Complement

Kerin Fresa-Dillon, Ph.D. Lecture 61 January 10, 2011 Objectives 1. To understand the key differences between the classical, alternate and lectin pathways for activating complement 2. To understand the critical role(s) of C3 in each complement cascade 3. To understand complement regulatory circuits 4. To identify the elements and activation cascade for the membrane attack complex 5. To correctly match individual elements of the complement cascade with their biologic activities What is complement? Complement is a group of approximately 30 proteins whose functions complement the antigen-binding function of antibodies. Complement is essentially a proteolytic cascade. Each complement component exists in the plasma in an inactive or zymogen form. When activated (usually by proteolytic cleavage), each component cleaves the next component in the cascade. This process leads to the deposition or fixing of complement components to the pathogen surface Major functions of complement 1. Opsonization 2. Direct lysis of target cells/pathogens 3. Chemoattraction and activation of inflammatory cells There are three pathways for activating complement (in order of activation) 1. Alternative pathway: This pathway does not involve immunoglobulin deposition on a pathogen surface. It is triggered at sites of microbial infection 2. Lectin pathway: This pathway also does not involve immunoglobulin deposition on a pathogen surface. It is activated by binding of a plasma protein to mannose-containing peptidoglycans on microbial surfaces 3. Classical pathway: triggered as an effector mechanism when certain isotypes of immunoglobulin or C-reactive protein bind to a pathogen

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Important point: the central role of C3 in all complement cascades The central event in complement activation is proteolysis of the complement protein C3 to generate biologically active products and the subsequent covalent attachment of a product of C3, called C3b, to microbial cell surfaces or to antibody bound to antigen. -Abbas and Lichtman. Cellular and Molecular Immunology. 6th Ed. p. 330, 2007. Why? 1. Patients lacking C3 are prone to successive severe infections 2. C3 represents an important amplification step in the complement cascade 3. When complement is activated by infection: leads to the cleavage of C3 into C3a and C3b a. C3b becomes covalently bound (fixed) to the pathogen surface i. Tags pathogen for destruction by phagocytes (opsonization) ii. Organizes the formation of protein complexes that damage the pathogens membrane (MAC) b. Soluble C3a fragment i. Chemoattractant to recruit effector cells (including phagocytes)

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How is complement activated? 1. Alternative pathway a. Triggered with a hydrolysis product of C3 is deposited on a pathogen surface 2. Lectin pathway a. Initiated when mannose binding protein binds to mannose on the pathogen surface b. MBP structurally similar to C1 in classical pathway c. How is complement activated? 3. Classical pathway a. IgM bound to targets on cell/pathogen surface b. IgG (two or more) bound to targets on cell/pathogen surface c. C reactive protein i. Pentameric protein similar to C1 Order of complement activation in classical pathway (commit this to memory) C1, C4, C2, C3, C5, C6, C7, C8, C9 C5b-C9 represents the common terminal pathway for all three pathways

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Classical pathway in detail C1 complex consists of three proteins 1. C1q 2. C1r 3. C1s

IgM has receptors for C1q

1. IgM in planar (soluble) configuration does not bind C1q 2. Upon binding to pathogen surface, IgM undergoes a conformational change to a staple form, which can then bind C1q at multiple sites 3. C reactive protein: another pentameric protein with multiple binding sites for C1q

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IgG also has receptors for C1q (IgG1 and IgG3 fix complement via the classical pathway better than IgG2)

Activation of C1 1. activated C1q activates C1r 2. activated C1r activates C1s 3. Activated C1s has proteolytic activity on C4 and C2 Regulation of C1 1. Proteolytic activity of C1r and C1s is inhibited by C1 INH 2. Deficiency of C1 INH causes hereditary angioneurotic edema a. Autosomal dominant b. Intermittent skin and mucosal edema i. Abdominal pain, vomiting, diarrhea, airway obstruction c. Caused by increased breakdown of C2 and C4 i. Proteolytic fragment of C2 (C2 kinin) is responsible for edema The C3 convertase 1. After cleavage of C4 and C2, C4b fragments that are covalently attached to the pathogen surface bind C2a 2. C4b2a is the classical C3 convertase

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3. Important note: There is a discrepancy among textbooks as to what they call the C2 cleavage product that becomes part of the C3 convertase and which one is released. For historical reasons, the small cleavage product of C2 is called C2b and the larger one C2a. Whereas for other complement components, the smaller cleavage component is called the a fragment (ex: C3a, C4a) and the larger one is called the b fragment (ex: C3b, C4b). I will never ask you if the C3 convertase is C4b2a or C4b2b (you should know that the C3 convertase is composed of cleavage products of C4 and C2) 4. C3 is activated by C3 convertase and is split into C3a and C3b a. important amplification step b. C3b becomes attached to the target cell membrane 5. C3b also binds to C4b2a to form active C3b2a4b complex which is called the C5 convertase C5 convertase

1. C5 convertase splits C5 into C5a and C5b 2. each have important biologic properties 3. C5a is an anaphylatoxin (so is C3a) a. Smooth muscle contraction b. degranulation of mast cells and basophils c. release of histamine and other vasoactive amines d. Increase vascular permeability 4. C5a is also a chemotaxin for neutrophils a. substances that attract phagocytic cells and cause their migration from areas of lower concentration to areas of higher concentration 5. C5b initiates the membrane attack complex

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Classical pathway: The membrane attack complex

Important point for standardized testing: Deficiency in terminal complement components is associated with an increased susceptibility to infection by Neisseria! The alternate pathway: General points 1. First to act 2. Initiated by hydrolysis of a thioester bond in C3 that occurs in the plasma at low level but is increased when pathogens are present a. When C3 is first made in the liver, a thioester bond is sequestered in the hydrophilic innards of the protein 3. In the aqueous environment of the plasma, conformational change occurs a. Thioester bond is exposed and hydrolysed b. Hydrolysis occurs at low rate c. Yields a molecule called iC3 (C3H20)

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The alternative pathway: Formation of the alternative C3 convertase 1. iC3 binds to the inactive serum factor B 2. Makes serum factor B susceptible to cleavage by Factor D a. Makes smaller Ba and larger Bb 3. Factor Bb has protease activity and cleaves more C3 into C3a and C3b a. iC3Bb is a soluble C3 convertase 4. C3b gets deposited on pathogen surface a. Recruits more factor B b. Facilitates the cleavage of Factor B by Factor D c. Resulting complex of C3bBb is the alternative C3 convertase Regulation of C3 in the alternative pathway 1. Properdin a. Increases the speed and power of complement activation by binding the C3 convertase C3bBb on the pathogen surface b. Prevents its degradation by proteases 2. Factor H counteracts properdin a. Facilitates cleavage of C3b to iC3b by plasma factor I b. iC3b cannot form the C3 convertase 3. Factor I deficiency a. Formation of the C3bBb (C3 convertase) is unchecked until depletes reservoir of C3 b. When faced with bacterial infection: Less C3b deposited c. Individuals are more susceptible to ear infections and abscesses caused by encapsulated bacteria i. These infections cleared more efficiently when opsonized by C3b 4. Another type of regulatory proteins prevent complement activation on human cell surfaces a. Decay accelerating factor: DAF binds to C3b in the alternative C3 convertase, causing dissociation and inactivation b. Membrane co-factor protein: MCP binding to C3b makes it susceptible to action of Factor I (so its similar to Factor H)

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The alternative C5 convertase

The C5 convertase is assembled when a C3b fragment created by C3bBb is added C3bBb: C3 convertase C3bC3bBb: C5 convertase
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The alternative pathway:membrane attack complex works just like the classical one! Complement receptors 1. CR1 a. Expressed on many cell types, including i. macrophages and neutrophils ii. erythrocytes (facilitates transport of immune complexes to the spleen for clearance) b. Bind C3b or C4b on pathogen surface (high affinity) c. Facilitate uptake and destruction of pathogen i. C3b and, to a lesser degree, C4b are acting as opsonins d. Markedly enhanced when Fc region of IgG also binds phagocyte via FcR or in presence of IFN- 2. CR2 a. Expressed on follicular dendritic cells and B cells b. Component of the B cell receptor complex (co-receptor) c. Binds to degradation products of C3b (C3d) on microbial surfaces i. Delivers activation signals to B cell ii. Innate to acquired immune response bridge d. Later: you will learn it is the surface receptor for Epstein-Barr virus 3. CR3 and CR4 a. -integrins i. CR3 (also called CD11b/CD18 and Mac-1) ii. CR4 (also called CD11c/CD18) b. Bind iC3b (proteolytic product of C3b) c. Both expressed on phagocytes i. Promotes phagocytosis of microbes opsonized with iC3b ii. Also involved in adherence of leukocytes to endothelial cells at sites of inflammation via binding to ICAM-1 (CR3, maybe CR4)

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