to 5b to form acceptor for C7.

C7 binds to C5bC6 to form an amphiphilic complex to be partially inserted into bilayer.

C8 binds to C5b67 and acts to initiate C9 polymerization.

C9 polymerises to C5b678 to form membrane spanning pore and thus cell lysis.

Question 20

Describe recruiment of inflammatory mediators to the site of infection

Answer 20

C3b is opsonin.

C5a and C3a are initiators of local inflammation, also called enaflotoxins as they can create an uncontrolled inflammatory response.

Question 21

Receptor that causes downstream signalling in cell of receptor. Complement receptor 1 (CR1)

Answer 21

Binds C3b, and inactive C4b and promotes their decay.

Stimulates phagocytosis and so acting as an opsonin.

Very important for erythocyte transport of immune complexes; CR1 binds complex and take them to spleen in order to remove them. Otherwise, complexes will cause immunodisease (Lupus).

CR1 is a control protein as it helps remove C3b and C4b when it binds to the wrong cell.

Found in Erythrocytes, macrophages, monocytes, B cells, leukocytes, FDC.

Question 22

Complement receptor 2 (CR2)

Answer 22

Not involved in the activation of complement; it sits in lymph nodes and allows cells to capture antigens for antigen presentation during the adaptive immune response.

(Binds to C3d, inactive C3b, C3dg, Epstein-barr virus.

Found in B cells, FDC).

Question 23

Complement receptor 3 (CR3) and Complement receptor 4 (CR4)

Answer 23

Binds to inactive C3b and simulates phagocytosis.

Found on Macrophages, monocytes, leukocytes, FDC (CR3) dendritic cells (CR4).

Question 24

What is the importance of C5a receptor?

Answer 24

The other receptors need this receptor to function. So there must be deposition of C3b but also cascade as C5a comes after C3; ie complement must move to cleavage of C5 before there is full phagocytosis.

Question 25

C5a receptor

Answer 25

Binds to C5a and activates G protein.

Found in endothelial cells, mast cells, phagocytes.

Question 26

C3a receptor

Answer 26

Binds to C3a and activates G protein.

Found on endothelial cells, mast cells and phagocytes.

Question 27

Describe steps leading to phagocytosis

Answer 27

Manose binding lectin or C1q bound to bacterium which in turn is bound to macrophage CR1 receptor. Second signal is the binding of C5a to C5a receptor. C5a receptor also bound to a G protein; the C5a binding activates G protein so cell phagocytose.

Full destruction needs both signals from complement.

Question 28

State the effects of C5a and C3a in terms of inflammatory response

Answer 28

They effect blood vessel membranes and change the permeability of epithelial cells so antibodies and complement allowed to pass through into tissue; increases the gaps between the cells and results in leakage.

They act as chemokines and allow chemotaxis (migration) of phagocytes from blood into tissue. They increase microbicidal activity of phagocytes by acting with CR1 to remove bacteria.

Inflammatory response needs effector cells, antibodies, complement from circulation of blood into the infected tissue.

Question 29

Control of complement: list the control proteins

Answer 29

C1 inhibitor -prevent C1q activation

stops C3 convertase forming and therefore prevents cascade moving forward: 
C4 binding protein (C4BP)
Complement receptor 1 (CR1)
Factor H
Factor I
Decay-accelerating factor (DAF)
Membrane cofactor protein (MCP) 

CD59 (Protectin) - prevents C9 binding and polymerisation.

Question 30

C1 inhibitor

Answer 30

Controls only C1; it binds to activated C1r, s and removes them from C1q and stops complement activation.

Question 31

C4BP

Answer 31

Main control in Classical and Mannose binding lectin pathway.

It binds to C4b and displaces C2a and then inactivates it using cofactor I to cleave C4b.

“cofactor I for inactivation”

Question 32

CRI control

Answer 32

In both Classical and alternative pathways. Binds to C4b or C3b to displace C2a or Bb (part that makes up enzymes) to stop the cascade using cofactor I.

Question 33

Factor H and DAF

Answer 33

Main control of Alternative pathway.

It stops C3 convertase alternative pathway forming along with cofactor I.

DAF displaces Bb from C3b and C2a from C4b.

Question 34

Factor I

Answer 34

Is a serine protease that cleaves C3b or C4b with the help of other control proteins H, MCP,CR1,C4BP.

Question 35

CD59

Answer 35

Prevents binding and polymerisation of C9 and therefore prevents formation of membrane attack complex

Question 36

MCP

Answer 36

inactivates C3b and C4b with cofactor I.

Question 37

Summary

Answer 37

3 pathways, different initiator molecules that all produced C3 convertase (at which point 80% of control is focused which stop convertase activating and proceding to cascade.) Where there is no control, it is a permissive surface, there is cascade of C5, 6, 7, 8, 9 which makes the membrane attack complex and cell lysis.

Labelling with C3b to be removed.Enhancement of these labels through molecules C5a and C3a peptide mediators of inflammation, allowing blood vessel permeability change and complement recruitment. They also improve phagocytosis in addition to CR1.

Question 38

Complement deficiencies in terminal part of complement (C5-C9: the membrane attack complex)

Answer 38

There are deficiencies for every part of this pathway with very specific diseases.

Present with is an increased susceptibility to serial infections. Terminal component are about getting rid of foreign organisms.

Question 39

Deficiencies in C3

Answer 39

very rare; increased infection with fever causing bacteria called pyogenic.

Also increased susceptibility to serial infections.

As well as susceptibility to immune complex mediated diseases like lupus

Question 40

Classical pathway deficiency

Answer 40

Leads to immune complex disease; the first three components C1, C2, C4 are important in labeling antigen-antibody complexes and them being removed.

Question 41

MBL pathway defciencies (MBL,MASP1, MASP2, C2, C4)

Answer 41

Increased susceptibility to certain bacterial infections. But this grown out of as the child gets older (about 4/5 years old), antibodies take over this pathway.

Question 42

Alternative pathway deficiency (Factor D, Factor P)

Answer 42

Increase infection with fever causing bacteria and serial infections (Neisseria spp.)

Not about removing complex from circulation as there is no immune complex diseases caused.

Question 43

Deficiency in control proteins: lack of C1 inhibitor

Answer 43

Condition called Hereditary angioedema, where minor trauma, with fluid rushing in to trauma, swelling and death.
This is because C1 inhibitor is the main inhibitor in the kinin pathway.

No immune problems

Question 44

Deficiency in control proteins: lack of CD55/indirect CD59

Answer 44

Causes condition called PNH (Paroxysmal nocturnal haemaglobinuria)

They lyse their red blood cells as red cells have no control proteins on their surface; they cant control alternative pathway as one of the control proteins has gone.