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Complement System (CS) Part II Lukas Menges 24.5.2016 Molecular Immunology CONTENTS ▪ ▪ ▪ ▪ ▪ ▪ 2 Most basic facts about the CS (repetition) Control of recognition Modulation of increasing Complement receptors (CRs) Small fragments C5a and C3a Regulation of membrane attacking 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Most basic facts about the complement system (repetition) ▪ Antibodies only detect antigens. The complement system is one system which complements the antibody function ▪ 3 modes of action: - complement fixation (binding and lysis of the cell membrane) - opsonisation (stimulation of phagocytosis) - enticement of phagocytes by triggering of inflammatory reaction ▪ CS consists of an cascade of approx. 20 zymogens (mostly serine proteases) activating each other 3 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Prevention of autoimmunity ▪ Complement activation should take place on pathogens, not on normal „harmless“ cells. How it is ensured? ▪ All activated zymogens bind nearby, otherwise they are rapidly inactivated by hydrolysis ▪ Complement regulatory proteins protect endogenous cells from complement activation on their surfaces 4 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Prevention of autoimmunity ▪ Complement activation is regulated by proteins that serve to protect cells from accidental damage ▪ These proteins act on different stages of the complement cascade, dissociating complexes or catalyzing the enzymatic degradation of covalently bound complement proteins Regulation of recognition 5 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Recognition unit The recognition unit C1 consits of C1q, C1r and C1s and binds to the Fc terms of IgM and IgG. Binding is only possible if IgM and IgG have bound to antigens. ▪ C1q is composed of 18 polypeptide chains (A6B6C6) forming 6 triple bundles (“bunch of tulips”) ▪ At least two C1q subunits must be linked to immunoglobulins to activate C1 („lower limit“) Prevention that one unspecific antibody activates CS ▪ Two IgGs or one IgM activate(s) C1. Why?! 6 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY EM-picture ▪ Stage at which complement activity is regulated C1q binding to antibodies: C1r and C1s are activated C1 inhibitor (C1NH) dissociates C1r and C1s from the active C1 complex C1r and C1s cannot propagate the complement cascade 7 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Regulation of increasing 8 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Important role of C3 ▪ Formation of C3 convertase: All pathways of complement activation converge. ▪ C3 most abundant complement protein in plasma (1.2 mg/ml) Approx. 100 proteins in plasma, abundance: 60-80 mg/ml ▪ C3 is cleaved into C3b and C3a. C3b binds to molecules on the pathogen surface ▪ C3b forms a covalently bonded coat that can signal the ultimate destruction of the pathogen by phagocytes (macrophages,…). 9 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Complement receptors (CRs) ▪ ▪ ▪ ▪ CRs bind pathogens opsonized with complement components C3b is an opsonin Receptor Specificity Functions Cell types CR1 (CD35) C3b, iC4b Promotes C3b and C4b decay. Stimulates phagocytosis (requires C5a) Erythrocyte transport of immune complexes Erythrocytes, macrophages, monocytes, polymorphonuclear leucocytes, B cells, FDC (Dendritic cell) CR2 (CD21) C3d, iC3b, EpsteinBarr virus Part of B-cell coreceptor B cells, FDC CR3 (Mac-1) iC3b Stimulates phagocytosis Macrophages, monocytes, polymorphonuclear leucocytes, FDC CR4 iC3b Stimulates phagocytosis Macrophages, monocytes, polymorphonuclear leucocytes, dendritic cells CRIg C3b, iC3b Phagocytosis of circulating pathogens Tissue-resident macrophages Hepatitic sinusoid macrophages C5a C5a Binding of C5a activates G protein Endothelial cells, Mast cells, phagocytes C3a C3a Binding of C3a activates G protein Endothelial cells, Mast cells, phagocytes C3b receptor is called CR1, specific to C3b CR1 promotes C3b decay. Stimulates phagocytosis (requires C5a) + C5a 10 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Stage at which complement activity is regulated C4b2a is the active C3 convertase, cleaving C3 into C3a and C3b DAF, C4BP and CR1 displace C2a from the C4b2a complex, C4b bound by C4BP, MCP or CR1 is cleaved by a soluble protease I to inactive forms C4d and C4c Destruction of the C3 convertase 11 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY C3b is another target of regulation ▪ 1. Competition. Decay-accelerating factor DAF, membrane-attached protein, competes with Bb for binding to C3b on the cell surface - preventing formation of the C3bBb C3 convertase ▪ 2. Cleaving. The plasma protease Factor I cleaves C3b to inactive iC3b. Needs cofactors (membrane cofactor of proteolysis = MCP or CD46) ▪ Further C3b binding proteins: Factor H, CR1 12 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Factor I deficiency ▪ ▪ ▪ Genetic disorder ▪ Symptoms: Repeated bacterial infections Uncontrolled complement activation Complement proteins rapidly become depleted because they are mostly active on “wrong” cells 13 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY A closer look at the pathway…increase 14 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY A closer look at the pathway ▪ ▪ ▪ ▪ ▪ Generation of the C5 convertases ▪ C5b and C5a propagate the complement cascade 15 Classical pathway: C4b2a3b Alternative pathway: C3b2Bb From different pathways, but same function C5 is cleaved into C5a and C5b. What is the small fragment? 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Stage at which complement activity is regulated The C5 convertases cleave C5 into C5a and C5b CR1 and H displace C3b from the convertase. CR1 and H act as cofactors in the cleavage of C3b by I Destruction of the C5 convertase 16 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY A closer look at the pathway…C3a and C5a Small fragments with a big effect 17 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY The small complement fragments C5a and C3a ▪ C5a and C3a produce local inflammatory responses by acting directly on local blood vessels, stimulating an increase in blood flow, increased vascular permeability and increased binding of phagocytes to endothelial cells. ▪ C5a also activates mast cells to release mediators, such as histamine that contributes to the inflammatory response enticement of phagocytes by triggering of inflammatory reaction 18 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY C5a is more active than C3a, which is more active than C4a Higher permeability of blood vessels Infiltrates get into the tissue Regulation of membrane-attacking 19 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Membrane attack complex (MAC) C5b is the cleavage product from the C5convertase and binds to C6 and C7 ▪ The C5b/C6/C7-complex (amphiphilic) binds to C8 and to (up to 18) C9-molecules forming a tube in the membrane ▪ ▪ The MAC is a pore with a diameter of 3-10 nm ▪ One single MAC is sufficient for killing a cell Cell loses electrolytes but no macromolecules (interior of the cell is hypertonic). Thereby the cell takes water and bursts („osmotic lysis“ or „colloidal osmotic lysis“) 20 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY EM-picture ▪ Stage at which complement activity is regulated The terminal components of complements form a membrane pore: The membrane attack complex CD59 prevents final assembly of the membrane-attack complex at the C8 to C9 stage The terminal complement proteins cannot polymerize to form a pore in membranes that kill pathogens 21 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Knowledge about CS helps us to develop immunotherapies ▪ The initiating Cells of the diffuse large B Cell Lymphoma (DLBCL) are white blood cells (B-lymphocytes) ▪ The B-lymphocyte-antigen CD20 is a glycosylated phosphoprotein, which is expressed on the membrane surface of B-cells. ▪ CD20 is a target of monoclonal antibodies due to cure B-celllymphoma and leukemia with the aid of CS https://www.youtube.com/watch?v=sINGPr6buvw 22 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Take home messages ▪ Complement activation must be regulated to protect cells from accidental damage ▪ Regulation by “lower limitation”, regulating proteins and rapid inactivation ▪ ▪ C3b receptor called: CR1 (effects phagocytosis) ▪ The membrane attacking complex (MAC) forms a pore in the membrane that kill pathogens The small complement fragments C5a and C3a produce local inflammatory responses 23 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY References ▪ ▪ ▪ ▪ ▪ 24 Janeway, 2009, pp. 59-71 Lecture: Biochemistry III, part 15, WS2015, Michael Hollmann Voeth & Voeth, 1995, figs 34.41 Alberts et al., 1994, fig. 23.27, 23.28, 23.29 https://www.youtube.com/watch?v=sINGPr6buvw, access 20.5.2017 24.5.2017 – LUKAS MENGES – MOLECULAR IMMUNOLOGY Thank you for attention! 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