Lecture 25 - Complement Proteins

Question 1

When and how were complement discovered?

Answer 1

1900.

A heat-labile factor in fresh serum that complements the function of antibodies is discovered

Question 2

What are complement proteins?

Answer 2

Inactive proteins (often pro-enzymes, zymogens) in serum activated by proteolysis to carry out a range of immune functions

Question 3

Complement protein functions
1)
2)
3)

Answer 3

1) Bacterial, infected cell, foreign cell lysis
2) Chemotaxis
3) Inflammation

Question 4

Number of C’ proteins

Answer 4

Over 30

Question 5

What produce C' proteins?
1)
2)
3)
4)

Answer 5

1) Hepatocytes
2) Macrophages/monocytes
3) Some epithelial cells
4) Neutrophils (less commonly)

Question 6

Proportion of globin plasma that is C’

Answer 6

10%

Question 7

Globin plasma

Answer 7

Protein component of plasma

Question 8

How are C’ activated?

Answer 8

Enzymatic cascade

Question 9

Significance of ‘a’ and ‘b’ fragments of a C’

Answer 9

‘a’ is smaller fragment, ‘b’ is larger fragment.

Exception is C2, where ‘a’ is larger

Question 10

Where does activation of C’ often occur?

Answer 10

Surface of pathogen

Question 11

How do host cells minimise self damage by C’?

Answer 11

Self cells have regulatory factors on their surfaces for reducing C’ activity.
Pathogens lack these

Question 12

Activity of soluble/fluid phase C’

Answer 12

Often transiently active, inactive.

Question 13

Which C’ pathway is an effector of humoral immunity?

Answer 13

Classical pathway

Question 14

Alternative pathway origins

Answer 14

Evolutionarily older than the lectin or classical pathways

Question 15

Antibody-independent pathways

Answer 15

Lectin, alternative

Question 16

Steps in C’ cascade
1)
2)
3)

Answer 16

1) Initiation
2) Early
3) Late

Question 17

Initiation
1)
2)
3)

Answer 17

1) 3 pathways for activation
2) Different pathways use different, but homologous components
3) Result in formation of different, but homologous C3 convertases

Question 18

Early stages
1)
2)

Answer 18

1) Cleavage of C3

2) Formation of C5 convertase

Question 19

How can C3 be cleaved?
1)
2)

Answer 19

1) C3 convertase (C4b/C2a, C3b/Bb)

2) Spontaneous hydrolysis of C3 (tickover)

Question 20

Types of C3 convertase
1)
2)

Answer 20

1) Classical/lectin - C4b/C2a

2) Alternative - C3b/Bb

Question 21

Late steps (effector phase)
1)
2)

Answer 21

1) After C3 cleavage, C5 convertases are formed

2) C5 activation results in pore formation, inflammation, cell lysis

Question 22

Common steps in complement activation
1)
2)
3)

Answer 22

1) C3 convertase cleaves C3 (C4b/C2a or C3b/Bb)
2) C3 is cleaved, C3a is an inflammatory mediator, C3b binds to the surface of microbe (acts as an opsonin)
3) C3 convertases form the C5 convertases (C4b/C2a/C3b or C3b/Bb/C3b)

Question 23

How does C3b bind to microbial surface?

Answer 23

Cleavage exposes reactive thioester groups on C3b

Reactive thioester groups bind amino and hydroxyl groups on microbial surface

Question 24

Two types of C5 convertase

Answer 24

1) Classical/lectin - C4b/C2a/C3b

2) Alternative - C3b/Bb/C3b

Question 25

Alternative pathway initiation
1)
2)
3)
4)
5)
6)

Answer 25

1) Low-levels of C3 hydrolysis initiate formation of active intermediates
2) Intermediates cleave C3 to C3a and C3b. In fluid, C3b is short-lived
3) C3b thioester group is revealed, C3b binds to microbial surface
4) B cleaved by factor D to Bb
5) C3b and Bb form C3 convertase on microbial surface
6) Properdin binds and stabilises C3 convertase on microbial surface

Question 26

What cleaves B into Bb?

Answer 26

Factor D

Question 27

What stabilises C3 convertase on microbial surface in alternative pathway?

Answer 27

Properdin

Question 28

How is C5 convertase formed in the alternative pathway?

Answer 28

When C3 convertase cleaves C3, C3b joins C3b/Bb on cell surface, forms C5 convertase

Question 29

Factor Bb
1)
2)
3)

Answer 29

1) Active form of factor B
2) Cleaved by factor D
3) Forms alternative C3 convertase with C3b, and alternative C5 convertase with two C3b’s

Question 30

Factor D
1)
2)

Answer 30

1) Serine protease

2) Cleaves factor B when bound to C3b

Question 31

How is alternative initial phase regulated?
1)
2)

Answer 31

1) Factors I and H rapidly degrade C3b in fluid phase

2) C3b can bind host-cell surfaces, but is rapidly inactivated by complement regulatory proteins on these cells

Question 32

Factor I and factor H role

Answer 32

Inactivate C3b in fluid phase

Question 33

What initiates the classical C’ pathway?

Answer 33

C1q binding to an antibody or C-reactive protein, bound to a pathogen

Question 34

C1 structure

Answer 34

A compex of C1q (6x collagen-like tails, 6x globular heads), C1r and C1s (serine proteases)

Question 35

What does C reactive protein bind?

Answer 35

Phosphocholine residues on bacterial surfaces

Question 36

How is C1 activated?

Answer 36

C1q binding activates C1r to cleave C1s, which forms an active serine protease

Question 37

IgGs that best activate classical pathway

Answer 37

IgG1, IgG3

Question 38

Antibody that best activates classical pathway

Answer 38

IgM

Question 39

Minimum number of Ig heavy chains that C1q must bind to activate classical pathway

Answer 39

2

Question 40

Can soluble IgM activate classical pathway?

Answer 40

No

Question 41

How does IgM/IgG activate classical pathway?

Answer 41

Binds to pathogen, this results in a conformational change.

IgM Fc region involved in C1q binding is exposed

Question 42

Classical complement pathway formation of C3 convertase
1)
2)
3)
4)
5)
6)
7)
8)

Answer 42

1) C1q binds antibody
2) C1r2, C1s2 are activated
3) C4 binds activated C1q
4) Activated C1s cleaves C4 into C4a, C4b
5) C4b binds covalently to cell surface
6) C4b binds C2
7) C2 is cleaved by C1s
8) C4b/C2a C3 convertase formed

Question 43

Classical pathway equivalent to alternative factor B

Answer 43

C2

Question 44

Classical pathway equivalent to alternative C3b

Answer 44

C4b

Question 45

Classical and alternative formation of C5 convertase
1)
2)

Answer 45

1) C3 convertase cleaves C3 into C3a, C3b

2) C3b binds to C3 convertase, forms C5 convertase

Question 46

C5a

Answer 46

Powerful inflammatory mediator

Question 47

C3a

Answer 47

Inflammatory mediator

Question 48

Lectin pathway formation of C3 convertase
1)
2)
3)
4)
5)
6)
7)

Answer 48

1) MBL binds to sugars on microbial surface
2) MBL-associated serine protease (MASP) binds to collagen-like region of MBL
3) MASP cleaves C4 into C4a, C4b (from here on, the same as classical pathway)
4) C4b covalently binds to cell surface
5) C4b binds C2
6) C2 cleaved by MASP into C2a, C2b
7) C4b and C2a form C3 convertase

Question 49

What is MBL equivalent to?

Answer 49

C1q

Question 50

Alternative pathway amplification loop
1)
2)
3)
4)

Answer 50

1) C3b produced by any pathway can interact with factors B and D
2) C3b binds to cell surface with thioester region
3) Factor B is cleaved by factor D
4) Bb binds to C3b, forms C3 convertase

Question 51

Formation of membrane attack complex
1)
2)
3)
4)
5)
6)
7)

Answer 51

1) C5b bound to cell membrane
2) C5b recruits C6, C7 (hydrophobic)
3) C5b/C6/C7 insert into cell membrane, recruit C8
4) C8 is a trimer. 1 unit inserts into cell
5) C5b/C6/C7/C8 capable of transiently lysing cells
6) C5b/C6/C7/C8 casuses polymerisation of C9
7) Polymerised C9 forms a 100 Angstrom hole

Question 52

Bacteria that is controlled with C9 pore formation

Answer 52

Neisseria spp

Question 53

Factors involved in forming classical C3 convertase

Answer 53

C1(q, r, s), C2a, C4b

Question 54

Factors involved in forming lectin C3 convertase

Answer 54

MBL, MASP, C2a, C4b

Question 55

Factors involved in forming alternative C3 convertase

Answer 55

C3b, Bb, factor D (cleaves B)