25 Complement System-I

Dr. M. N. Gupta

1. Objectives

•  To understand complement as a cascade system of reactions

•  To learn about how to measure and detect complement

•  To explain how classical pathway of the complement works

•  To understand how an alternative pathway operates which unlike the classical pathway is not dependent upon humoral immunity.

2. Concept Map

3. Description

Complement system is a set of serum enzymes. It is an important illustration of how cascade system can amplify a biological signal.

You would have come across similar cascades in metabolic controls and blood clot formation.

The major form of complement system are:

Initiating inflammation, attraction of neutrophils, opsonisation of the microbe and killing of the microbe by lysis.

The complement system is common to all vertebrates. It is one of the serum enzyme systems. It is a cascade system in which one reaction leads to form the active enzyme for the next. The result is a domino effect where a small stimulus can get amplified down the line

The complement proteins

In humans, complement contain 20 soluble glycoproteins. Many of these are produced by hepatocytes and monocytes. These are constitutively present in blood.

These are designed as C1,C2 etc or as factor B….etc.

Why cascade reactions are useful ?

In many cases, uncontrolled activity can have disastrous consequences. Yet when needed, it needs to be in place when required.

A known example is that of blood clot formation. Blood clots can be fatal when formed inside the body. However, once bleeding starts a slow clot formation would lead to an individual bleeding to death.

The strategy is to limit such activities by producing them in small quantities and with short half lives so that high amount of activity is generated transiently.

Cascade system like complement achieve this beautifully. Complement system was discovered by Belgian scientist Bordat. It is a system whose activation results in disruption of cell membrane. There are various pathways by which complement system operates.

In the classical pathway, the trigger is an immune complex of Ag and Ab

The properties of antibody complexed with antigen are very different from free antibody. Firstly the network of antigen and antibody creates a spatial array of molecules. Secondly antigen binding to antibody via Fab generates a conformational change which leads to Fc region being more available for many interactions.

C1 can be stimulated by IgM bound to antibody or pair of IgG bound to an antbody but located closely on the antibody surface Although the complement components are numbered, the sequence of their participation is not in numerical order . Thus C1 acts on C4 and so on. Polymeric IgM activates complement more effectively than IgG.

This table shows components of all the pathways of the complement activation . In the classical pathway, as we have seen, only C1 , C4 , C2 , C3 are involved.

The five components in the complex C1q(C1r)2(C1s)2 are bound to each other non covalently and stabilised by Ca2+.

C1q itself consists of 18 polypeptide chains in which three polypeptides A,B, and C form a subunit . C1 is made up of one molecule of C1q and two molecule of C1r and C1s each.

The chains are linked by S-S bonds. 80 residues at the N-terminal have a collagen like structure with glycine occupying 3rd position in a sequence rich in Hydroxyproline, Hydroxylysine and of course Glycine . In fact , Collagenase can digest part of C1q .

The complex (C1r)2 (C1s)2 can occur as such and has an extended form. Upon binding with C1q, it turns into a more compact form twisted with shape of 8. Each C1r and C1s has one catalytic domain and one interaction domain which is responsible for binding with each other as well as with C1q. Binding of C1q brings catalytic domains of C1q and C1s together. The globular heads of C1q (the C-terminals which do not have collagen like structure!) is responsible for binding to the Fc portion of the antibody (which in turn is bound to an antigen!). Each C1q molecule has to bind to at least two Fc sites for a stable complex formation to take place. This is the molecular mechanism of the trigger which requires either 2Ig molecules closely or an (IgM bound to antigen).

Also, in circulating IgM, C1q binding sites on Fc are not exposed. Hence, unless bound to an antigen, IgM cannot initiate complement cascade reactions.

C4 is the next component which comes in play. It is glycoprotein with three chains α, β and γ. Their molecular weights are 93kDa, 78kDa and 33kDa respectively. C1r and C1s potential catalytic domains coming together results in C1r getting activated and splitting C1s. This exposes catalytic sites on C1s. C1s cleaves C4. The active C1s in fact cleaves α-chain of C4 to form two fragments: Small fragment from the N-terminal called C4a and a major fragment C4b.

C4b is unstable in free form and can be rapidly broken down to an inactive product

Activated C1s can also split C2. C2 is a glycoprotein of 117 KDa

C2a can combine with C4b.

C4bC2a is called classical C3 convertase as it activates C3.

C4bC2a can further complex with C3b to form C4bC2aC3b which is called C5 convertase. C3

It has molecular weight of 190kDa and has one α chain (120kDa) and one β-chain (70 kDa) linked by 2 S-S bonds.

Complement pathways

C3 has the highest concentration among all complement components in the serum. In a way C3 activation is the most critical step of the complement action. C3 can also be activated by other pathways. In fact, all pathways differ in this early stage with characteristic way C3 is activated. The late stage of all the pathways is similar in which large multimolecular complexes of other complement components cause cell lysis by damaging membranes

Alternative pathway

C3 actually also breaks down spontaneously in plasma and as a result C3b is generated in normal serum at small concentrations all the time. C3b can combine with another protein to form alternative pathway C3 convertase. Thus, through this route C3b can form in adequate amount. A protein called factor H which can destroy C3b very fast. For alternate pathways to operate factor H activity has to be controlled. Factor H is a glycoprotein that can bind to α-chain of C3b. This makes α-chain of C3b susceptible to proteolytic attack by factor I.

Factor H requires surface (on the antigen) rich in sialic acid to be able to bind C3b.

For example, if some bacterial surface do not have enough sialic acid, factor H cannot bind to C3b. In that case C3b is able to bind to factor B. This leads to progressive activation of the complement pathway by positive feedback loop.

Factor B

Factor B is a large protein of 100 kDa molecular weight. Its binding to C3b is Mg++ dependent.

C3bB has weak C3 convertase activity. It can however aquire potent C3 convertase activity. upon cleavage by factor D.

C3bBb is a potent C3 convertase. Factor D, is a serine esterase which actually cleaves B only when it is bound to C3b. So, C3b supply regulates this process.

C3bBb has a very short half life of 5 minnutes. Its half life is extended to 30 minutes if it binds to another protein called factor P or properdine. Factor P is a γ-globuln of 220 kDa molecular weight and can bind to C3bBb reversibly. Hence sometime alternative complement pathway has been referred to properdine pathway by some people.

The alternative pathway requires low level breakdown of C3. This cleavage generates a thioester bond in C3b which is reactive enough to bind to many molecule or cells. If C3b binds to a self cell, there are regulating molecules which can inactivate it. If it binds to the invading microbe, Bb binds to this bound C3b. The alternative complement pathway can protect from infection unaided by lymphocytes. Hence, alternative complement pathway is older pathway in terms of evolution. It may be recollected that antibody formation takes some time to form. Hence during this period alternative pathway is the first line of defense. Classical pathway in fact evolved much later after the evolution of humoral immunity.

The terminal part of the complement pathway

C3b, formed from either classical pathway or alternative pathway initiate the late phase or terminal complement pathway. C5 convertase is formed from either of the pathway by binding of the components.

C5 convertase of the classical pathway: C4b2a3b.

C5 convertase of the alternative pathway: PC3b2Bb

C5 is a protein of molecular weight 206 kDa with an α chain (mol. wt. 141 kDa) and a β chain (mol. wt. 83 kDa) linked by a S-S bond.

If unbound C5b is unstable.

C6 is a single chain protein of 95kDa mol. wt.

C7 is a protein of 120 kDa mol. wt. which can bind to C5bC6 complex either in free solution or when the later is bound to a membrane.

Membrane bound C5bC6C7 binds to C8, a three chain protein of mol. wt. 163kDa.

C5bC6C7C8 initiates polymerization of C9

Cell lysis

C9 is a perforin, its oligomers of 12-15 monomers form a polyC9 which can damage the integrity of the cell membrane by insertion.

The central hole of poly C9 becomes a transmembrane channel resulting in osmotic lysis of the cell.

The alterate pathway was discovered by Pillemer in the 1950s. Unlike classical pathway it does not require antibody bound to the microbe. So what can trigger it? It can get triggered by presence of many substances. We will also see that apart from these two, there is yet another pathway which can activate the complement.

The alternative pathway can be triggered by:

• Microbial polysaccharides like inulin
• Lipopolyssaccharides of of gram negative bacteria
• Teichoic acid of gram negative bacteria
• Some bacteria and parasite such as schistosoma mansoni

So, encounter with a foreign surface can trigger it. Deficiency of complement components is associated with many diseases.

Evaluation of the complement system

(A) Assay for the total hemolytic complement (CH50).

Antibody coated RBC are added to the test sample of the serum. Normally sheep RBC are used. The classical pathway is activated and antibody coated sheep RBC are lysed. The sera as a source of haemolytic complement vary in their efficiency. Guinea pig complement is highly haemolytic, horse complement is non haemolytic.

One CH50 unit is the amount of complement required to lyse 50% of a standard preparation of sheep RBC.

In the presence of Ca++ chelators like EDTA, C1 is inactivated and classical pathway is blocked. It necessary with such samples, the alternative pathway is activated by adding an endotoxin or Ig aggregate.

The above test is based upon the nonspecific nature of complement. Complement from one species can work with antibodies (& antigens!) from another species.

Complement content does not change upon immunization. All immunization does it is to induce the activation of the pathways.

The lytic property of complement is thermolabile. Heating at 56ºC for few minutes destroy its lytic activity. This is useful. To rule out the artefact in determining CH50, the serum sample can be heated and test redone to obtain a contral zero value in the CH50 determination.

(B) The individual complement compound can be quantified using immunoassays. As deficiency of each complement component is how know to be responsible for specific diseases, this serves as a useful diagnostic test.

The complement in the test serum of the patient can be used as a chemo attractant and measuring neutrophil chemotaxis. This evaluates chemotactic factors such as C5α.

In fact, this attraction of neutrophils to C5α leads them to the site of attraction and constitute one of the major functions of the complement.

( C) The complement system is powerful weapon as a part of our immune defence system. It has many biological consequences. It also needs to be regulated. In this lecture, we have focussed on its activation and lytic property only.

Summary

•  Complement can destroy a microbe with or without the help of lymphocytes.

•  The earlier evolved pathway, the alternative pathway is activated by the presence of foreign surfaces.

•  The classical pathway is triggered by antibody coated a microbes.

•  Various assays for detecting lytic activity of complement or estimating its individual components have been developed.