Complement system functions and nomenclature, University of Portsmouth

Complement System Functions and Nomenclature

Learning Objectives

On completion of this session you should be able to

1. Define complement system and understand the important function of complement system as a crucial part of the innate immune response.
2. Learn the naming conventions and numbering system of complement proteins.

Complement System Overview

1. A set of serum proteins that act in a cascade to attack and kill extracellular pathogens
2. Approximately 30 components
3. Most of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogene)
4. Activation is induced by proteolytic cleavage
5. Cooperates with both the innate and the adaptive immune systems to eliminate pathogens, dying cells, and immune complexes from the body

Functions of Complement

1. Complement components have 3 functions: a) to opsonise bacteria and promote phagocytosis, b) to generate pores in bacterial membranes (membrane attack complex) to kill bacteria c) to mediate the inflammatory response

Complement Functions: Lysis, Opsonization, Activation, Clearance

LYSIS OPSONIZATION ACTIVATION OF CLEARANCE OF INFLAMMATORY RESPONSE IMMUNE COMPLEXES Complement receptor Bacteria Complement 5 Degranulation Extravasation Tissue Blood Target cell Phagocyte Ag-Ab complex Phagocyte Figure 7-1 Kuby IMMUNOLOGY, Sixth Edition @ 2007 W.H.Freeman and Company

Components of Complement System

1. Soluble proteins and glycoproteins
2. Synthesised mainly by liver hepatocytes and other cell types
3. 5% of serum globulins
4. Circulate as inactive proenzymes - proteolytic cleavage removes inhibitory fragments and exposes active site

Complement Nomenclature

1. Designated by numerals, letter symbols, or trivial names
2. Examples: C1-C9, factor D, homologous restriction factor
3. Not numbered according to sequence of activation, which is as follows: C1, C4, C2, C3, C5, C6, C7, C8, C9
4. Components are activated by cleavage to give 2 fragments designated a and b

Complement Fragment Naming

1. Large fragments are denoted as b (big fragment!) and small as a (except for C2): C3 -> C3b + C3a
2. Components unique to alternative pathway and some regulatory proteins are designated by uppercase letters e.g. B and D
3. Cleavage products also designated by lowercase e.g B -> Bb + Ba.

Summary of Complement System

1. The complement system is a key part of the innate immune response, critical for pathogen clearance and immune defense.
2. Learning the naming conventions and numbering system of complement proteins, such as C1, C3, and their activated fragments like C3a and C3b, is essential for grasping the complexity and organisation of this system.

Complement Activation Pathways

Learning Objectives for Complement Activation

On completion of this session you should be able to

1. Comprehend the classical, lectin, and alternative pathways of complement activation, including their unique triggers and components involved.
2. Explain the sequence of events in the complement activation cascade for each pathway and how they converge at the formation of C3 convertase.
3. Recognise the key proteins and complexes involved in each pathway, and their roles in the activation process.

Complement Activation Pathways Overview

1. Three pathways by which complement is activated: · Classical Pathway - activated by antibody:antigen (Ab:Ag) complexes (adaptive) and pentraxins · Mannan binding lectin (MBL) pathway by binding to carbohydrate on pathogen surface (innate) · Alternative Pathway - part of innate immune response and amplifies classical and MBL pathways
2. Final steps leading to membrane-attack complex (MAC) are identical in all 3 pathways

Complement Activation Overview Diagram

CLASSICAL MB-LECTIN PATHWAY ALTERNATIVE AB:AG COMPLEX PATHOGEN SURFACE ACTIVATION OF COMPLEMENT PROTEINS RECRUITMENT OF INFLAMMATORY CELLS, ACTIVATION OF ENDOTHELIUM ETC OPSONISATION OF PATHOGEN FORMATION OF MEMBRANE ATTACK COMPLEX (MAC)

Classical Pathway Activation

1. Activated by Ab:Ag interactions (adaptive response, most commonly IgM and IgG)
2. First step is the binding of C1 to antibody on the surface of the pathogen
3. C1 has three subunits - C1q, C1r and C1s
4. One C1q is associated with two molecules each of C1r and C1s i.e. (C1r:C1s)2
5. C1q binds to antibody and undergoes a conformational change

Classical Pathway Cascade

1. This change activates C1r which cleaves C1s
2. C1s is activated and cleaves C4 to give C4b and C4a
3. C4b binds covalently to the pathogen surface
4. Bound C4b binds C2, localising it on the pathogen surface and allowing it to be cleaved by C1s
5. This generates the complex C4b2a which is the C3 convertase of the classical pathway

Classical Pathway: C1q Binding

1 C1q binds antigen-bound antibody. C1r activates auto-catalytically and activates the second C1r; both activate C1s. C1qr2S2 C1q C1r2S2 Antibody FC Figure 7-5 part 1 Kuby IMMUNOLOGY, Sixth Edition 2007 W.H.Freeman and Company

Classical Pathway: C4 and C2 Cleavage

2 C1s cleaves C4 and C2. Cleaving C4 exposes the binding site for C2. C4 binds the surface near C1 and C2 binds C4, forming C3 convertase. C4 C2 D C4a C2b C4b2a C3 convertase Figure 7-5 part 2 Kuby IMMUNOLOGY, Sixth Edition 2007 W.H. Freeman and Company

Classical Pathway: C3 Hydrolysis and C5 Convertase Formation

3 C3 convertase hydrolyzes many C3 molecules. Some combine with C3 convertase to form C5 convertase. + C3b C3a C3 C4b2a C4b2a3b C5 convertase Figure 7-5 part 3 Kuby IMMUNOLOGY, Sixth Edition @ 2007 W.H.Freeman and Company

Classical Pathway: C5 Cleavage

4 The C3b component of C5 convertase binds C5, permitting C4b2a to cleave C5. + C5b C5a C5 C5 convertase Figure 7-5 part 4 Kuby IMMUNOLOGY, Sixth Edition @ 2007 W.H.Freeman and Company

Classical Pathway: Membrane Attack Complex Initiation

5 C5b binds C6, initiating the formation of the membrane-attack complex. C6 C7 C8 C5b C5b67 C5b678 C5b678 C9 Poly-C9 Membrane-attack complex Figure 7-5 part 5 Kuby IMMUNOLOGY, Sixth Edition @ 2007 W.H.Freeman and Company

C3 Complement Protein

1. Most abundant complement protein in plasma
2. Up to 1000 molecules can bind in vicinity of C3 convertase
3. Main effect of complement activation is the deposition of large quantities of C3b on pathogen surface (opsonin)
4. C3b binds C3 convertase allowing it to bind and activate C5 (i.e generates C5 convertases - C4b2a3b and C3bBb3b)
5. C5b initiates formation of MAC
6. Thus, all three pathways result in the activation of C3 and, to a lesser extent C5
7. These components are central to the effector mechanisms of the complement pathway

Membrane Attack Complex (MAC) Function

1. Final stage of classical pathway involves formation of MAC which is also called lytic unit
2. Form pores in cell membrane
3. Ions and small molecules can freely pass through pores
4. Cells cannot maintain osmotic stability, leading to swelling and cell lysis

Membrane Attack Complex (MAC) Formation

C5b binds C6 and C7 C5b67 complexes bind to membrane via C7 C8 binds to the complex and inserts into the cell membrane C9 molecules bind to the complex and polymerize 1-16 molecules of C9 bind to form a pore in the membrane C6 C7 C8 1 C9 C5b C5b67 complex lipid bilayer Pathogen Membrane lesions-end on (rings) Membrane lesions-side on (tubes) Schematic representation of the membrane-attack complex pore 15nm 3nm 10 nm Figure 2-35 Immunobiology, 6/e. ( Garland Science 2005)

MBL Pathway Activation

1. Antibody-Independent
2. However, proceeds more like classical pathway
3. Uses C4 and C2
4. MBL is present in low concentrations in plasma and increased during acute phase response
5. MBL forms a complex with two enzymes called MASP-1 and MASP-2 (mannan binding lectin associated serine proteases)
6. First step is the binding of MBL to mannose residues on the surface of the pathogen
7. On binding MBL undergoes a conformational change that activates MASP-2

MBL Pathway Cascade

1. MASP-2 cleaves a second MASP-2 to generate active protease
2. The role of MASP-1 is unclear
3. Activated MASP-2 cleaves plasma protein C4 to give C4b which binds covalently to the surface of the pathogen
4. C4b binds C2 making it susceptible to cleavage by MASP-2
5. MASP-2 cleaves C2 to give C2b which remains bound to C4b
6. The complex C4b2b is also the C3 convertase of the MB lectin pathway
7. Role of C3 convertase is activation of C3 to C3a and C3b
8. Thus pathogen becomes coated with C3b
9. C3 convertase is covalently bound to pathogen so activation of C3 is localised

Alternative Pathway Initiation

1. Antibody-Independent
2. Initiated by cell surface constituents foreign to host
3. E.g. Gram+ or Gram- bacteria

TABLE 7-1 Initiators of the alternative path- way of complement activation PATHOGENS AND PARTICLES OF MICROBIAL ORIGIN Many strains of gram-negative bacteria Lipopolysaccharides from gram-negative bacteria Many strains of gram-positive bacteria Teichoic acid from gram-positive cell walls Fungal and yeast cell walls (zymosan) Some viruses and virus-infected cells Some tumor cells (Raji) Parasites (trypanosomes) NONPATHOGENS Human IgG, IgA, and IgE in complexes Rabbit and guinea pig IgG in complexes Cobra venom factor Heterologous erythrocytes (rabbit, mouse, chicken) Anionic polymers (dextran sulfate) Pure carbohydrates (agarose, inulin) SOURCE: Adapted from M. K. Pangburn, 1986, in Immunobiology of the Complement System, G. Ross, ed., Academic Press, Orlando. Table 7-1 Kuby IMMUNOLOGY, Sixth Edition 2007 W.H. Freeman and Company

Alternative Pathway Cascade

1. Activated by C3b bound to pathogen
2. C3b generated by classical pathway, MB lectin pathway or spontaneous cleavage
3. Bound C3b binds factor B which induces its cleavage to generate Bb, by the protease factor D
4. C3bBb is the C3 convertase of the alternative pathway
5. C3bBb cleaves more C3 to coat pathogen with C3b
6. Again, cleavage is localised