Chapter 6

Complement

The end of 19 century

Jules Bordet (1870-1961)

• Fresh serum containing an antibacterial antibody was added to the bacteria at physiologic temperature (37℃）， bacteria were lysed.

• If the serum was heated to 56℃ or more , it lost its lytic capacity.

• This loss of lytic capacity was not due to decay of antibody activity because antibodies are heat-stable and even heated serum was capable of agglutinating the bacteria.

• Bordet concluded that the serum must contain another , heat-labile component that assists the lytic function of antibodies, and this component was later given the name “complement”

Definition of complement systemDefinition of complement system ：：

A system of serum and cell surface proteins （ including more than 30 proteins ) that interact with one another and with other molecules of the immune system to generate important effectors of innate and adaptive immune response.

Contents

• Part The components and propertiesⅠ of complement system

• Part Activation of complement systemⅡ• Part Regulation of complement systemⅢ• Part Ⅳ Complement receptors

• Part Ⅴ Biological functions of complement

• Part Complement and diseaseⅥ

PartⅠ The components and properties of complement system

(I) The components and nomenclature(I) The components and nomenclature of complement systemof complement system

(II) Physical and chemical features of complement(II) Physical and chemical features of complement

(I). The components and nomenclature of (I). The components and nomenclature of complement systemcomplement system

1.1. Components participating complement activationComponents participating complement activation

• Classical pathway: C1 (C1q,C1r,C1s)Classical pathway: C1 (C1q,C1r,C1s) ，， C2C2 ， ， C3C3 ， ， C4C4

• MBLMBL （（ mannan-binding lectin) pathway: MBL, MASP( MBL-associated mannan-binding lectin) pathway: MBL, MASP( MBL-associated serine protease) serine protease)

• Alternative pathway: factor B, factor DAlternative pathway: factor B, factor D

• Common terminal pathwayCommon terminal pathway ：： C5C5 ，， C6C6 ，， C7C7 ，， C8C8 ，， C9C9

2. Regulatory components of complement system

factor I, factor H, S protein, properdin(factor P), C1 inhibitor(C1 INH) , C4-binding protein(C4BP), SP40/40, membrane cofactor protein(MCP), decay accelerating factor(DAF), homologous restriction factor(HRF), membrane inhibitor of reactive lysis(MIRL)

3. Complement receptors

CR1~CR5, C3aR, C2aR, C4aR, etc

Nomenclature of the complement system• Intrinsic components in classical pathway:C1~C9• Intrinsic components in alternative pathway: factor D• Regulatory proteins:C1INH,C4BP• Cleaved fragments:C3a, C3b; C2a, C2b• Activated components:C1• Inactivated components:iC3b• Complement receptor:CR

（Ⅱ） . The physical and chemical features of complement

1. Synthesized sites: liver, macrophage, small intestine epithelium 2. The concentration of complement in serum is stable( 10% of

serum proteins) , C3 is the highest in all of complement components:1~2 g/L

3. Heat–labile feature: 56℃ 30min—inactivation 0~10℃ for 3~5 days 4. The concentration of complement is the highest in the serum of guinea pig.

humoural complements name Molecule weight （kDa） concentration（g/ml）

460

80

83 50 83 50 200 600 102 20

Classical pathway C1q C1r C1s C4 C2 C3 185 1300

24

Alternative pathway D factor B factor 90

1

210

MBL pathway MBP

30 x 3

1

Terminal pathway C5

204

70

C6 120 65

C7 120 55 C8 160 55

C9 70 60

Regulatory protein C1 INH

105

200

C4BP 550 250

Factor H 150 480

Factor I 88 35

Factor P 4 x 56 20

Part AⅡ ctivation of the complement system

• The soluble proteins of the complement system are synthesized in the liver and are secreted as non-active forms called zymogens.

Zymogen: The cleavage is required for activation

• The cleavage of a zymogen usually produces a large active fragment with enzymatic activity and a small fragment with inflammatory effects.

Three different pathwayspathways of complement activation：

1.Classical pathway: from C1 by Ag-Ab

2.Alternative pathway: from C3 by the surface of microbe

3.MBL pathway: from C4 and C2 by binding of MBL mannan on the surface of microbes

Terminal pathway : formation of MAC (membrane

attack complex), same in the three pathways

(C4b,2a)

(C4b,2a,3b)

Ⅰ. The Classical pathway of complement activation

1.Initiating substances: antigen-antibody complex or immune complex

2.Components: C1q ,C1r,C1s,C4,C2,C3,5,6,7,8,9

3.Process:

C1q,C1r,C1s C4,C2,C3,5,6,7,8,9

The process of complement activation

in classical pathway:

1. Initiation step: recognizing unit (C1qrs),

activated C1

2. Activation step: activating unit( C4,C2,C3),

C3 convertase and C5 convertase

3. Effector step: membrane-attack complex (MAC) ,

C5~9

1. Initiation step

recognizing unit (C1qrs) --- activated C1

• IgG1~3 and IgM can activate complement by classical pathway

• The complement component C1 binds to the Fc part of the antibodies(CH2 of IgG or CH3 of IgM), and then is turned into activated C1 (C1)

monomer

pentamer

C1q=6

2 C1r+2C1s+6C1q = C1

6

The first protein in the classical pathway is C1

2 C1s2 C1r

C1q

• IgG

The C1 must bind to at least two IgG molecules that are close enough together so that it can bind to both of them at the same time.

• IgMThe C1 must bind at least 2 CH3 domains of one IgM molecule to be activated.

IgM is the best complement activator because it is a pentamer.

Bacteria with antigenic proteins on the surface

After antibody binds to antigen on the surface of a pathogen ， complement-binding sites are exposed

IgM is the best activator of complement.

C1 binds to at least two IgG molecules to be activated

Binding of two or more of the C1q globular domains causes activation of C1r, which then make C1s activated.

2. Activation step

Formation of C3 convertase （ C4b2a)

and C5 convertase(C4b2a3b)

（ 1 ） Formation of C3 convertase （ C4b2a)

C1

C4 ， C2 C4b2a (C3 convertase) C4a,C2b

The activated C1 cleaves C4 into C4b and C4a.

C4

C4aC4b

C1 cleaves C4 to produce C4b and C4a.

The C4b fragment binds to the surface of the pathogen.

C1 then cleaves C2 into C2b and C2a.

C2

C4b

C2b

C2a

C4b

The activated C1 then cleaves C2 into C2b and C2a.

C4b and C2b together form the C3 convertase(C4bC2a)

C3 convertase

C2a

C4b

(2) Formation of C5 convertase

C1 C4 ， C2 C4b2a (C3 convertase)

C4a,C2b

C3 C4b2a3b (C5 convertase) C3a

• This is the most important step in the classical pathway

C3 convertase

C3

C3

C3

C3 convertase cleaves C3 to C3a and C3b

C3 convertase

C3

C3b

C3

C3 convertase cleaves C3 into C3a and C3b.

C3a

C3b

The C3b covalently binds to C4b2a to form C4b2a3b complex,C5 convertase

C3b

C3a

C3a

C5 convertase

4b2a

C2b

C4b2a

C4b,2a,3b

C4b,2a,3b C4b,2a,3b

C4b2a

C2a

C4b2a

（ C5 convertase ）

Classical pathway

IgM/IgG –Ag complexIgM/IgG –Ag complex

C1q : r : s

C4 C4b + C2

C4a C2b

C4b2a

C3 C3b

C3a

Ca++

Mg++

Ca++

（ C3 convertase）

C4b2a3b

3. Effector step: Common terminal pathway

Formation of the Membrane Attack Complex (MAC)

MAC:MAC: a lytic complex of the terminal components of the complement cascade, including C5,6,7,8 and multiple copies of C9, that forms in the membrane of target cells. The MAC causes lethal ionic and osmotic changes in cells.

C5 convertase cleaves C5 into C5a and C5b.

C5 convertase

C5b

C5a

C5b

C5b binds to the surface and C6 binds to C5b,stabilizing it.

C6

C5b

C6C7

Then C7 binds. C7 inserts into the phospholipid bilayer of the plasma membrane.

C5b

C6

C7

Then C8 binds to the complex and alsoinserts into the bilayer.

C8

C5b

C6

C7

Finally, C9 molecules bind to the complex and polymerize. Twelve to fifteen C9 molecules form a pore in the membrane.

C8

C9

C5b

C6

C7

Twelve to fifteen C9 molecules form a pore in the membrane.

C8

C5b

C6

C7

The membrane attack complex is a pore in the plasma membrane.

C8

Effect of MAC

• On the surface of cell: lyse the cell

• In the serum:SC5b~7, SC5b~8, SC5b~9

Ⅱ. Alternative pathway

1.The initiating substances:

some components of microbial cell surface

aggregated IgA or IgG4

---providing a surface for binding of complement

2.Components and process: factor D, factor B, C3,5,6,7,8,9

3.Function: participate in non–specific immunity

• Normally, C3 in plasma is being continuously cleaved at a low rate to generate C3b in a process.

• A small amount of the C3b in the fluid phase

is unstable and inactive.

• C3b may become covalently attached to the surfaces of cells, including microbes.

The process of complement activation

in alternative pathway:

1. Initiation step: C3b binds to microbial surface,

binds factor B, and forms C3 convertase.

2. Activation step: form C5 convertase

3. Effector step: membrane-attack complex (MAC) ,

C5-9

Spontaneous conversion or from classical pathway

C3b

B factor

D factor

C3bBb C3bBbP（ C3

convertase）

C3

C3b

C3bnBb （ C5 convertase）enlarge

P factor

C3bC3bB C3C3

positive feedback loop of C3

D factor

C3b

Bb

B factor

Important characteristics

In alternative pathway, complement can recognize self from nonself----complement regulatory protein

Alternative pathway is the important enlarge mechanism of complement

Ⅲ. MBL or Lectin Pathway

1.Initiating substances: MBL combine with mannose on the surface of microbe.

2.Components: Mannose-binding lectin (MBL) , MBL-associated serine protease (MASP) ， C4,C2,C3,5,6,7,8,9

3.Process:

MBL-mannose-MASP----C4,C2,C3,5,6,7,8,9

• The MBL and c-reactive protein were produced by liver after microbe infection

• MBL binds to mannose residues on polysaccharide of microbe

• The MBL, structurally similar to C1q.

The MBL activate MASP( mannan-associated serine protease) and then MASP activate C4,C2(MASP is similar to C1r and C1s)

Mannose-binding lectin

MBL is Structurally similar to C1q

MBL binds to mannose on glycoproteinson the surface of microorganisms. Then MASPsbind to it.

MASP-1MASP-1

MASP-2MASP-2

MASP = mannose associated serine protease

mannose

MBL

mannose

MASP

C4 C4a + C4b

C2 C2a + C2b

C4b2a(C3

convertase)

+ MASP

(C4b,2a)

(C4b,2a,3b)

Part Ⅲ Regulation of complement system

• The explosive potential of the complement system requires that it is kept under tight control.

• At least there are 12 proteins known that do this.

Mechanism of complement regulation

Ⅰ. Regulation of self-inactive :decay

Ⅱ. Action of regulatory factors

Ⅰ. Regulation of self-inactive : decay

Spontaneous decay of complement

Cleaved components C3b,C4b,C5b

C3 convertase (C4b2a,C3bBb)

C5 convertase (C4b2a3b,C3bnBb)

Ⅱ. Action of regulatory factors

1. Regulation of C1 activation:   C1 inhibitor (C1INH) binds to sites on

activated C1r and C1s shutting down their proteolytic activity.

Inhibition of C1 activation

2. Regulation of C3 convertase formation

Factor H removes Bb from the alternative pathway C3 convertase,breaking the positive feedback loop.

Factor I inactivates C3b and C4b DAF -----binds to C4b and C2a, preventing

formation of C3 convertase

H and I factors depredate C3b

Inhibition of the C3 convertase formation

Inhibition of the C3 convertase formation

3. Regulation of MAC

Homologous restriction factor(HRF):

HRF(C8bp )------interferes with binding of C9 and C8, prevents formation of MAC

Membrane inhibitor of reactive lysis(MIRL): MIRL(CD59)------interferes with binding of C5b6 complex and C7,C8, and prevents formation of MAC

Regulation of terminal pathway

Part Ⅳ Complement receptors

1.CR1(CD35,C3b/C4bR): Mainly express on blood cells Combine with C3b and C4b with high

affinity Participate in opsonization, immune

adhesion et al

2. CR2(CD21) ：Mainly express on B cellCombine with C3dg,C3d and iC3bPromote B cell to be activated Act as the receptor of EBV(epstein-barr

virus)

3. CR3 ：• Combine with iC3b• Neutrophil,phagocyte,B cell,NK

4. CR4(CD11c/CD18), CR5 ：• Combine with iC3b and C3dg• Neutrophil,phagocyte

PartⅤ Biological functions of complement

Ⅰ. Lyse bacteria and cells

Ⅱ. Opsonization

Ⅲ. Elimination of immune complex

Ⅳ. Induce inflammation reaction

Ⅴ. Regulation of immunity

I. Lyse bacteria and cells

Complement activation

by classical pathway, MBL pathway or alternative pathway leads to the formation of MAC.MAC mediates lysis of target cell.

C3b,C4b ---- CR1

Ⅱ. Opsonization:

C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.

Ⅲ. Elimination of immune complex

Ag-Ab-C3b complex can adhere to the C3bR which exists on the surface of RBC or platelet.This facilitates phagocytosis of immune complex by phagocytes.

Clearance of immune complex -----Immune adherence C3b,C4b----CR1

C3b coats immune complexes facilitating binding to CR1 on erythrocytes, which carry the immune complexes to the liver or spleen where they can be removed and phagocytized

C4b,2a,3b

Ⅳ. Induce inflammatory reaction

Kinin-like action: C2a

Anaphylatoxins:C3a,C4a,C5a

Chemokine-like action: C5a

Ⅴ. Regulation of immunity

Help APC to present Ag

Promote the proliferation and differentiation of B cell

Enhance ADCC by CR1

Chemotaxis effect

Lyse target cell degranulation

Part Complement and diseaseⅥ

1. Congenital defects of the complement system• C1INH defect C2a,C3a,C5a hereditary

angioneurotic edema(HAE)

2. Hypercomplementemia• Acute inflammation, cancer

3. Hypocomplementemia• Chronic inflammation, some autoimmune disease