Chapter 6 Complement

Section I introduction Discovery of complement The end of 19 century Jules Bordent ( ),

Fresh serum containing an antibacterial antibody was added to the bacteria at physiologic temperature (37  C ）， bacteria were lysed If the serum was heated to 56  C 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 Border concluded that the serum must contain another, heat-labile component that assists,or complements, the lytic function of antibodies, and this component was later given the name “ complement”

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.

I. The constitution and name of complement system (I). The constitution of complement system (II) The name of complement system

(I). The constitution of complement system 1. Intrinsic components of complement Classical pathway: C1 (C1q,C1r,C1s),C2 ， C4 Classical pathway: C1 (C1q,C1r,C1s),C2 ， C4 MBL （ mannan-binding lectin 甘露糖结合凝集素 ) pathway: MBL, MASP( MBL-associated serine proteinase, MBL 相关丝氨酸蛋白酶 ) MBL （ mannan-binding lectin 甘露糖结合凝集素 ) pathway: MBL, MASP( MBL-associated serine proteinase, MBL 相关丝氨酸蛋白酶 ) Alternative pathway: B factor, D factor Alternative pathway: B factor, D factor Common terminal pathway ： C3 ， C5 ， C6-9 Common terminal pathway ： C3 ， C5 ， C6-9

2. complement regulatory proteins C1 inhibitor,I factor,C4-binding protein, H factor, S protein 3. Complement receptors CR1-CR5

(II). The name of complement system Intrinsic Intrinsic components : C1-C9 Other components: H factor,D factor, ect. Activation : C3, C3b, C3a; Bb, Ba Inactivation : iC3

II. physical and chemical properties of complement 1. The concentration of complement in serum is stable( 10% of serum proteins), C3 is highest in all of complement components: g/L 2. Heat –liable feature 56  C 30 min—inactivation 0-10  C for 3-4 days 3. Synthesized sites: liver, macrophage 4. The concentration of complement is highest in the serum of guinea pig.

Section II The activation of complement The soluble proteins of the complement system are synthesized in the liver and are secreted as non- active forms called zymogens( 酶原）. 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.

The three different pathways of complement activation 1.classical pathway :from C1 activated by by Ag-Ab 2. Alternative pathway: from C3 by the surface of microbe 3. MBL pathway: from C4 and C2 by binding of MBL to mannan on surface of microbes Terminal pathway : formation of MAC (membrane attack complex) same in the three pathway

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. Phase of recognition (initiation ): recognizing unit (C1qrs) :activated C1 2. Phase of activation: activating unit( C4,C2,C3) C3 convertase and C5 convertase 3. Phase of attack( effector): membrane-attack complex (MAC),C5-9

1.Recognition phase 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 activated into activated C1 (C1)

IgM The C1 must bind at least 2 CH3 domains of one IgM molecules to be activated. IgM is the best complement activator because it is a pentamer 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.

C1q=6 2 C1r+2C1s+ C1q = C1 6

The first protein in the classical pathway is C1 2 C1s 2 C1r C1q

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 cleave C1s to activate it.

2. Activation phage Formation of C3 convertase （ C4b2a) and C5 convertase(C4b2aC3b)

（ 1 ） Formation of C3 convertase （ C4b2a) C1 C4 ， C2 C4b2a (C3 convertase) C4a,C2b

The activated C1 cleaves C4 into C4b and C4a. C4

C4a C4b 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 pathway

C3 convertase 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 covanlently binds to C4b2b to form C4b2b3b complex,C5 convertase C3b C3a C5 convertase

Classical pathway IgM/IgG –Ag complex C1q : r : s C4 C4b + C2 C4a C2b C4b2a C3C3b C3a Ca ++ Mg ++ Ca ++ （ C3 convertase ） C4b2a3b （ C5 convertase ）

3. Effector Phase (Common terminal pathway) (1)Formation of the Membrane Attack Complex (MAC) (2) Effect of 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 MAC: C56789

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 C6 C7 Then C7 binds. C7 inserts into the phospholipid bilayer of the membrane.

C5b C6 C7 Then C8 binds to the complex and also inserts into the bilayer. C8

C5b C6 C7 Finally, C9 molecules bind to the complex and polymerize. Ten to sixteen C9 molecules form a pore in the membrane. C8 C9

C5b C6 C7 Ten to sixteen C9 molecules form a pore in the membrane. C8

C5b C6 C7 The membrane attack complex is a pore in the membrane. C8

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: C3,5,6,7,8,9 (1) initiating step: C3 convertase (C3bBb) (2)activation step: C5 convertase (C3bnBb) (3) common terminal effector step ( C56789)

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

Spontaneous conversion or from classical pathway C3b B factor D factor C3bB C3bBb C3bBbP （ C3 convertase ） C3 C3b C3bnBb （ C5 convertase ） enlarge P factor Ba

3.Function: participate in non–specific immunity 4.Features ： Spontaneous cleavaged C3b make individual in status of pre-activation Important amplified mechanism of complement Recongnize self and non-self

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 4. Function: participate in non–specific immunity

The MBL and c-reactive protein were produced after microbe infection MBL binds to mannan residues on polysaccharide of microbe The MBL, structurally similar to C1q. The MBL activate C1r-C1s complex and then C1s cleaves C4,C2 or The MBL activate MASP( mannan-associated serine proteinase) and then MASP activate C4,C2

Mannose-binding lectin MBL is Structurally similar to C1q

MBL binds to mannose on glycoproteins on the surface of microorganisms. Then MASPs bind to it. MASP-1 MASP-2 MASP = mannose associated serine protease C1q-like Similar to C1r,C1s

mannan MBL mannan MASP C4 C4a + C4b C2C2a + C2b C4b2a C3 convertase + MASP MBL Microbe

Complement system 1. Introduction : Definition and composition of complement system 2. Activation of the complement system Three pathway of complement-activating 3.Regulation of Complement activation 4. Complement receptors 5. Biological functions of complement

Section III. regulation of Complement system The explosive potential of the complement system requires that it be kept under tight control. At least 12 proteins are 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 C3 convertase: C4b2a or C3bBb C5 convertase : C4b2a3b or C3bnBb C5b negative feedback

II. action of regulatory factors (I) The regulation for classical pathway (II) The regulation of alternative pathway (III) The regulation of MAC formation

1. C1 inhibitor ( C1INH) 2. Inhibit the C3 convertase C4 binding protein (C4bp) Complement receptor 1(CR1) C4bp and CR1 bind to C4b and then inhibit the binding of C4b to C2a I factor I factor: C3b/4b C3c/4c and C3d/4d H factor : bind to B or Bb decay-accelerating factor （ DAF): separate C3b from C3bBb or C4b from C4bC2a

1. Inhibition of C1 activation

CD59

1. C1 inhibitor ( C1INH) 2. Inhibit the C3 convertase C4 binding protein (C4bp) Complement receptor 1(CR1) C4bp and CR1 bind to C4b and then inhibit the binding of C4b to C2a I factor I factor: C3b/4b C3c/4c and C3d/4d H factor : bind to B or Bb decay-accelerating factor （ DAF): separate C3b from C3bBb or C4b from C4bC2a

2. Inhibition of the C3 convertase formation C4 binding protein (C4bp) I factor

2.Inhibition of the formation C3 convertase

Complement receptor 1(CR1) Decay-accelerating factor （ DAF):

H and I factor depredate C3b

3. The Regulation of common terminal pathway: inhibiting the formation of MAC C8 –binding protein ( homologous restriction factor, HRF) interfere the binding of C8 to C9 Membrane inhibitor of reactive lysis, MIRL(CD59) block binding of C7,C8 to C5b-6 complex

Regulation of terminal pathway

Section IV Complement receptors 1. CR1 ( CD35 and C3b/C4bR): Macrophage: opsonization B cells : proliferation RBC: immune adhesion 2. CR2 (CD21) B cells : coreceptor of B cells 3. CR3 (iC3bR) 4.CR4 ( CD11c/CD18) : iC3bR and C3dgR 5. CR5: C3dgR C3dR

Opsonization: C3b,C4b ----CR1

Section V Biological functions of Complement

1. Lyse cells, bacteria and virus The complement system lyses target cells bacteria,virus and other microbes through alternative pathway and MBL pathway of complement-activating in absence of antibody and through classical pathway in presence of antibodies Actions of complement system

2. Opsonization The phenomenon that phagocytosis of phagophage are enhanced by antibody or complement binding to microbial surface. The bound C3b,C4b to CR1 on macrophage and neutrophils enhances their actions

Opsonization: C3b,C4b ----CR1

3. Clearance of immune complex -----Immune adherence C3b,C4b----CR1 C3b or C4b 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

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

4. Mediate inflammatory reaction: Anaphylatoxins : C3a, C4a,C5a ( 过敏毒素） Kinin-like action （激肽样作用） : C2b Chemokine-like action （趋化因子样作用） C3a, C5a

5. participate in immunologic regulation component of co-receptor for B cell activation

Section VI Abnormal complement system and disease

What you should know by the end of this lecture  Terms: MAC  Definition and composition of complement system  Three pathway of complement-activating  Biological functions of complement  Regulation of Complement activation