ZOOLOGY Immunology Complement System Development Team

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Paper No. : 10 Immunology Module : 23 Complement System

Development Team

Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi

Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator: Prof. Anju Srivastava Department of Zoology, University of Delhi

Content Writer: Dr. Sudhir Verma Deen Dayal Upadhyaya College, University of Delhi

Content Reviewer: Prof. Sukhmahendra Singh Banaras Hindu University

ZOOLOGY Immunology Complement System

Description of Module

Subject Name ZOOLOGY

Paper Name Immunology; Zool 010

Module Name/Title Complement System

Module ID 23; Complement System

Keywords Complement, Opsonization, Immune Clearance, Cascade, Anaphylatoxin, Classical Pathway, Lectin Pathway, Alternative Pathway, Membrane Attack Complex, Innocent bystander.

Contents

1. Learning Outcomes 2. Introduction 3. Historical Perspective 4. Functions 5. Nomenclature 6. Pathways 6.1 Classical Pathway 6.2 Alternative pathway 6.3 Lectin Pathway 6.4 Terminal Pathway: Membrane Attack Complex (MAC) 7. Receptors for Complement Proteins 8. Regulation of Complement System 9. Diseases due to Complement Deficiency 10. Complement Assay Systems 11. Microbial Evasion Strategies from Complement Mediated Damage 12. Summary

ZOOLOGY Immunology Complement System

1. Learning Outcomes

After studying this module, you shall be able to

 Know: What complement system is? Why some individuals face recurrent bacterial infections more as compared to others?  Learn: How complement system provides immunity to us? Also, how it links innate immunity with adaptive immunity?  Identify: The components that constitute and regulate the complement system.  Evaluate: The non-specific yet highly organized component of our immunity, the complement cascade system.  Analyze: The underlying beauty of our innate immune system in terms of complement. 2. Introduction

 Complement system was first discovered in 1890 by Jules Bordet as a heat-labile component of serum that „complemented‟ or „augmented‟ its bactericidal properties.  It is known to be comprised of around 30 soluble proteins, together which constitute around 10% of total serum proteins.  Most of these proteins are synthesized by liver and remain in inactive form in the absence of infection.  Evolutionarily, complement system is as ancient as adaptive immune system as even worms and starfishes also have complement system.  Complement system is the major component of innate system which is encountered by pathogens that enter our body after breaching physical barriers and antimicrobial defenses.  It consists of three different pathways i.e. classical pathway, alternative pathway and lectin pathway. All these pathways ultimately lead to a common terminal pathway, leading to formation of membrane attack complex which subsequently lyses the target cell.  Complement system broadly performs three types of functions: host defense against various infections, interface between innate and adaptive immunity and clearance of waste.  For many of these biological activities of complement system, complement fragments bind various receptors present on different cell types. Additionally, these receptors regulate the complement activity.  Besides these receptors, various other regulatory proteins are there that keep a check on the activity of complement system.  The importance of complement system in providing immunity to us lies in the fact that a number of diseases result due to genetic or functional deficiency of different complement proteins.  Still there are a number of pathogens which have developed evading strategies from complement mediated damage. The mechanism of evasion strategy and type of microbes possessing these are discussed in later part of this unit. 3. Historical Perspective

In 1890s, Jules Bordet at the Pasteur Institute in Paris showed that sheep antiserum to bacterium Vibrio cholera causes lysis of the bacteria. On heating the antiserum this bacteriolytic activity gets lost. But, the same can be restored to the heated serum by adding fresh serum. This fresh serum contained no antibodies directed against the bacterium and was unable to kill the bacterium by itself. Bordet reasoned that this bacteriolytic activity utilizes two different substances: first, the specific antibacterial antibodies, which survive the heating process, and a second, heat-sensitive component 3

ZOOLOGY Immunology Complement System

responsible for the lytic activity. Similar experiments were carried out by Paul Ehrlich in Berlin independently and he defined this phenomenon as “the activity of blood serum that completes the action of antibody.” He coined the term complement for the same. Later it was found that complement is interactive composition of large and complex group of proteins.

Value Addition

Dr. Jules Bordet was honoured with Nobel Prize in physiology and medicine (1919) for his discovery of „complement mediated bacteriolysis‟.

Jules Jean Baptiste Vincent Bordet Source: Wellcome Library, London. Public domain, CC BY 4.0, via Wikimedia Commons http://www.nobelprize.org/nobel_prizes/medicine/laureates/1919/bordet-photo.html 4. Functions

The functions of complement system are broadly categorized as:

Figure 1: Physiological roles of complement system Source: Author

ZOOLOGY Immunology Complement System

5. Nomenclature

 Complement proteins can be named by: i) Numerals e.g. C1, C2, C3…C9, ii) Letter symbols e.g. factor B, factor D, iii) Trivial names e.g. Decay accelerating factor (DAF), homologous restriction factor (HRF)  The peptide fragments generated upon cleavage or activation of zymogen complement are denoted by small letters. The smaller fragment resulting from cleavage of a component is designated “a” and the larger fragment is designated as “b” (e.g., C3a, C3b). C2 is an exception to this rule as smaller fragment is called C2b whereas C2a is the larger cleavage fragment. The larger fragments bind to the target near the site of activation, and the smaller fragments diffuse from the site and act as anaphylatoxin.  Functional complexes are formed by interaction among complement factors. The complexes having enzymatic activity are designated by a bar over the number or symbol e.g. C4b2a (C3 convertase in classical pathway). 6. Pathways

There are three pathways for activation of complement system, which culminate to formation of C5b. These pathways are named as classical pathway, alternative pathway and lectin pathway. The final steps for the formation of membrane attack complex (MAC) are common for all the pathways, termed as terminal pathway.

6.1 Classical Pathway

The classical pathway of complement activation starts when the complement C1 recognizes a microbial surface directly or indirectly via antibody bound to pathogen. C1 complement is a complex, made up of a large subunit C1q, which senses the pathogen, and two zymogen subunits C1r and C1s that act as serine proteases. One molecule of C1q and two molecules each of C1r and C1s are stabilized together as C1qr2s2 by Calcium ions. C1q is a hexamer of trimers, each monomer of which contain a globular domain at its amino terminal and collagen like domain on its carboxy terminal. The pathogen recognition activity of C1 lies within the globular domains of C1q. When these globular heads interact with a particular ligand, a conformational change is caused in C1r:C1s, the associated members of C1. It further leads to activation of autocatalytic activity in C1r. The resulting active form of C1r then cleaves C1s to generate active serine protease.

C1s acts on two substrates i.e. C4 and C2. C4 is a glycoprotein that consists of three polypeptide chains i.e. α, β and γ. C1s hydrolyzes the C4a i.e. small fragment of C4 from amino terminal of α polypeptide chain and exposes a binding site on remaining C4b component. C2 attaches itself to the exposed binding site on C4b and is cleaved by C1s to C2a and C2b. The smaller fragment C2b (As mentioned earlier, C2 is the exception in nomenclature, C2b being smaller fragment) diffuses away and C2a attaches itself to target surface along with C4b. The resulting C4b2a complex is called as classical C3 convertase enzyme as it activates C3 from its zymogen form to active form.

C3 complement consists of two polypeptide chains α and β. C3 convertase hydrolyzes C3a from amino terminal of α chain of C3 and generates C3b. The C3b thus generated, binds to C4b2a complex, giving rise to trimolecular C4b2a3b complex which is termed as C5 convertase. The C5 is cleaved by C5 convertase to C5a that diffuses away and C5b which binds to C6 and starts the membrane attack complex formation (Figure 1).

Various proteins involved in classical pathway of complement activation are listed in table 1 below. 5

ZOOLOGY Immunology Complement System

Table 1: List of various proteins involved in classical pathway of complement activation

Source: Author

Figure 2: Overview of the complement activation by classical pathway, leading to formation of C5b, which eventually leads to formation of membrane attack complex. A) C1qr2s2 complex approaches and gets activated (B) upon binding to antigen-antibody complex. (C) C4 is cleaved by activated C1qr2s2 to form C4b which binds with target surface, whereas C4a diffuses away. (D) C2 is cleaved to C2a and C2b. C2a binds with C4b, forming C4b2a i.e. C3 convertase. (E) C3 is cleaved by C3 convertase to form C3a and C3b. C3b binds with C4b2a to form C4b2a3b i.e. C5 convertase. (F) C5 convertase acts upon C5 to form C5b and C5a. C5b initiates formation of membrane attack complex by terminal pathway.

Source: Author 6

ZOOLOGY Immunology Complement System

Value Addition

Why circulating IgM does not activate complement system whereas antigen bound IgM does so?

Because circulating IgM exists in planer configuration in which C1q-binding sites are not exposed. Whereas, when pentameric IgM is bound to antigen on its target surface, it acquires staple configuration in which three binding sites for C1q are exposed. Hence, antigen bound IgM can bind to C1q and activate complement system, whereas circulating IgM cannot.

Why IgG is less efficient in activation of classical complement pathway as compared to IgM?

C1 molecule must bind by its C1q globular heads to atleast two Fc sites for a stable C1-antibody interaction to take place. An IgG molecule contains only one C1q binding site. Thus, for a stable interaction, atleast two IgG molecules should be in close vicinity of 30-40 nm distance. IgM having atleast three C1q binding sites, binds and activates complement more efficiently than IgG.

6.2 Alternative Pathway

Alternative pathway is antibody-independent, innate immune system component. It is activated by foreign cell surface and does not require antibody to initiate. In alternative pathway, serum C3, containing an unstable thioester bond, undergoes slow, spontaneous hydrolysis to generate C3a and C3b. The C3b component cannot differentiate host surface from foreign surface but because of high sialic acid present on host surface, it rapidly deactivates and does not lead to further cascade. Whereas, on foreign surfaces such as bacteria, yeast and virus; low sialic acid is present and thus C3b remains active. C3b then binds factor B by a Mg2+ dependent bond which provides a surface for serum protein D to bind. Factor D cleaves factor B, releasing Ba. Remaining complex C3bBb possesses C3 convertase activity but it is very unstable. Another factor, properdin stabilizes this complex. The C3 convertase further activates C3 to generate more C3b leading to formation of C5 convertase, C3bBb3b. The C5 is cleaved by C5 convertase to C5a that diffuses away and C5b, which binds to C6 and starts the membrane attack complex formation (Figure 2).

Various proteins involved in alternative pathway of complement activation are listed in table 2 below.

Table 2: List of various proteins involved in alternative pathway of complement activation

Source: Author

ZOOLOGY Immunology Complement System

Figure 3: Overview of the complement activation by alternative pathway, leading to formation of C5b, which eventually leads to formation of membrane attack complex. (A) Self and slow activation of small amount of C3 results in C3b formation that binds on microbial surface. (B) Factor B is cleaved to Bb and Ba in the presence of factor D. Bb binds with C3b to form c3 convertase. (C) Properdin stabilizes the C3bBb i.e. C3 convertase. (D) C3 is cleaved by C3 convertase, resulting in formation of C5 convertase (C3bBb3b). (E) C5 convertase cleaves C5 to C5b and C5a. Futher, C5b participates in membrane attack complex formation by terminal pathway.

Source: Author

6.3 Lectin Pathway

Lectin pathway is also antibody-independent, innate immune system component. Lectins are the proteins that bind to carbohydrates. This pathway initiates with binding of mannose binding lectin (MBL) to mannose residues on surface of microorganisms. MBL is an acute phase protein, analogous to C1q of classical pathway. MBL binding leads to binding of MBL-associated serine protease (MASP) which further cleaves and activates C4. MASP is structurally similar to C1r and C1s. Rest of the mechanism is similar to classical pathway for generating C3- and C5-convertase leading to membrane attack complex formation (figure 3).

Various proteins involved in lectin pathway of complement activation are listed in table 3 below.

ZOOLOGY Immunology Complement System

Table 3: List of various proteins involved in lectin pathway of complement activation

Source: Author

Figure 4: Overview of the complement activation by lectin pathway, leading to formation of C5b, which eventually leads to formation of membrane attack complex.(A) Mannose binding lectin (MBL) binds to mannose present on microbial surface. (B) Activation of MBL associated serine protease (MASP) takes place. (C) to (F) MBL activated MASP acts like C1qr2s2 of classical pathway and rest all the steps are like classical pathway only.

Source: Author 9

ZOOLOGY Immunology Complement System

6.4 Terminal Pathway: Membrane Attack Complex

C5 convertase upon activation in all the three pathways, cleaves C5 into C5a and C5b. C5a diffuses away whereas C5b binds to the target surface. C5b being very labile, is stabilized by C6. Upon binding of C7, C5b67 complex undergoes hydrophilic-amphiphilic structural transition exposing hydrophobic regions that serve as binding sites for membrane phospholipids. If this reaction takes place on target cell surface, C5b67 inserts into phospholopid bilayer. Whereas, if it occurs on immune complexes, then the released C5b67 complex enters into membrane of nearby cells causing „Innocent Bystander Lysis‟, an autoimmune damage.

Next C8 binds to C5b67, which creates a small pore of 10Å. 10-17 molecules of C9 polymerize around C5b678 complexand increase the pore size from 10Å to 70-100Å. This pore causes osmotic instability in the target cell because of water influx and loss of electrolytes, ultimately leading to cell lysis.

Various proteins involved in terminal pathway of complement activation are listed in table 4 below.

Table 4: List of various proteins involved in terminal pathway of complement activation

Source: Author

ZOOLOGY Immunology Complement System

Different proteins involved in complement system can be broadly categorized in the categories as depicted in figure 5 below.

Figure 5: Categories of functional proteins involved in complement system Source: Modified from Janeway‟s Immunobiology 8th ed. By Kenneth Murphy

ZOOLOGY Immunology Complement System

Table 5: Comparative view of three activation pathways of complement system

Source: Modified from Kuby‟s Immunology 4th ed. by R. A. Goldsby, T. J. Kindt and B. A. Osborne

Figure 6: Summary of Complement pathways Source: Author

ZOOLOGY Immunology Complement System

7. Receptors for Complement Proteins

The biological activity of complement system depends on their binding to various receptors present on different cell types. These receptors not only mediate biological function of complement proteins, but also regulate them by inactivating the activated form of complement proteins upon binding. Some of the major receptors and the cells which they are present on, along with their ligand and their function are mentioned in table 6 below.

Table 6: Various cell receptors for complement components

Source: Modified from: Modified from Janeway‟s Immunobiology 8th ed. by Kenneth Murphy & Kuby‟s Immunology 4th ed. by R. A. Goldsby, T. J. Kindt and B. A. Osborne

8. Regulation of Complement System

Elaborate regulatory system has evolved to restrict the non-specific activity of complement system on host cells. A general way of regulation is achieved by labile nature of all the complement components which undergo spontaneous inactivation on diffusing away from target cells. A number of regulatory proteins also serve as regulators of complement system as listed in table below. Many of these proteins are encoded at a single location on chromosome 1 in humans and known as regulators of complement activation (RCA) gene cluster. The regulatory proteins operate at all types and levels including classical, alternative, lectin and terminal pathway and also at effector level.

ZOOLOGY Immunology Complement System

Table 7: Regulatory proteins for complement components

Source: Modified from: Modified from Janeway‟s Immunobiology 8th ed. by Kenneth Murphy & Kuby‟s Immunology 4th ed. by R. A. Goldsby, T. J. Kindt and B. A. Osborne 9. Diseases due to Complement Deficiency

Deficiencies in various complement components result in number of diseases which illustrate the homeostatic roles of complement. Generally, complement deficiencies are rare but some are very common in specific racial groups. Some of the common impacts/ diseases arising due to complement deficiency are:

 Deficiencies of classical pathway: Deficiency of any of the classical pathway component i.e. C1, C3, C4, C2 predisposes an individual to a condition, similar to systemic lupus erythematosus

ZOOLOGY Immunology Complement System

(SLE). In SLE, immune complexes i.e. antigen-antibody complexes gets deposited in skin, kidney, blood vessels etc.  Deficiencies of alternative pathway: Deficiency of factor B or factor D reduces the efficiency of opsonization of pathogens. It leads to recurrent bacterial infections in the affected individuals.  MBL (or Lectin pathway) deficiency: MBL deficiency in infants makes them susceptible to bacterial infections. But as the age progresses, this susceptibility disappears as other immune mechanisms mature. In adults, MBL deficiency doesn‟t lead to any serious consequences unless accompanied with other immunosuppression/ immunodeficiency.  Deficiencies of terminal pathway: Deficiency of any of the terminal pathway component i.e. C5, C6, C7, C8 or C9, predisposes the person to Gram-negative bacteria infection particularly Neisseria genus. Gonnorhoea and meningococcal meningitis are common diseases caused by Neisseria.  C1Inh deficiency: C1Inh is a classical pathway regulator whose deficiency leads to hereditary angioedema which is an autosomal dominant disease.  Properdin deficiency: Properdin, which is a stabilizer of alternative pathway C3 convertase, shows X-linked inheritance in its deficiency. Thus, properdin deficiency is exclusive in males only. Males deficient in properdin shows meningococcal meningitis. 10. Complement Deficiency Assay Systems

The diagnostic approaches for detecting structural and/ or functional deficiency in complement system are broadly classified as:

 Global assays for identification of lack of pathway function: Either hemolytic assays or ELISA with IgM, Mannan, LPS coated plates is used for such analysis. The total functional activity or functional activity of single complement components can be performed in such assays.  Protein chemical, cytofluorometric and functional assay for individual component and regulators, irrespective of their functional activity: By radial immunodiffusion, nephelometry or ELISA.  Molecular analysis to identify genetic alterations: Polymorphism or mutation in complement components can be studied by genetic mapping. 11. Microbial Evasion Strategies from Complement Mediated Damage

Though complement system is very effective in providing immunity against various pathogen but microbes have also evolved a number of strategies to breach this system. Complement system is more effective against Gram negative bacteria as compared to Gram positive ones. A few Gram negative and most Gram positive bacteria adopt different survival strategies by fooling our complement system. Some of their common evading mechanisms along with examples are mentioned in table below:

ZOOLOGY Immunology Complement System

Figure 7: Some of the evading strategies adopted by microbes to survive from complement mediated damage. Source: Modified from Kuby‟s Immunology 4th ed. by R. A. Goldsby, T. J. Kindt and B. A. Osborne 12. Summary

 Complement system is a group of around 30 soluble proteins synthesized by liver and present in inactive form in serum in the absence of infection.  They were first discovered by Jules Bordet as a heat-labile component of serum that „complemented‟ or „augmented‟ its bactericidal properties.  Complement system is the major component of innate system which is encountered by pathogens that enter our body after breaching physical barriers and antimicrobial defenses.  Moreover, it provides a link between two different branches of immunity i.e. innate and adaptive.  There are three different pathways for activation of complement i.e. classical pathway, alternative pathway and lectin pathway. All these pathways ultimately lead to a common terminal pathway, leading to formation of membrane attack complex which subsequently lyses the target cell.  Three broad categories of functions that complement system performs are: host defense against various infections, interface between innate and adaptive immunity and clearance of waste.  The biological activities of complement system are governed upon binding of complement components and fragments with various receptors, present on different cell types. Additionally, these receptors regulate the complement activity.  Various regulatory proteins are there that keep a check on the activity of complement system to avoid the damage to host itself.  Genetic or functional deficiency of different complement proteins result in number of diseases which illustrates the homeostatic role of complement system.  Different diagnostic methods are available now to detect the overall complement deficiency or deficiency of any specific component.  A number of pathogens have evolved evading strategies from complement system. Particularly, Gram positive bacteria and some Gram-negative bacteria and viruses are among these smart evaders.

ZOOLOGY Immunology Complement System