COMPLEMENT

ACTIVATION

By
JITENDRA KUMAR
 INTRODUCTION

• Complement system comprises a vital component of innate immunity system.

• Discovered in 1896 by " Bordet " as a heat labile component of serum. It was named for its
ability to complement the antibacterial properties of antibody in the heat-stabile fraction of
serum.
• First it was thought to be a vertebrate feature but genomic and functional studies on
complement effectors molecules have discovered complement analogs in ancient phyla
like cephalochordates(lancets), urochordates (tunicates), and Echinodermata (sea
urchins). Most recently , functional C3 identified in horseshoe crab and cnidarian
anthozoans Nematostella (miller et al 2007).
• Complement is a system of 30 proteins in plasma & on cell surface , approx. more than
15% of globular fraction of plasma.
• This array of proteins is organized into a hierarchy of proteolytic cascades. It can elicit
highly regulated inflammatory and cytolytic immune response to bacteria, virus , parasites
and tissue damaged by physical, chemical or neoplastic insults, and other surfaces
identified as nonself.
• Complement system also interface with and influence T- cells and B-cells biology.
• It has 4 important functions : lysis, opsonization , activation of inflammatory response,
clearance of immune complex.
 Pathways of complement Activation

Complement system can be activated through 3 different pathways :

A ) Clasical pathway
B ) Lectin/Bypass pathway
C) Alternative pathway
 CLASSICAL PATHWAY

• Antibody directed
• Initiates with C1 complex
• Human C1 -
(1) C1q, the biggest molecular weight, composed of 18
polypeptide chains that associate to form six collagen-
like triple helical arm (pentamolecule-c1q fragment with
6 domain + 2Xc1r+2c1s)
(2) the only component of complement that circulate in
the serum in functionally active forms
(3) For activation, it need to bind to at least two IgG or
one IgM and need the presence of Ca2+.
(4) Free or soluble Ab can not bind to the complement;
the formation of an antigen-antibody complex induces
conformational changes in the Fc portion of the Ab
molecule
(5) C1 complex undergo conformational change results
autocatalysis of c1r, ultimately activation of c1s.
 Generation of C3 convertase  
----Activated C1s enzymatically cleaves C4  into C4a and C4b.
----Activated C1s enzymatically cleaves C2  into C2a and C2b.
----C4b2b complex binding to the membrane, is known as C3 convertase .

Generation of C5 convertase
    ----C3 convertase cleaves C3 into C3a and C3b. 
    -----C3b binds to the membrane to form C4b2b3b complex.
    -----C4b2b3b complex functions as C5 convertase .

  Effector step
C5 convertase cleaves C5 into C5a and C5b. C5b binds the membrane.
C5b binds C6 and  C7 to yield a hydrophobic C5b67 complex which attaches quickly
to the cell membrane. 
 C8 binds to this complex and causes the insertion of several C9
molecules.C5b6789 complex is known as MAC which  leads to formation of a hole in
the membrane resulting in cell lysis.
Figure.1 Classical pathway
 LECTIN PATHWAY

• It employs pattern recognition receptors (PRRs),such as mannose binding lectin

(MBL) and Ficolins,to conduct nonself recognition .
• Few highly conserved structures present in large group of microorganisms popularly
reffered to as PAMP (pathogen associated molecular patterns) e.g. Endotoxin /LPS of
Gram-ve bacteria,lipoteichoic acid of Gram+ve Bacteria and Beta glucagon of fungi.
• MBL (receptor of collectin family) is complexed with MBL associated serine
proteases(MASP)-1,2 &3, (which are functionally and structurally similar to c1s and
c1r) such that binding of MBL to pathogenic surface leads to activation of MASPs,
cleavage of C2 and C4, and ultimately to the generation of the C3 convertase of both
classical and lectin pathways,C4bC2a.
JCI, May 2006
 Alternative pathway

Initiated by the spontaneous hydrolysis of C3 to the C3b analog C3(H2O) ( contains

an activated thioester bond) , which binds to Factor B.

In turn allowing cleavage of Factor B in to Bb and Ba by Factor D and forming the AP C3

convertase C3(H2O)Bb.

This C3 convertase amplify the reaction by converting C3 in to C3b and C3a. C3b generated
can bind to surfaces in the in the vicinity and associate with Factor B, which inturn be activated
By Factor D to form C3bBb..

This complex is stabilized by Properidin , which helps to amplify AP activation .

Figure3. Alternative pathway The central molecule C3 consists of 13 domains, shown by the different colours
in the left-most structure. C3 is cleaved by the C3 convertase (generated either through the classical and lectin
pathways or through the alternative pathway). Cleavage results in release of the small anaphylatoxin C3a and
the large opsonin C3b. The activated C3b exposes its thioester and covalently binds to hydroxyls on adjacent
surfaces. C3b binds pro-enzyme factor B, which consists of five domains: the Von Willebrand factor A-type
domain (green), the serine protease domain (dark blue) and the domains comprising fragment Ba (red).
Binding of factor B to C3b is indicated by the complex formed by factor B and the C3b homologue cobra venom
factor (CVF). Next, factor B is cleaved by factor D, yielding C3bBb, which is the active C3 convertase of the
alternative pathway. In the scheme, the putative binding of C3 to C3bBb is indicated (here, C3 is in grey, with
the C3a domain in red; C3b is shown in light blue); the factor B protease fragment Bb (dark blue and green) is
associated with the carboxyl terminus of the C3 substrate. The C3 convertase cleaves additional C3 molecules
into C3a and C3b, thereby amplifying complement activation. Host cells are protected by complement
regulators such as factor H. Factor H dissociates the C3bBb complex by binding to C3b (shown is the complex
of C3b and factor H domains CCP1–CCP4). subsequent binding of protease factor I cleaves C3b into inactive
C3b (iC3b) and the further degradation products C3dg and C3c.
NATURE REVIEWS MICROBIOLOGY, June 2010
Figure 3 Three major complement activation pathways
Cell research Jan. 2010
 Misclleneous pathways

• It is reported that Properidin could promote denovo C3 convertase assembly when

immobilized to an inert surface and initiate C3 convertase formation on microbial
surface such as Nisseria Gonorrhoeae.(Atkinson, 2007).
• Also reported that C3 and C5 can be directly cleaved by proteases unrelated to the
complement cascade, such as kallikrein and thrombin (which are involved in
coagulation), leading to an additional source of anaphylatoxins (C5a and C3a) and
establishing a novel and potentially important connection between thcomplement and
coagulation cascades (lambris J,D 2007).
 Regulatory pathways
• Due to the des truc ti v e potential of c ompl ement ac tiv ati on, es pec ial ly i n li ght of the potent feedbac k ampli fic ati on abi lity of the AP, c ompl ement ac ti vi ti es need to be c onfined to appropri ate pathogeni c surfac es, and generation of potent effec tors mus t be ti ghtl y regulated to prev ent c oll ateral damage to heal thy host tis s ue.
• Complement regulati on oc c urs predomi nantl y at two s teps wi th i n the c as cades ,at the
Lev el of the c onv ertas es , both in thei r as s embly and i n their enzy matic ac tiv i ty , and during as s embly of the MAC.
• Upon generation of C4b and C 3b fragments & their c ovalent li nkage to c el lular s urfac e,they ex perienc e one of the two fates :
A) Catabol is m of C3b and C4b vi a c onstitutiv ely ac ti v e s erine proteas e Fac torI, whi ch c an c l eav e C3b and C4b into inac tiv e fragments , s uch as i C3b, C3c and C3dg.To prev ent nons pec i fic C3b degradati on, for ins tanc e in the c as e of proper complem ent activ ati on, Fac tor I requi res c ofac tors for i ts proteol y tic ac ti v ity. Thes e cofac tors inc lude membrane c ofac tor protein (MCP; CD46), c om plement receptor 1 (CR1), and
Fac tor H whic h are ei ther intrins ic m embrane proteins on hos t c ell s or hav e v ari ous mec hanis ms to ens ure preferenti al c ofac tor activ ity on hos t s urfac es , and thereby limi t c om pl em ent ac tiv ation in these contex ts and prev ent by s tander tis s ue dam age.
B) To prevent unregulated C3 c onv ertase formation in the event that C3b depos iti on c annot be controll ed, there are s ev eral c omplement i nhibitors that pos s es s inhibitory and/or dec ay-ac celerating ac ti vi ty for c onv ertas es. A primary ex ampl e of this c ategory i s DAF;CD55), whic h s erv es to inhi bit as sembly of new C3 conv ertas es,thereby li miting there as sembly to partic ipate i n further c ompl ement ac tiv ati on. Others c opl ement inhibitors wi th DAA is CR1,Fac tor H, C4bp etc .

• ……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………… of c omp le men t in hib itor s is d eca y-a cc eler atin g

• fa cto r ( DAF ; CD55 ), wh ic h s erv es to i nhib it as sembly
• of ne w C3 c on ver tase s an d sh ort en the ha lf-l ife o f p refo rme d
• co nv er tase s, the reby li mit ing the ir a bilit y t o p arti cip ate
• in fu rth er c omp lement ac tiv atio n [ 36, 38] .
• Recent studies has solved the cstructure of first 4 domains of Factor-H in complex
with C3b,reported that FactorH destabilizes the AP C3 convertase through
competition and also provides binding platform for Factor I mediated proteolysis of
C3b, threr by limiting the AP.(Ricklin.D 2009).

•The final level of complement control, necessary in situations in which there is rampant
complement activation, is to inhibit the assembly of the lytic MAC via of membrane
bound (CD59) or fluid-phase (vitronectin, S protein) inhibitors .
Figure 5.Effectors of the complement system. The function of complement in innate host defense is
accomplished through three broad effector pathways; lysis, inflammation, and opsonization/phagocytosis.
(A) Complement activation and the generation of C5 convertases lead to the liberation of the C5 product,
C5b. C5b forms the basis of the MAC assembly. C5b first associates with C6, C7, and the targeted
surface. C8 associates with this complex and is partially inserted into the membrane. This allows C9 to
insert into the lipid bilayer, where approximately 12-15 C9 molecules will form a stably inserted pore with
~10nm diameter. Formation of the pore leads to the targeted lysis of the surface upon which it is
assembled, accompanied with a dysregulation of ion concentrations across the membrane and loss of
mitochondrial polarity. (B) Anaphylatoxins are potent proinflammatory molecules generated from the
cleavage of C4, C3, and C5 into C4a, C3a, and C5a (C4a not shown), respectively. Binding of
anaphylatoxins to the N-terminal region of their cognate receptors, C3aR and C5aR, allows
conformational changes to the intracellular domains to induce G-protein coupling and downstream
signaling. The effects of this binding depend on the cell type on which the anaphylatoxin receptor is
expressed; and some of the most important cell types and effects in the innate immune response are
summarized. (C) Generation of the C3b fragments by C3 convertases of all three activation pathways
initiates the opsonization pathway of complement, an important effector in the ‘tagging’ and clearance of
foreign bodies. Opsonic fragments, such as C3b and its cleavage products, are recognized by
complement receptors 1-4 (CR1-4) and CRIg. Fc receptors bind to the Fc region of antibody. Binding of
the complement receptors to opsonized bodies mediates their sequestration and uptake by phagocytic
cells, most commonly macrophages and neutrophils.
SUMMARY
Figure 6 Detailed view of complement activation,
amplification, signaling and regulation. (a) A network of soluble and surface-bound proteins enables
the recognition, tagging and elimination of microbial intruders and foreign cells (a) and stimulates
downstream immune responses. In the classical pathway, C1q recognizes pathogen- or damage-
associated PRMs (such as IgG, IgM and CRP) on foreign or apoptotic cells, inducing the formation of
the classical pathway C3 convertase (C4b2b) through cleavage of C2 and C4 by C1s. Detection of
carbohydrate patches by MBL or ficolins associated with MASP via the lectin pathway forms the same
convertase, which activates the plasma protein C3, generating its active fragments C3a and C3b.
Covalent deposition of C3b on nearby surfaces (opsonization) leads to the binding of factor B and
conversion into the alternative pathway C3 convertase (C3bBb), which cleaves more C3 into C3b and
thereby amplifies the complement response. In addition, a low level of complement activation is
maintained in solution (tick-over), and resulting C3b or C3 convertases can be recruited to foreign
surfaces and stabilized by properdin (P). Increasing surface densities of C3b lead to a gradual
substrate shift of the convertases from C3 to C5. Cleavage of C5 into C5a and C5b initiates the
assembly of the lytic TCC on susceptible cells. Opsonization by C1q and C3b, and its degradation
products iC3b, C3c and C3d, induces phagocytosis by complement receptors. The anaphylatoxins C3a
and C5a cause strong proinflammatory signaling through their GPCRs. C5a also co-regulates
immunoglobulin (Ig)-mediated phagocytosis of immune complexes (IC) by modulating the differential
expression of activating (FcγRI/III) and deactivating (FcγRIIB) Fcγ receptors. On B cells, binding of
C3dg to the CR2/CD19 co-receptor complex lowers the threshold of activation by several orders of
magnitude and has an important role in their maturation. Close crosstalk between TLRs, complement
receptors and regulators modulates IL-12 in APCs and thereby influences the activation and
differentiation of T cells. (b) On healthy human cells (b), any complement activation or amplification is
attenuated by surface-bound regulators that accelerate decay of the convertases (CR1, DAF), act as a
cofactor for the fI-mediated degradation of C3b and C4b (CR1, MCP), or prevent the formation of the
TCC (CD59). Soluble regulators such as C4BP, FH and FHL-1 recognize self-surface pattern-like
glycosaminoglycans and further impair activation. Finally, regulators enable control at the level of
initiation (C1-INH, MAP-1, sMAP, C2 receptor inhibitor trispanning, FHR-4), the C5 convertases (FHR-
1, CRIg) or TCC (FHR-1, VTN, clusterin).
THANKS